**Meet the editor**

Rita Payan-Carreira earned her veterinary medical degree from the Faculty of Veterinary Medicine in Lisbon, Portugal, in 1985. She joined the University of Trás-os-Montes and Alto Douro (UTAD) in 1986 and is now an Associated Professor with Habilitation for the field of Physiology of Reproduction and Reproductive Medicine. She also entered the private practice of Small

Animal Internal Medicine in the Vila Real area. She frequently supervises students during the preparation of their final graduation (MSc.) thesis in the field of Theriogenology. She has participated in several conjoint academic initiatives with foreign Universities and is now the Adjunct Deputy for the Animal and Veterinary Research Center (CECAV), UTAD. Her main interests are focused on embryo-maternal interactions and fertility in domestic species.

Contents

**Preface VII**

Chapter 1 **Dermatology in Dogs and Cats 3**

and Evandro Silva Favarato

**Mammary Carcinoma 35** Anna M. Badowska-Kozakiewicz

**Section 2 Current Topics in Infectious Diseases 89**

Matos and Maria dos Anjos Pires

Chapter 4 **Mycobacterium avium Complex in Domestic and**

**Nairobi, Kenya 49**

Peter M. F. Mbithi

**Wild Animals 91**

Chapter 5 **Psittacosis 129**

**Section 1 Current Topics in Mammal Diseases and Welfare 1**

Elisa Bourguignon, Luciana Diegues Guimarães, Tássia Sell Ferreira

**Proliferating Cell Nuclear Antigen in Canine Inflammatory**

James Nguhiu-Mwangi, Joshua W. Aleri, Eddy G. M. Mogoa and

Ana Cláudia Coelho, Maria de Lurdes Pinto, Ana Matos, Manuela

João Morais, Ana Cláudia Coelho and Maria dos Anjos Pires

Chapter 2 **Immunohistochemical Analysis of Progesterone Receptor and**

Chapter 3 **Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of**

## Contents

**Preface XI**

#### **Section 1 Current Topics in Mammal Diseases and Welfare 1**


#### **Section 3 Current Topics on Hormones and Hormone-Related Diseases 163**


Preface

chapters of this book.

It is my pleasure to present a new book that integrates a continuing series on Veterinary Medicine. Titled "Insights from Veterinary Medicine", this book presents original research results and reviews flashing several distinctive aspects of the Veterinary Medicine Sciences,

Veterinary and Human Medicine have been developed in close association, in part as a re‐ flection of the intertwined relationship found between animals and humans since the estab‐ lishment of first civilizations. Human and animals share common mechanisms of disease, thus serving as spontaneous models for the study of particular disorders, such as tumors and cardiac diseases. Furthermore, concerns regarding the deleterious side-effects of con‐ taminants, in particular over the endocrine axis regulating different body functions and fer‐ tility, are on the table, as humans share the same environment with animals and are subject to the same influences, but also because humans can be exposed through the food chain.

The Veterinary Sciences are often at the vanguard of research in human diseases and behind improvements in human health. Several and distinctive reasons may be found for that asso‐ ciation, such as the epidemiological, since many human pathogens have their origins in ani‐ mal hosts, or the physiological, once animals used as models of disease allowed to elucidate the underlying mechanisms of many diseases. In addition, animals are a major contribution to human welfare: the quality of the animal life with regards to both health and welfare also contributes to the quality of human life, and food-animal health and safety are safeguards against the disruption of the food chain. These topics are also brought to you in some of the

Advances in tests and procedures made possible through research changed the animal world and both pets and farm animals enjoy a better quality of life today. Accompanying its tremendous development, the Veterinary Medicine also broadened its scope of action and nowadays it encompasses much more than the veterinary medical practice. Similarly, a wide field of knowledge has been covered in this book. Even so, it was our goal to provide you

Veterinary research is also essential to the health and increased longevity of service and

This book includes different chapters on new developments covering a wide range of sub‐ jects from animal medicine and welfare to public health concerns and environmental endo‐ crine disruptors. The chapters are organized into four parts or sections, according to main subjects. The initial section presents both educational and research chapters in major areas

with current advanced resources in different veterinary science disciplines.

companion animals and thereby reduces stress in both animals and owners.

in which the knowledge has continuously increased over the past decades.

Some of these subjects are presented within the book chapters.


## Preface

**Section 3 Current Topics on Hormones and Hormone-Related**

Helena Vala, João Rodrigo Mesquita, Fernando Esteves, Carla Santos, Rita Cruz, Cristina Mega and Carmen Nóbrega

Yolanda Millán, Silvia Guil-Luna, Carlos Reymundo, Raquel Sánchez-

**Amphibians and Fish: The Effects of Endocrine Disruption 241**

**Capsicum annum Seeds from South Western Uganda 257**

Chapter 7 **Sex Steroid Hormones and Tumors in Domestic Animals 191**

Chapter 6 **The Endocrine Glands in the Dog: From the Cell**

Céspedes and Juana Martín de las Mulas

Chapter 8 **Feline Mammary Fibroepithelial Hyperplasia: A Clinical**

Chapter 9 **Diseases of Thyroid in Animals and Their Management 233**

Chapter 10 **Crossregulation of the Thyroid Hormone and Corticosteroids in**

**Diseases 163**

**VI** Contents

**to Hormone 165**

**Approach 215** Rita Payan-Carreira

R. Singh and S. A. Beigh

Xavier Terrien and Patrick Prunet

**Section 4 Highlights for Homeopathic Therapeuthicals 255**

Charles Lagu and Frederick I. B. Kayanja

**Extracts on Ascaridia galli 269** Charles Lagu and FIB Kayanja

Chapter 11 **Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of**

Chapter 12 **The in vitro Antihelminthic Efficacy of Erythrina Abyssinica**

It is my pleasure to present a new book that integrates a continuing series on Veterinary Medicine. Titled "Insights from Veterinary Medicine", this book presents original research results and reviews flashing several distinctive aspects of the Veterinary Medicine Sciences, in which the knowledge has continuously increased over the past decades.

Veterinary and Human Medicine have been developed in close association, in part as a re‐ flection of the intertwined relationship found between animals and humans since the estab‐ lishment of first civilizations. Human and animals share common mechanisms of disease, thus serving as spontaneous models for the study of particular disorders, such as tumors and cardiac diseases. Furthermore, concerns regarding the deleterious side-effects of con‐ taminants, in particular over the endocrine axis regulating different body functions and fer‐ tility, are on the table, as humans share the same environment with animals and are subject to the same influences, but also because humans can be exposed through the food chain. Some of these subjects are presented within the book chapters.

The Veterinary Sciences are often at the vanguard of research in human diseases and behind improvements in human health. Several and distinctive reasons may be found for that asso‐ ciation, such as the epidemiological, since many human pathogens have their origins in ani‐ mal hosts, or the physiological, once animals used as models of disease allowed to elucidate the underlying mechanisms of many diseases. In addition, animals are a major contribution to human welfare: the quality of the animal life with regards to both health and welfare also contributes to the quality of human life, and food-animal health and safety are safeguards against the disruption of the food chain. These topics are also brought to you in some of the chapters of this book.

Advances in tests and procedures made possible through research changed the animal world and both pets and farm animals enjoy a better quality of life today. Accompanying its tremendous development, the Veterinary Medicine also broadened its scope of action and nowadays it encompasses much more than the veterinary medical practice. Similarly, a wide field of knowledge has been covered in this book. Even so, it was our goal to provide you with current advanced resources in different veterinary science disciplines.

Veterinary research is also essential to the health and increased longevity of service and companion animals and thereby reduces stress in both animals and owners.

This book includes different chapters on new developments covering a wide range of sub‐ jects from animal medicine and welfare to public health concerns and environmental endo‐ crine disruptors. The chapters are organized into four parts or sections, according to main subjects. The initial section presents both educational and research chapters in major areas

of bovine and pet internal medicine, ending with a paper highlighting important issues on dairy cattle welfare in the tropics. The second section presents information pertaining to the field of public health and discusses important zoonosis evolving on domestic and wild ani‐ mals worldwide. The third section highlights major aspects of hormones and hormone relat‐ ed diseases in mammals and aquatic organisms, including aspects associated to environmental endocrine disruptors. Other materials related to the pharmacological and toxicological effects of natural explants used as natural therapeutics in particular areas of the globe are included in the final section.

I wish to thank all the authors for their contribution and efforts in improving the quality of the book content, as well as the support by the members of InTech editorial and produc‐ tion team, for providing a stimulating and pleasant atmosphere during the book production.

To the reader, I hope you find this to be an interesting book well worth reading by any prac‐ titioner, graduating student or researcher searching for either highlights or an overview on current topics in the Veterinary Science field.

> **Rita Payan-Carreira** CECAV [Veterinary and Animal Research Centre] University of Trás-os-Montes and Alto Douro, Dept. Zootechnics, Vila Real, Portugal

**Section 1**

**Current Topics in Mammal Diseases and Welfare**

**Current Topics in Mammal Diseases and Welfare**

of bovine and pet internal medicine, ending with a paper highlighting important issues on dairy cattle welfare in the tropics. The second section presents information pertaining to the field of public health and discusses important zoonosis evolving on domestic and wild ani‐ mals worldwide. The third section highlights major aspects of hormones and hormone relat‐ ed diseases in mammals and aquatic organisms, including aspects associated to environmental endocrine disruptors. Other materials related to the pharmacological and toxicological effects of natural explants used as natural therapeutics in particular areas of the

I wish to thank all the authors for their contribution and efforts in improving the quality of the book content, as well as the support by the members of InTech editorial and produc‐ tion team, for providing a stimulating and pleasant atmosphere during the book production. To the reader, I hope you find this to be an interesting book well worth reading by any prac‐ titioner, graduating student or researcher searching for either highlights or an overview on

**Rita Payan-Carreira**

Dept. Zootechnics, Vila Real, Portugal

CECAV [Veterinary and Animal Research Centre] University of Trás-os-Montes and Alto Douro,

globe are included in the final section.

VIII Preface

current topics in the Veterinary Science field.

**Chapter 1**

**Dermatology in Dogs and Cats**

Elisa Bourguignon, Luciana Diegues Guimarães, Tássia Sell Ferreira and Evandro Silva Favarato

The skin is the largest organ of the body with many different functions as thermoregulation, immune protection, sensory perception, vitamin D production and it acts as a barrier be‐ tween the animal and the environment. Besides all of these important functions and the dis‐ eases that affect directly the skin, it may also share or reflect pathologic processes from other tissues. Due to these characteristics, dermatologic problems are among the most commonly seen disorders in veterinary hospitals. It is important for the veterinarian to know and un‐ derstand about the physiology of the skin and about the most common dermatologic disor‐

The skin is divided in three layers: epidermis, dermis and hypodermis (Figure 1). Epider‐ mis, the outermost layer of the skin, is composed by keratinocytes, melanocytes and Langer‐ hans cells. Keratinocytes are also disposed in layers in the epidermis. The deepest one, the stratum basale, is formed by a single row of germinative keratinocytes and also contains melanocytes. These germinative keratinocytes generate the other layers by cell division and differentiation. The next layer, stratum spinosum, differs from stratum basale by the pres‐ ence of intercellular junctions. Langerhans cells are also present in this layer. Stratum granu‐ losum is characterized by a large amount of keratohyaline granules inside keratinocytes which are important in the skin keratinization process. Stratum corneum, the outer epider‐ mis layer, is composed by keratinocytes in their maximal differentiation degree (corneo‐

> © 2013 Bourguignon et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Bourguignon et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons

ders that affects dogs and cats, which will be addressed in this chapter.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53660

**1. Introduction**

**2. Skin structure**

cytes), interspersed in lipid matrix [1].

**Chapter 1**

## **Dermatology in Dogs and Cats**

Elisa Bourguignon, Luciana Diegues Guimarães, Tássia Sell Ferreira and Evandro Silva Favarato

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53660

**1. Introduction**

The skin is the largest organ of the body with many different functions as thermoregulation, immune protection, sensory perception, vitamin D production and it acts as a barrier be‐ tween the animal and the environment. Besides all of these important functions and the dis‐ eases that affect directly the skin, it may also share or reflect pathologic processes from other tissues. Due to these characteristics, dermatologic problems are among the most commonly seen disorders in veterinary hospitals. It is important for the veterinarian to know and un‐ derstand about the physiology of the skin and about the most common dermatologic disor‐ ders that affects dogs and cats, which will be addressed in this chapter.

## **2. Skin structure**

The skin is divided in three layers: epidermis, dermis and hypodermis (Figure 1). Epider‐ mis, the outermost layer of the skin, is composed by keratinocytes, melanocytes and Langer‐ hans cells. Keratinocytes are also disposed in layers in the epidermis. The deepest one, the stratum basale, is formed by a single row of germinative keratinocytes and also contains melanocytes. These germinative keratinocytes generate the other layers by cell division and differentiation. The next layer, stratum spinosum, differs from stratum basale by the pres‐ ence of intercellular junctions. Langerhans cells are also present in this layer. Stratum granu‐ losum is characterized by a large amount of keratohyaline granules inside keratinocytes which are important in the skin keratinization process. Stratum corneum, the outer epider‐ mis layer, is composed by keratinocytes in their maximal differentiation degree (corneo‐ cytes), interspersed in lipid matrix [1].

© 2013 Bourguignon et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Bourguignon et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Dermis, the layer under the epidermis, is composed by a conjunctive matrix where reticular, elastic and collagen fibers are found. Dermis cellular structure is composed by fibroblasts, mast cells and histiocytes. It also contains epidermal appendages (hair, nails, sebaceous and sweat glands), arrector pili muscles and blood and lymph vessels. Hypodermis or subcuta‐ neous tissue provides support and cushioning against physical trauma. It is composed by a loose connective tissue and elastic fibers interspersed by adipocytes [1]. The hair follicles ex‐ hibit activity cycles that result in hair formation. Anagen is a period of active growth when a new hair is being formed. Catagen is when the hair growth stops and degenerative changes occur in the base of the follicle. Telogen represents a period of follicle inactivity, when the hair is shed so that a new one may start to grow [1]. Hair cycle activity, in some dog breeds, is strongly related to temperature variation and photoperiod, leading to decreased hair den‐ sity in the warmer months, which helps the heat loss in these animals [2].

ed in pyoderma refractory to initial therapy or when rods are observed in cytology [4]. The direct examination of the hairs, the trichogram, is performed by pulling the hairs from the affected area, followed by microscopic evaluation. With this technique it is possible to deter‐ minate hair growth phase abnormalities and the presence of follicles parasites and dermato‐ phytes [3]. Cytology is the analysis of tissue cells and it is a highly efficient and valuable exam to evaluate a lesion, after which it is possible to establish the next step in the diagnos‐ tic approach. The sample may be obtained by fine-needle aspiration, swab, skin scrapings or lesion imprint [5]. Histopathology associated with clinical findings usually leads to defini‐ tive diagnosis. Skin biopsy is recommended in unusual lesions, possibly neoplastic nodules, dermatosis with expensive therapy or when it represents a risk to the patient health, with poor response to previous therapy and to exclude differential diagnoses [4]. To perform the histopathological exam at least three representative samples should be obtained by punch or

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 5

Pyoderma is a bacterial skin infection and it is among the most common causes of skin dis‐ eases in dogs [6], however, it is less common in cats [7]. Lesions may be superficial and in‐ volve only the epidermis or they may affect deeper structures in the dermis or subcutaneous tissue, and it is therefore divided into surface, superficial and deep pyoderma [6]. *Staphylo‐ coccus pseudintermedius* is the most commonly isolated bacteria from dog's skin [8] and it is

Surface pyodermas are characterized by superficial erosions of the stratum corneum. The presence of alopecia, erythema and pruritus are common findings [10]. Intertrigo is a surface pyoderma that affects the skin folds found in lips, face, vulva, tail and mammary glands of some breeds, and it may also affect the skin folds of obese animals. Acute moist dermatitis, also known as pyotraumatic dermatitis or hot spots, is of acute onset and rarely occurs as a primary disease in healthy skin, being usually secondary to other diseases [11]. Probably, lo‐ cal irritation due to an underlying cause leads to self-inflicted trauma, which quickly be‐

Superficial pyodermas are the most common causes of cutaneous bacterial infection in dogs [6]. They affect the superficial portion of the hair follicles (bacterial folliculitis) or the epider‐ mis (impetigo), causing pustules [10, 11]. The most common lesions are crusted papules due to the transient nature of canine pustules. Pruritus, epidermal collarettes, hyperpigmenta‐ tion and alopecia are also common findings [6]. Impetigo affects sexually immature dogs that may present subcorneal pustules formed in inguinal and axillary areas [10]. Superficial bacterial folliculitis is the most common form of pyoderma in dogs [12]. Papules, pustules associated with hair follicles, epidermal collarettes, alopecia and hyperpigmentation are

Deep pyoderma does not occur spontaneously, often starting as superficial pyoderma [6]. Other organisms such as *Proteus* spp., *Pseudomonas* spp. and *E. coli* may be involved [12-14].

among the main reasons for antimicrobial use in these animals [9].

surgical resection [3].

**4. Bacterial skin diseases**

comes extensive areas of skin damage [10].

commonly found [10] (Figure 2).

**Figure 1.** Structure of the skin.

## **3. Diagnosis techniques**

Diagnosis approach for skin diseases depends on obtaining detailed history with thorough physical and dermatological examination. The evaluation offers precious information and guides the investigative process. Specific tests and, occasionally, therapeutic trials should be performed [3].

Skin scrapings, trichogram, fungal and bacterial culture, cytological evaluation and skin bi‐ opsy are important diagnosis techniques in dermatology. Skin scrapings are applied in mite detection, and it may be superficial or deep depending on the mite that is suspected. The sample obtained is evaluated under optical microscopy [3]. Fungal culture is recommended for patients that fungal diseases are suspected. Hair samples and skin scales should be col‐ lected from the lesion margin. Bacterial culture is not often performed and it is recommend‐ ed in pyoderma refractory to initial therapy or when rods are observed in cytology [4]. The direct examination of the hairs, the trichogram, is performed by pulling the hairs from the affected area, followed by microscopic evaluation. With this technique it is possible to deter‐ minate hair growth phase abnormalities and the presence of follicles parasites and dermato‐ phytes [3]. Cytology is the analysis of tissue cells and it is a highly efficient and valuable exam to evaluate a lesion, after which it is possible to establish the next step in the diagnos‐ tic approach. The sample may be obtained by fine-needle aspiration, swab, skin scrapings or lesion imprint [5]. Histopathology associated with clinical findings usually leads to defini‐ tive diagnosis. Skin biopsy is recommended in unusual lesions, possibly neoplastic nodules, dermatosis with expensive therapy or when it represents a risk to the patient health, with poor response to previous therapy and to exclude differential diagnoses [4]. To perform the histopathological exam at least three representative samples should be obtained by punch or surgical resection [3].

## **4. Bacterial skin diseases**

Dermis, the layer under the epidermis, is composed by a conjunctive matrix where reticular, elastic and collagen fibers are found. Dermis cellular structure is composed by fibroblasts, mast cells and histiocytes. It also contains epidermal appendages (hair, nails, sebaceous and sweat glands), arrector pili muscles and blood and lymph vessels. Hypodermis or subcuta‐ neous tissue provides support and cushioning against physical trauma. It is composed by a loose connective tissue and elastic fibers interspersed by adipocytes [1]. The hair follicles ex‐ hibit activity cycles that result in hair formation. Anagen is a period of active growth when a new hair is being formed. Catagen is when the hair growth stops and degenerative changes occur in the base of the follicle. Telogen represents a period of follicle inactivity, when the hair is shed so that a new one may start to grow [1]. Hair cycle activity, in some dog breeds, is strongly related to temperature variation and photoperiod, leading to decreased hair den‐

Diagnosis approach for skin diseases depends on obtaining detailed history with thorough physical and dermatological examination. The evaluation offers precious information and guides the investigative process. Specific tests and, occasionally, therapeutic trials should be

Skin scrapings, trichogram, fungal and bacterial culture, cytological evaluation and skin bi‐ opsy are important diagnosis techniques in dermatology. Skin scrapings are applied in mite detection, and it may be superficial or deep depending on the mite that is suspected. The sample obtained is evaluated under optical microscopy [3]. Fungal culture is recommended for patients that fungal diseases are suspected. Hair samples and skin scales should be col‐ lected from the lesion margin. Bacterial culture is not often performed and it is recommend‐

sity in the warmer months, which helps the heat loss in these animals [2].

**Figure 1.** Structure of the skin.

4 Insights from Veterinary Medicine

performed [3].

**3. Diagnosis techniques**

Pyoderma is a bacterial skin infection and it is among the most common causes of skin dis‐ eases in dogs [6], however, it is less common in cats [7]. Lesions may be superficial and in‐ volve only the epidermis or they may affect deeper structures in the dermis or subcutaneous tissue, and it is therefore divided into surface, superficial and deep pyoderma [6]. *Staphylo‐ coccus pseudintermedius* is the most commonly isolated bacteria from dog's skin [8] and it is among the main reasons for antimicrobial use in these animals [9].

Surface pyodermas are characterized by superficial erosions of the stratum corneum. The presence of alopecia, erythema and pruritus are common findings [10]. Intertrigo is a surface pyoderma that affects the skin folds found in lips, face, vulva, tail and mammary glands of some breeds, and it may also affect the skin folds of obese animals. Acute moist dermatitis, also known as pyotraumatic dermatitis or hot spots, is of acute onset and rarely occurs as a primary disease in healthy skin, being usually secondary to other diseases [11]. Probably, lo‐ cal irritation due to an underlying cause leads to self-inflicted trauma, which quickly be‐ comes extensive areas of skin damage [10].

Superficial pyodermas are the most common causes of cutaneous bacterial infection in dogs [6]. They affect the superficial portion of the hair follicles (bacterial folliculitis) or the epider‐ mis (impetigo), causing pustules [10, 11]. The most common lesions are crusted papules due to the transient nature of canine pustules. Pruritus, epidermal collarettes, hyperpigmenta‐ tion and alopecia are also common findings [6]. Impetigo affects sexually immature dogs that may present subcorneal pustules formed in inguinal and axillary areas [10]. Superficial bacterial folliculitis is the most common form of pyoderma in dogs [12]. Papules, pustules associated with hair follicles, epidermal collarettes, alopecia and hyperpigmentation are commonly found [10] (Figure 2).

Deep pyoderma does not occur spontaneously, often starting as superficial pyoderma [6]. Other organisms such as *Proteus* spp., *Pseudomonas* spp. and *E. coli* may be involved [12-14]. The bacterial infection affects the deepest portion of the hair follicle (deep folliculitis), that may lead to follicular wall rupture and to bacterial product release in the dermis (furunculo‐ sis), or it can also affect the deeper portion of the dermis and subcutaneous tissue (cellulitis) [10, 15]. The affected skin appears erythematous, hyperpigmented, with the presence of se‐ ropurulent debris from the ruptured pustules; variable pruritus, swelling, skin stiffness and evident pain are also noted [6].

require keratin for their growth [19]. Dermatophytes are classified into three groups based on their habitat: zoophilic, mostly found in animals, occurring transmission to other ani‐ mals or to humans; anthropophilic, mostly found in humans, transmitted between hu‐ mans and rarely to animals and geophilic, dermatophytes, found in the soil, infects humans and animals [20]. *M. canis* is the most frequently isolated fungal species in dogs

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 7

Clinically, canine and feline fungal infections differ. Infections in dogs often produce le‐ sions, while it is possible to isolate dermatophytes from clinically healthy cats, which can act as a conidia reservoir of the fungus [22]. The affected animals usually have alopecic, scaly, crusted, erythematous and papular lesions, especially in the face and limbs. Occasionally, dermatophytes may be presented in a nodular form known as kerions. This form of derma‐ tophytosis is characterized by deep, inflammatory and suppurative lesions [23]. Pruritus

The direct microscopic examination of hairs and scales can reveal the presence of fungal hy‐ phae or spores. The fungal culture is the diagnostic test of choice and the sample may be

The best strategy for the treatment of dermatophytes is the association of systemic and topi‐ cal antifungal therapy. The aims of the treatment with topical products are the elimination of the fungi present at the epidermis and hair surface, while systemic treatment aims to eliminate infection within the hair shafts [26]. Lime sulfur rinse at 6.5 % twice a week showed good results in cats infected with *M. canis* [27, 28]. Systemic treatment options in‐ clude itraconazole orally at 10 mg/kg once a day, griseofulvin 50 mg/kg once a day or terbi‐ nafine 5 mg/kg once a day [27-29]. The treatment must be extended over 2 to 4 weeks after

*Malassezia pachydermatis* is a commensal skin yeast, commonly isolated from lips, interdigital skin, anal mucosa and external auditory canal [30]. It is an opportunistic yeast, which usual‐ ly manifests itself after the installation of other diseases. It is very common in dogs and least frequent in cats [31]. Previous antibiotic therapy is associated with the development of cuta‐ neous *M. pachydermatis* over growth in dogs [32], as well as disorders of keratinization and hypersensitivity diseases [33]. Basset Hounds, Cocker Spaniels and West Highland White

The most common clinical manifestation of *Malassezia* dermatitis is the presence of moderate to intense pruritus [34]. Erythema, lichenification, oily skin, malodor, alopecia and erosions

Cytology is the diagnostic method of choice and allows microscopic identification of the in‐ creased number of yeasts. Samples can be obtained by skin scrapings, swabs, direct imprint or by tape preps. Fungal culture is not recommended as a diagnostic procedure because it is

are also common clinical findings [35] that can be generalized or localized [34].

obtained by brushing the animal with a toothbrush or by skin scrapings [25].

clinical cure and after obtaining two or more negative fungal cultures [23].

Terriers are more predisposed to this type of infection [32].

and cats [21].

may vary from absent to severe [24].

**5.2.** *Malassezia* **dermatitis**

not a quantitative assessment [36].

Diagnosis is obtained through the evaluation of clinical signs, presence of characteristic skin lesions, elimination of other possible causes of folliculitis and by cytological evaluation of the intact pustules content, exudative lesions and skin debris. In the management of pyoder‐ ma, it is important to identify the possible underlying disorder, which may be done through skin scrapings, scabies therapeutic trial, allergy tests, endocrinopathies screenings, hypoal‐ lergenic diet trials, strict ectoparasites control and skin biopsies. [16].

The treatment varies depending on presented lesions. Local surface and superficial pyoder‐ mas may be treated only with topical antibiotics such as silver sulphadiazine, neomycin or 2% mupirocin ointments applied twice daily over the affected areas. Generalized lesions and deep pyodermas require a combination of oral and topical antibiotics. In patients with se‐ vere pruritus it is recommended to use anti-inflammatory doses of prednisone orally for up to two weeks [17]. The antibiotics of choice for oral use include cephalexin (22-33 mg / kg q12h) and amoxicillin associated with clavulanic acid (22 mg/kg q12h) [18]. Recurrent cases require culture and susceptibility testing to access resistance [16].

**Figure 2.** Superficial bacterial folliculitis in a Dachshund. Multiple areas of alopecia and erythema are seen in the trunk area.

## **5. Fungal diseases**

#### **5.1. Dermatophytosis**

Dermatophytosis is a superficial mycosis caused by *Microsporum*, *Trichophyton* or *Epidermo‐ phyton* fungi genera. These fungi are isolated from hair, nails and skin surface since they require keratin for their growth [19]. Dermatophytes are classified into three groups based on their habitat: zoophilic, mostly found in animals, occurring transmission to other ani‐ mals or to humans; anthropophilic, mostly found in humans, transmitted between hu‐ mans and rarely to animals and geophilic, dermatophytes, found in the soil, infects humans and animals [20]. *M. canis* is the most frequently isolated fungal species in dogs and cats [21].

Clinically, canine and feline fungal infections differ. Infections in dogs often produce le‐ sions, while it is possible to isolate dermatophytes from clinically healthy cats, which can act as a conidia reservoir of the fungus [22]. The affected animals usually have alopecic, scaly, crusted, erythematous and papular lesions, especially in the face and limbs. Occasionally, dermatophytes may be presented in a nodular form known as kerions. This form of derma‐ tophytosis is characterized by deep, inflammatory and suppurative lesions [23]. Pruritus may vary from absent to severe [24].

The direct microscopic examination of hairs and scales can reveal the presence of fungal hy‐ phae or spores. The fungal culture is the diagnostic test of choice and the sample may be obtained by brushing the animal with a toothbrush or by skin scrapings [25].

The best strategy for the treatment of dermatophytes is the association of systemic and topi‐ cal antifungal therapy. The aims of the treatment with topical products are the elimination of the fungi present at the epidermis and hair surface, while systemic treatment aims to eliminate infection within the hair shafts [26]. Lime sulfur rinse at 6.5 % twice a week showed good results in cats infected with *M. canis* [27, 28]. Systemic treatment options in‐ clude itraconazole orally at 10 mg/kg once a day, griseofulvin 50 mg/kg once a day or terbi‐ nafine 5 mg/kg once a day [27-29]. The treatment must be extended over 2 to 4 weeks after clinical cure and after obtaining two or more negative fungal cultures [23].

#### **5.2.** *Malassezia* **dermatitis**

The bacterial infection affects the deepest portion of the hair follicle (deep folliculitis), that may lead to follicular wall rupture and to bacterial product release in the dermis (furunculo‐ sis), or it can also affect the deeper portion of the dermis and subcutaneous tissue (cellulitis) [10, 15]. The affected skin appears erythematous, hyperpigmented, with the presence of se‐ ropurulent debris from the ruptured pustules; variable pruritus, swelling, skin stiffness and

Diagnosis is obtained through the evaluation of clinical signs, presence of characteristic skin lesions, elimination of other possible causes of folliculitis and by cytological evaluation of the intact pustules content, exudative lesions and skin debris. In the management of pyoder‐ ma, it is important to identify the possible underlying disorder, which may be done through skin scrapings, scabies therapeutic trial, allergy tests, endocrinopathies screenings, hypoal‐

The treatment varies depending on presented lesions. Local surface and superficial pyoder‐ mas may be treated only with topical antibiotics such as silver sulphadiazine, neomycin or 2% mupirocin ointments applied twice daily over the affected areas. Generalized lesions and deep pyodermas require a combination of oral and topical antibiotics. In patients with se‐ vere pruritus it is recommended to use anti-inflammatory doses of prednisone orally for up to two weeks [17]. The antibiotics of choice for oral use include cephalexin (22-33 mg / kg q12h) and amoxicillin associated with clavulanic acid (22 mg/kg q12h) [18]. Recurrent cases

**Figure 2.** Superficial bacterial folliculitis in a Dachshund. Multiple areas of alopecia and erythema are seen in the

Dermatophytosis is a superficial mycosis caused by *Microsporum*, *Trichophyton* or *Epidermo‐ phyton* fungi genera. These fungi are isolated from hair, nails and skin surface since they

lergenic diet trials, strict ectoparasites control and skin biopsies. [16].

require culture and susceptibility testing to access resistance [16].

evident pain are also noted [6].

6 Insights from Veterinary Medicine

trunk area.

**5. Fungal diseases**

**5.1. Dermatophytosis**

*Malassezia pachydermatis* is a commensal skin yeast, commonly isolated from lips, interdigital skin, anal mucosa and external auditory canal [30]. It is an opportunistic yeast, which usual‐ ly manifests itself after the installation of other diseases. It is very common in dogs and least frequent in cats [31]. Previous antibiotic therapy is associated with the development of cuta‐ neous *M. pachydermatis* over growth in dogs [32], as well as disorders of keratinization and hypersensitivity diseases [33]. Basset Hounds, Cocker Spaniels and West Highland White Terriers are more predisposed to this type of infection [32].

The most common clinical manifestation of *Malassezia* dermatitis is the presence of moderate to intense pruritus [34]. Erythema, lichenification, oily skin, malodor, alopecia and erosions are also common clinical findings [35] that can be generalized or localized [34].

Cytology is the diagnostic method of choice and allows microscopic identification of the in‐ creased number of yeasts. Samples can be obtained by skin scrapings, swabs, direct imprint or by tape preps. Fungal culture is not recommended as a diagnostic procedure because it is not a quantitative assessment [36].

Identification and correction of the predisposing diseases are essential for the successful treatment of *Malassezia* dermatitis [23]. Mild cases can be treated with miconazole, chlorhexi‐ dine, ketoconazole or acetic acid shampoos. Baths should be given, two times per week, for a minimum of six weeks [36]. Patients requiring systemic treatment should receive oral anti‐ fungals such as ketoconazole, itraconazole or fluconazole 5 mg/kg once a day for a mini‐ mum of 30 days [36]. Terbinafine 30 mg /kg every 24 hours on two consecutive days in a week, for six weeks, may also be an effective treatment [37]. In order to prevent recurrences of the disease, regular maintenance therapy may be needed in many dogs [23].

munodeficiency virus (FIV) cryptococcosis tends to manifest itself in a disseminated or ad‐

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 9

Cryptococcosis diagnosis is based on fungal culture, cytological, histological and serological exams [49]. Cytological examination may reveal the presence of leukocytes, macrophages

Drug therapy leads to patient healing in most cases [49] and it consists of oral antifungal use until complete remission of clinical signs, usually in 3 to 12 months. It is strongly recom‐ mended not to interrupt the treatment until the titers of antibodies against cryptococcosis are reduced to zero [52]. Drugs commonly used include fluconazole 50 mg/cat every 12 hours [48] and itraconazole 50-100 mg/cat every 24 hours [52], or 10mg/kg for dogs and cats every 24 hours [49]. Patients with nasopharyngeal masses benefit from surgical resection, for

**Figure 3.** Feline cryptococcosis. Figure on the left: a cat presenting a nasal masse (red arrow). Figure on the right: Cy‐ tology by fine needle aspirate of the nasal masse showing several encapsulated structures (yellow arrows) compatible

Demodicosis is a very common skin disease in dogs but rare in cats [53]. It is an inflammato‐ ry disease, in which large amounts of *Demodex* mites are found in the skin [54]. Mites prolif‐ erate in the hair follicles and sebaceous glands causing the disease [55]. It is a common condition and it is often serious in dogs. Besides *Demodex canis*, two less common species were reported [54, 55], *Demodex* sp. *cornei* and *Demodex injai* [55]. Feline demodicosis may be

caused by two different kinds of mites, *Demodex cati* and *Demodex gatoi* [53].

with *C. neoformans* (microscopic image viewed with a 100x oil objective).

**6. Parasitic skin diseases**

**6.1. Demodicosis**

and numerous encapsulated structures (yeast) of different sizes (Figure 3) [50, 51].

upper airway patency and reduction of infected tissue to be treated medically [52].

vanced form [48].

#### **5.3. Sporotrichosis**

Sporotrichosis is a subcutaneous mycosis caused by a dimorphic fungus, *Sporothrix schenckii*, which can infect animals and humans [39]. It is a zoonotic disease and transmission to hu‐ mans occurs through bites or scratches, and contact with cats ulcers [40]. *S. schenckii* is present in the decaying vegetation and soil and animal contamination occurs by skin open lesions such as perforations, bites and scratches. Once in the host organism, the fungus may cause local lesions and possibly systemic signs [41].

Clinically, sporotrichosis has three forms: cutaneous, lymphocutaneous and generalized, and more than one form can occur simultaneously in the same animal. The cutaneous form is usually confined to the area of fungus inoculation and manifests after an incubation peri‐ od of one month. If this lesion is not treated, the progression to lymphocutaneous form can occur. The lymphocutaneous form is characterized by the development of nodules that evolve into ulcers, affecting skin, subcutaneous tissue, lymph vessels and regional lymph nodes [41]. History of lethargy, anorexia, depression and fever on physical examination sug‐ gest the presence of the disseminated form [42].

The diagnosis is based on clinical history, physical examination, cytological evaluation, fun‐ gal culture and histopathological findings [43]. Cytological evaluation usually reveals oval to elongate yeast cells consistent with *S. schenckii* form and inflammatory cells may also be present [45]. On histopathology, the presence of deep pyogranulomatous dermatitis, cellular infiltration of polymorphonuclear and mononuclear cells, and the presence of PAS positive structures compatible with *S. schenckii* may be observed [44]. Oral administration of itraco‐ nazole 10 mg/kg every 24 hours is the treatment of choice [46].

#### **5.4. Cryptococcosis**

*Cryptococcus* spp. is a saprophytic fungus present in the environment and in the feces of pi‐ geons, capable of causing systemic infection in dogs and cats, with a higher incidence in fe‐ lines. The species of interest in veterinary medicine are *C. neoformans*, which has a global distribution and *C. gattii* that has a limited distribution [47]. *C. neoformans* typically infects animals by inhalation and may cause ophthalmic, upper respiratory tract and central nerv‐ ous system lesions. Ulcerative lesions in the nasal, oral or pharyngeal mucosae, or a nasal masse may be present (Figure 3) [31]. Mycotic rhinitis and cutaneous nasal bridge and nasal plan involvement are the most frequent findings. In cats with positive serology for feline im‐ munodeficiency virus (FIV) cryptococcosis tends to manifest itself in a disseminated or ad‐ vanced form [48].

Cryptococcosis diagnosis is based on fungal culture, cytological, histological and serological exams [49]. Cytological examination may reveal the presence of leukocytes, macrophages and numerous encapsulated structures (yeast) of different sizes (Figure 3) [50, 51].

Drug therapy leads to patient healing in most cases [49] and it consists of oral antifungal use until complete remission of clinical signs, usually in 3 to 12 months. It is strongly recom‐ mended not to interrupt the treatment until the titers of antibodies against cryptococcosis are reduced to zero [52]. Drugs commonly used include fluconazole 50 mg/cat every 12 hours [48] and itraconazole 50-100 mg/cat every 24 hours [52], or 10mg/kg for dogs and cats every 24 hours [49]. Patients with nasopharyngeal masses benefit from surgical resection, for upper airway patency and reduction of infected tissue to be treated medically [52].

**Figure 3.** Feline cryptococcosis. Figure on the left: a cat presenting a nasal masse (red arrow). Figure on the right: Cy‐ tology by fine needle aspirate of the nasal masse showing several encapsulated structures (yellow arrows) compatible with *C. neoformans* (microscopic image viewed with a 100x oil objective).

## **6. Parasitic skin diseases**

#### **6.1. Demodicosis**

Identification and correction of the predisposing diseases are essential for the successful treatment of *Malassezia* dermatitis [23]. Mild cases can be treated with miconazole, chlorhexi‐ dine, ketoconazole or acetic acid shampoos. Baths should be given, two times per week, for a minimum of six weeks [36]. Patients requiring systemic treatment should receive oral anti‐ fungals such as ketoconazole, itraconazole or fluconazole 5 mg/kg once a day for a mini‐ mum of 30 days [36]. Terbinafine 30 mg /kg every 24 hours on two consecutive days in a week, for six weeks, may also be an effective treatment [37]. In order to prevent recurrences

Sporotrichosis is a subcutaneous mycosis caused by a dimorphic fungus, *Sporothrix schenckii*, which can infect animals and humans [39]. It is a zoonotic disease and transmission to hu‐ mans occurs through bites or scratches, and contact with cats ulcers [40]. *S. schenckii* is present in the decaying vegetation and soil and animal contamination occurs by skin open lesions such as perforations, bites and scratches. Once in the host organism, the fungus may

Clinically, sporotrichosis has three forms: cutaneous, lymphocutaneous and generalized, and more than one form can occur simultaneously in the same animal. The cutaneous form is usually confined to the area of fungus inoculation and manifests after an incubation peri‐ od of one month. If this lesion is not treated, the progression to lymphocutaneous form can occur. The lymphocutaneous form is characterized by the development of nodules that evolve into ulcers, affecting skin, subcutaneous tissue, lymph vessels and regional lymph nodes [41]. History of lethargy, anorexia, depression and fever on physical examination sug‐

The diagnosis is based on clinical history, physical examination, cytological evaluation, fun‐ gal culture and histopathological findings [43]. Cytological evaluation usually reveals oval to elongate yeast cells consistent with *S. schenckii* form and inflammatory cells may also be present [45]. On histopathology, the presence of deep pyogranulomatous dermatitis, cellular infiltration of polymorphonuclear and mononuclear cells, and the presence of PAS positive structures compatible with *S. schenckii* may be observed [44]. Oral administration of itraco‐

*Cryptococcus* spp. is a saprophytic fungus present in the environment and in the feces of pi‐ geons, capable of causing systemic infection in dogs and cats, with a higher incidence in fe‐ lines. The species of interest in veterinary medicine are *C. neoformans*, which has a global distribution and *C. gattii* that has a limited distribution [47]. *C. neoformans* typically infects animals by inhalation and may cause ophthalmic, upper respiratory tract and central nerv‐ ous system lesions. Ulcerative lesions in the nasal, oral or pharyngeal mucosae, or a nasal masse may be present (Figure 3) [31]. Mycotic rhinitis and cutaneous nasal bridge and nasal plan involvement are the most frequent findings. In cats with positive serology for feline im‐

of the disease, regular maintenance therapy may be needed in many dogs [23].

cause local lesions and possibly systemic signs [41].

gest the presence of the disseminated form [42].

nazole 10 mg/kg every 24 hours is the treatment of choice [46].

**5.3. Sporotrichosis**

8 Insights from Veterinary Medicine

**5.4. Cryptococcosis**

Demodicosis is a very common skin disease in dogs but rare in cats [53]. It is an inflammato‐ ry disease, in which large amounts of *Demodex* mites are found in the skin [54]. Mites prolif‐ erate in the hair follicles and sebaceous glands causing the disease [55]. It is a common condition and it is often serious in dogs. Besides *Demodex canis*, two less common species were reported [54, 55], *Demodex* sp. *cornei* and *Demodex injai* [55]. Feline demodicosis may be caused by two different kinds of mites, *Demodex cati* and *Demodex gatoi* [53].

The mites are transferred from the mother to the offspring in the early life [56]. *D. canis* is considered a commensal in canine skin. It is believed that this disease is a consequence of a specific immunosuppression, which allows the proliferation of the mites [57]. Genetic fac‐ tors are probably very important in the development of generalized disease and therefore, breeding of affected animals is contraindicated [54].

Skin scraping with microscopic identification of *S. scabiei* is a valuable diagnostic method, although mites are hardly seen in many cases. Diagnosis is usually based on the animal's history, clinical signs and a positive pinnal-pedal reflex (pinna margings are gently scratched and the dog will reflexively use an ipsilateral hind limb to scratch the source of

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 11

The treatment is recommended to the patient and other animals in their household. Bathing with 0.025% amitraz solution once weekly or twice weekly is recommended as an effective treatment. The therapy should be continued for two weeks after clinical signs remission. Ad‐ verse side effects should be monitored during the treatment [64]. Fipronil spray 0.25% can be applied three times with three weeks intervals on puppies [65]. Ivermectin can be admin‐ istered at 0.2 to 0.4 mg/kg, orally every seven days, or subcutaneously every 14 days. The treatment usually takes four to six weeks [66].This drug is contraindicated in Collies and their crosses [67]. An effective alternative treatment of scabies is a spot-on application of se‐ lamectin at 6-12 mg/kg every 15 to 30 days, for at least three applications and it is apparent‐ ly, well tolerated in different breeds [67, 68]. Milbemycin oxime at 2mg/kg in a weekly dose,

NOTE: The permission for the use of macrocyclic lactones in the treatment of cats and dogs is different for each country. The rules concerning its use should be checked before treat‐ ment institution and the owners must be warned in order to authorize any extra-label use of

**Figure 4.** Scabies. Figure on the left: a dog with erythematous, alopecic and lichenified pruritic lesions in the distal aspect of the hind limbs. Figure on the right: the animal's owner presenting abdominal pruritic papular lesions, high‐

administrated for up to five times has also a good efficacy [69].

the irritation) [62, 63].

these medications.

lighting the zoonotic aspect of scabies.

Erythema, comedones, scaling, partial or complete alopecia, papules, follicular casts, pus‐ tules, and in severe cases, furunculosis, crusting, exudation and ulceration with focal drain‐ ing tracts can be clinical sings. Generally the lesions begin on the face and limbs, but they may become generalized. Demodicosis can be classified into generalized or localized. The involvement of one complete body region, five or more focal areas, or the involvement of the legs is considered generalized demodicosis. The diagnosis is made by deep skin scrap‐ ings or trichogram. In some rare cases, in the legs or certain breeds such as Shar-peis, these tests may be negative, requiring biopsies for mite detection [58].

In most dogs, localized demodicosis resolves spontaneously, thus mite-specific therapy is not necessary until the disease generalizes [53]. The treatment of generalized demodicosis involves several approaches. In addition to the acaricidal treatment, the concurrent secon‐ dary infections and underlying diseases should be also accessed [58]. As an acaricidal treatment, ivermectin is recommended at 0.3 to 0.6 mg/kg orally once a day, however, it is recommended to begin the treatment with a lower dose and gradually increase the amount of drug administered. The animal should be monitored for the appearance of ad‐ verse effects. Therapy with moxidectin 0.2-0.5 mg/kg orally once a day and doramectin 0.6 mg/kg orally or subcutaneously once a week were proven effective in the treatment of generalized demodicosis. The same careful institution of a gradual dose taken with iver‐ mectin should be applied for these two other drugs. Milbemycin oxime can also be used at 1 to 2 mg/kg orally, once a day, with good results [58]. In cats weekly baths with 2% lime sulfur are indicated for the treatment of demodicosis. There is remote evidence to in‐ dicate weekly application of 0.025% amitraz and for the use of doramectin 0.6 mg/kg once a week subcutaneously [53]. Treatment should continue for one month after getting the third consecutive negative scraping [58].

#### **6.2. Canine scabies**

Canine scabies is a common condition in dogs and humans, but rare in cats, in which the skin is colonized by *Sarcoptes scabiei* mite after contact with a donor host. The infestation with this mites results in intense pruritus [59, 60]. Female mites dig galleries in the stratum corneum in order to lay their eggs that hatch, releasing larvae forms that migrate to the skin surface, where they reach the adult stage [61].

Extremely irritating and pruritic papular eruption, skin thickening, erythema, alopecia, exudation with crust formation and secondary bacterial infection with pustules are com‐ mon clinical findings (Figure 4). Chronic lesions are usually confined to the margins of the pinna, elbows and hocks that may present skin thickening, minimal crust formation and persistent pruritus [61].

Skin scraping with microscopic identification of *S. scabiei* is a valuable diagnostic method, although mites are hardly seen in many cases. Diagnosis is usually based on the animal's history, clinical signs and a positive pinnal-pedal reflex (pinna margings are gently scratched and the dog will reflexively use an ipsilateral hind limb to scratch the source of the irritation) [62, 63].

The mites are transferred from the mother to the offspring in the early life [56]. *D. canis* is considered a commensal in canine skin. It is believed that this disease is a consequence of a specific immunosuppression, which allows the proliferation of the mites [57]. Genetic fac‐ tors are probably very important in the development of generalized disease and therefore,

Erythema, comedones, scaling, partial or complete alopecia, papules, follicular casts, pus‐ tules, and in severe cases, furunculosis, crusting, exudation and ulceration with focal drain‐ ing tracts can be clinical sings. Generally the lesions begin on the face and limbs, but they may become generalized. Demodicosis can be classified into generalized or localized. The involvement of one complete body region, five or more focal areas, or the involvement of the legs is considered generalized demodicosis. The diagnosis is made by deep skin scrap‐ ings or trichogram. In some rare cases, in the legs or certain breeds such as Shar-peis, these

In most dogs, localized demodicosis resolves spontaneously, thus mite-specific therapy is not necessary until the disease generalizes [53]. The treatment of generalized demodicosis involves several approaches. In addition to the acaricidal treatment, the concurrent secon‐ dary infections and underlying diseases should be also accessed [58]. As an acaricidal treatment, ivermectin is recommended at 0.3 to 0.6 mg/kg orally once a day, however, it is recommended to begin the treatment with a lower dose and gradually increase the amount of drug administered. The animal should be monitored for the appearance of ad‐ verse effects. Therapy with moxidectin 0.2-0.5 mg/kg orally once a day and doramectin 0.6 mg/kg orally or subcutaneously once a week were proven effective in the treatment of generalized demodicosis. The same careful institution of a gradual dose taken with iver‐ mectin should be applied for these two other drugs. Milbemycin oxime can also be used at 1 to 2 mg/kg orally, once a day, with good results [58]. In cats weekly baths with 2% lime sulfur are indicated for the treatment of demodicosis. There is remote evidence to in‐ dicate weekly application of 0.025% amitraz and for the use of doramectin 0.6 mg/kg once a week subcutaneously [53]. Treatment should continue for one month after getting the

Canine scabies is a common condition in dogs and humans, but rare in cats, in which the skin is colonized by *Sarcoptes scabiei* mite after contact with a donor host. The infestation with this mites results in intense pruritus [59, 60]. Female mites dig galleries in the stratum corneum in order to lay their eggs that hatch, releasing larvae forms that migrate to the skin

Extremely irritating and pruritic papular eruption, skin thickening, erythema, alopecia, exudation with crust formation and secondary bacterial infection with pustules are com‐ mon clinical findings (Figure 4). Chronic lesions are usually confined to the margins of the pinna, elbows and hocks that may present skin thickening, minimal crust formation

breeding of affected animals is contraindicated [54].

10 Insights from Veterinary Medicine

third consecutive negative scraping [58].

surface, where they reach the adult stage [61].

**6.2. Canine scabies**

and persistent pruritus [61].

tests may be negative, requiring biopsies for mite detection [58].

The treatment is recommended to the patient and other animals in their household. Bathing with 0.025% amitraz solution once weekly or twice weekly is recommended as an effective treatment. The therapy should be continued for two weeks after clinical signs remission. Ad‐ verse side effects should be monitored during the treatment [64]. Fipronil spray 0.25% can be applied three times with three weeks intervals on puppies [65]. Ivermectin can be admin‐ istered at 0.2 to 0.4 mg/kg, orally every seven days, or subcutaneously every 14 days. The treatment usually takes four to six weeks [66].This drug is contraindicated in Collies and their crosses [67]. An effective alternative treatment of scabies is a spot-on application of se‐ lamectin at 6-12 mg/kg every 15 to 30 days, for at least three applications and it is apparent‐ ly, well tolerated in different breeds [67, 68]. Milbemycin oxime at 2mg/kg in a weekly dose, administrated for up to five times has also a good efficacy [69].

NOTE: The permission for the use of macrocyclic lactones in the treatment of cats and dogs is different for each country. The rules concerning its use should be checked before treat‐ ment institution and the owners must be warned in order to authorize any extra-label use of these medications.

**Figure 4.** Scabies. Figure on the left: a dog with erythematous, alopecic and lichenified pruritic lesions in the distal aspect of the hind limbs. Figure on the right: the animal's owner presenting abdominal pruritic papular lesions, high‐ lighting the zoonotic aspect of scabies.

## **7. Allergic diseases**

#### **7.1. Flea allergy dermatitis**

*Ctenocephalides felis felis* is the most important ectoparasite in dogs and cats in several coun‐ tries [70]. Its infestation may cause intense pruritus, self-inflicted trauma and even severe symptoms as anemia. Some animals will develop a severe condition known as flea allergy dermatitis (FAD). After the sensitization, the lesions may be initiated by only a few flea bites [71]. Flea allergy is one of the most frequent conditions in dogs, especially in humid and warm weather countries, where fleas are commonly found [72]. The most common clinical signs are erythema, excoriation, crusting, and pustules that usually affect the medial caudal aspect of forelegs and ventral abdomen; pyotraumatic dermatitis may also be observed [73].

least six weeks. Homemade diets are more appropriate for the CAFR trial described above,

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 13

Prescription diets, commercially available in some countries, are made of an unusual protein source and a non-allergenic carbohydrate source such as potato or oat meal. Lamb, duck, rabbit and Kangaroo meat are protein sources usually found in these diets [77]. Another op‐ tion considered really hypoallergenic is the hydrolyzed diet. For humans it is known that most food allergens are glycoproteins with molecular weight higher than 12,000 d. The hy‐ drolyzed food has smaller peptides what makes them potentially less allergenic [75]. How‐ ever, a small percentage of allergic dogs show poor response to hydrolyzed food. The possibility of adverse food reaction to hydrolyzed food in sensitive dogs is rare but should

CAFR differential diagnoses are other hypersensitivities as FAD, atopic dermatitis, diseases caused by ectoparasites, and yeast or bacterial infections. These infections can also appear as complications of a pruritic process. Therapy should include secondary infections and otitis externa. Some cases also demand severe pruritus control while the diet trial is applied. Any drug therapy should be interrupted at least three weeks before the end of the diet trial, so that the clinician can access the animal response to the diet [75]. It is still unknown if ani‐ mals develop tolerance to food allergens after a long period without contact, as reported in

Canine atopic dermatitis (CAD) is an allergic, hereditary, inflammatory and pruritic skin disease, with characteristic clinical signs associated with immunoglobulin E (IgE) produc‐ tion against environmental allergens [80]. Atopic dermatitis also affects cats, but its inci‐

Most dogs with atopic dermatitis begin to manifest signs between six months and three years of age [82, 83]. There is no sex predisposition and clinical signs may or not be seasonal, depending on the allergen involved. Usually, patients have a history of pruritus with or without secondary skin or ear infections. Primary lesions include macules and papules, but frequently, patients are presented with secondary lesions from self-inflicted trauma as ex‐ coriations, alopecia, lichenification and hyperpigmentation. Lesions affect the face, concave part of the pinna, ventral aspect of the neck, axilla, groin, abdomen, perineum, ventral as‐

In dog as in human beings there is no pathognomonic sign of atopic dermatitis that could provide a diagnosis based only in history and physical examination. Diagnosis depends on patient fitting in several criteria associated with the condition and on elimination of differ‐ ential diagnoses. Following clinical diagnosis, laboratory or clinical tests as allergy tests and histopathology, reinforces the diagnosis. However, these tests should not be used to estab‐ lish the diagnosis but to confirm it [82]. It is important to highlight the fact that some dogs with inflammatory and pruritic skin diseases, displaying clinical signs identical to those found in CAD, may have no IgE production in response to environmental allergens. This

pect of the tail, limbs joints, medial aspects of limb extremities, feet and ears [85].

humans, however, natural hyposensitization is apparently rare [77].

but there are also commercially available prescription and hydrolyzed diets [75].

be considered [79].

**7.3. Atopic dermatitis in dogs**

dence is lower than in dogs [81].

Diagnosis is based on circumstantial evidence, such as clinical response to flea control. A successful treatment for FAD depends on eliminating the allergy source, the flea. Therapy goals are the total elimination of flea population in the patient's body and environment, as well as in contact animals. It is also important to prevent new infestation, what is not always simple, once fleas have a high reproductive capacity and a complex life cycle [71]. Flea con‐ trol advances with modern insecticides and insect growth regulators markedly decreased the number of affected animals. Products as fipronil, imidacloprid, metaflumizone, nintem‐ piram, selamectin e spinosad proved to be efficient to control fleas in animals with FAD [74]. These products are available in several presentations as shampoo, collars, spray, powder, spot on and oral medication [71].

#### **7.2. Adverse food reaction**

Adverse food reactions are described in veterinary medicine since 1920, reporting the occur‐ rence of gastrointestinal signs and skin reactions in response to food allergens [75]. Adverse food reaction refers to any abnormal clinical response assigned to consumption of food or its additives [76]. This reaction is classified as food allergy (immune-mediated) or food intoler‐ ance (non-immune-mediated). The majority of reactions in animals are food intolerances and they can be of pharmacological or metabolic origin, poisoning, idiosyncrasy [76, 77], toxicity or anaphylactic reaction to the food [77]. There are some dogs with pruritic skin dis‐ eases or otitis which resolves with restrictive diet, but it remains unproved the immunologic cause or hypersensitivity (allergy) associated with cutaneous adverse food reactions (CAFR). Once the etiopathogenesis was not elucidated yet, the term food allergy should be avoided, and CAFR is more appropriate [76, 78]

There are no breed, sex or age predisposition to the occurrence of clinical signs. Pruritus is the most important sign reported and it affects mainly the face, perineum and ears (otitis ex‐ terna). Gastrointestinal signs as vomiting and diarrhea can also be observed. The best diag‐ nostic approach for CAFR in dogs and cats is feeding them with a diet, with only one source of protein that the animal has never been in contact before (novel protein). The diagnosis is obtained with the resolution of clinical signs after the diet trial and with the return of these signs when the previous diet is offered again. The diet trial should be implemented for at least six weeks. Homemade diets are more appropriate for the CAFR trial described above, but there are also commercially available prescription and hydrolyzed diets [75].

Prescription diets, commercially available in some countries, are made of an unusual protein source and a non-allergenic carbohydrate source such as potato or oat meal. Lamb, duck, rabbit and Kangaroo meat are protein sources usually found in these diets [77]. Another op‐ tion considered really hypoallergenic is the hydrolyzed diet. For humans it is known that most food allergens are glycoproteins with molecular weight higher than 12,000 d. The hy‐ drolyzed food has smaller peptides what makes them potentially less allergenic [75]. How‐ ever, a small percentage of allergic dogs show poor response to hydrolyzed food. The possibility of adverse food reaction to hydrolyzed food in sensitive dogs is rare but should be considered [79].

CAFR differential diagnoses are other hypersensitivities as FAD, atopic dermatitis, diseases caused by ectoparasites, and yeast or bacterial infections. These infections can also appear as complications of a pruritic process. Therapy should include secondary infections and otitis externa. Some cases also demand severe pruritus control while the diet trial is applied. Any drug therapy should be interrupted at least three weeks before the end of the diet trial, so that the clinician can access the animal response to the diet [75]. It is still unknown if ani‐ mals develop tolerance to food allergens after a long period without contact, as reported in humans, however, natural hyposensitization is apparently rare [77].

#### **7.3. Atopic dermatitis in dogs**

**7. Allergic diseases**

12 Insights from Veterinary Medicine

**7.1. Flea allergy dermatitis**

spot on and oral medication [71].

avoided, and CAFR is more appropriate [76, 78]

**7.2. Adverse food reaction**

*Ctenocephalides felis felis* is the most important ectoparasite in dogs and cats in several coun‐ tries [70]. Its infestation may cause intense pruritus, self-inflicted trauma and even severe symptoms as anemia. Some animals will develop a severe condition known as flea allergy dermatitis (FAD). After the sensitization, the lesions may be initiated by only a few flea bites [71]. Flea allergy is one of the most frequent conditions in dogs, especially in humid and warm weather countries, where fleas are commonly found [72]. The most common clinical signs are erythema, excoriation, crusting, and pustules that usually affect the medial caudal aspect of forelegs and ventral abdomen; pyotraumatic dermatitis may also be observed [73].

Diagnosis is based on circumstantial evidence, such as clinical response to flea control. A successful treatment for FAD depends on eliminating the allergy source, the flea. Therapy goals are the total elimination of flea population in the patient's body and environment, as well as in contact animals. It is also important to prevent new infestation, what is not always simple, once fleas have a high reproductive capacity and a complex life cycle [71]. Flea con‐ trol advances with modern insecticides and insect growth regulators markedly decreased the number of affected animals. Products as fipronil, imidacloprid, metaflumizone, nintem‐ piram, selamectin e spinosad proved to be efficient to control fleas in animals with FAD [74]. These products are available in several presentations as shampoo, collars, spray, powder,

Adverse food reactions are described in veterinary medicine since 1920, reporting the occur‐ rence of gastrointestinal signs and skin reactions in response to food allergens [75]. Adverse food reaction refers to any abnormal clinical response assigned to consumption of food or its additives [76]. This reaction is classified as food allergy (immune-mediated) or food intoler‐ ance (non-immune-mediated). The majority of reactions in animals are food intolerances and they can be of pharmacological or metabolic origin, poisoning, idiosyncrasy [76, 77], toxicity or anaphylactic reaction to the food [77]. There are some dogs with pruritic skin dis‐ eases or otitis which resolves with restrictive diet, but it remains unproved the immunologic cause or hypersensitivity (allergy) associated with cutaneous adverse food reactions (CAFR). Once the etiopathogenesis was not elucidated yet, the term food allergy should be

There are no breed, sex or age predisposition to the occurrence of clinical signs. Pruritus is the most important sign reported and it affects mainly the face, perineum and ears (otitis ex‐ terna). Gastrointestinal signs as vomiting and diarrhea can also be observed. The best diag‐ nostic approach for CAFR in dogs and cats is feeding them with a diet, with only one source of protein that the animal has never been in contact before (novel protein). The diagnosis is obtained with the resolution of clinical signs after the diet trial and with the return of these signs when the previous diet is offered again. The diet trial should be implemented for at

Canine atopic dermatitis (CAD) is an allergic, hereditary, inflammatory and pruritic skin disease, with characteristic clinical signs associated with immunoglobulin E (IgE) produc‐ tion against environmental allergens [80]. Atopic dermatitis also affects cats, but its inci‐ dence is lower than in dogs [81].

Most dogs with atopic dermatitis begin to manifest signs between six months and three years of age [82, 83]. There is no sex predisposition and clinical signs may or not be seasonal, depending on the allergen involved. Usually, patients have a history of pruritus with or without secondary skin or ear infections. Primary lesions include macules and papules, but frequently, patients are presented with secondary lesions from self-inflicted trauma as ex‐ coriations, alopecia, lichenification and hyperpigmentation. Lesions affect the face, concave part of the pinna, ventral aspect of the neck, axilla, groin, abdomen, perineum, ventral as‐ pect of the tail, limbs joints, medial aspects of limb extremities, feet and ears [85].

In dog as in human beings there is no pathognomonic sign of atopic dermatitis that could provide a diagnosis based only in history and physical examination. Diagnosis depends on patient fitting in several criteria associated with the condition and on elimination of differ‐ ential diagnoses. Following clinical diagnosis, laboratory or clinical tests as allergy tests and histopathology, reinforces the diagnosis. However, these tests should not be used to estab‐ lish the diagnosis but to confirm it [82]. It is important to highlight the fact that some dogs with inflammatory and pruritic skin diseases, displaying clinical signs identical to those found in CAD, may have no IgE production in response to environmental allergens. This condition is known as "canine atopic like dermatitis" [80]. Other dermatosis might share similar signs with canine atopic dermatitis or could be concurrent diseases and it is why they should be eliminated or controlled before the conclusive diagnose of CAD. Differential diagnoses for CAD are FAD, CAFR, pruritic parasitic diseases, bacterial pyoderma, *Malasse‐ zia* dermatitis and other allergic diseases [82, 83]. Allergy tests goals are to determinate aller‐ gens, to contact avoidance and to be included in the allergy specific immunotherapy [83]. No allergy test is completely sensitive or specific, therefore, clinically normal animals can have positive responses and animals with negative results can have clinical characteristics of the disease [82]. Allergens generally related to CAD pathogenesis are domestic dust mites, mold spores, trees and grasses pollens, antigens from insects and epidermis [84].

treatment of atopic dermatitis. They are commercially available in capsules, liquid or en‐ riched diet. The benefits of fatty acids therapy for CAD, is not clear despite the many studies in the area, however, they might reduce the corticosteroids dose, after 8 weeks of concurrent

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 15

Pemphigus complex diseases result from the production of autoantibodies directed against epidermal intercellular content, resulting in cell separation with intraepidermal bullae for‐ mation. The deposition of antibodies promotes the physical separation of the cells; more‐ over, it is believed that the release of cell proteases occur, which digests the intercellular substance, further aggravating the condition. Among the forms observed in dogs and cats are pemphigus erythematosus, pemphigus vulgaris, pemphigus vegetans and pemphigus foliaceus, the latter being the most common autoimmune skin disorder in these species [92],

Pemphigus foliaceus is observed more frequently in middle-aged animals, especially in do‐ mestic short haired cats and in dogs of some predisposed breeds such as Bearded Collies, Japanese Akitas, Chow Chows, Doberman Pinschers and Newfoundlands [93, 94]. It is a vesiculobullous, erosive disorder, which produces evident footpad hyperkeratosis. The face, trunk and abdomen are also frequently affected [95, 96]. Pustules, crusted lesions, erythema, alopecia and secondary pyoderma may also be present [94, 96]. Cachexia and sepsis secon‐

When present, pustular lesions can be evaluated cytologically and can reveal non degen‐ erated granulocytes and acanthocytes [94]. Histologically intraepidermal and intrafollicu‐ lar pustules are observed, with the presence of superficial perivascular dermatitis and acanthocytes [92, 93, 98]. The histologic presence of acantholysis is the Hallmark of the

Therapy requires the use of immunosuppressant or immunomodulators. Prednisone, pre‐ dnisolone and methylprednisolone are commonly used in the treatment of pemphigus. Ini‐ tially it is recommended a dose of 2.2 to 4.4 mg/kg, every 24 hours, for all three drugs. If therapeutic response is seen in 14 days, the dosage should be reduced gradually over 30 to 40 days. After this, an alternate day dose should be implemented, reaching a final protocol of 1 mg/kg every 48 hours or less [98]. In resistant cases of canine pemphigus, the use of pre‐ dnisolone can be combined with azathioprine (2.2 mg/kg q12h or q24h) or cyclophospha‐ mide (50 mg/m2 q48h). Feline pemphigus normally responds well to prednisolone, although resistant cases may benefit from a combination of prednisolone and chlorambucil (0.1-0.2 mg/kg every 24 hours or every other day) [94]. Topical glucocorticoids can be used as a sin‐ gle therapy, in localized forms of pemphigus or used for persistent lesions, as adjunctive therapy to the systemic treatment [98]. The occurrence of deaths in cases of pemphigus folia‐

therapy [89].

**8. Immunologic skin diseases**

which will be discussed in this chapter.

dary to infection may be observed in severe cases [97].

**8.1. Pemphigus complex**

pemphigus complex [98].

Atopic dermatitis is one of the most common skin diseases in dogs, however, its pathogene‐ sis is not completely understood, so there is no curative therapy available yet [85]. Diverse topical and systemic therapies are currently available for CAD treatment such as specific al‐ lergy immunotherapy, corticosteroids, calcineurin inhibitors, anti-histamines and essential fatty acids. Unfortunately, these therapies are not effective in all cases or show adverse ef‐ fects with long term use [86]. For most dogs with atopic dermatitis, the elimination or pre‐ vention of the contact with allergens is extremely difficult and drug therapy is not always satisfactory, in these cases the possibility to modulate the immune response to allergens is a good option. Specific allergy immunotherapy, also known as hyposensitization, desensitiza‐ tion or allergy vaccines [85], is defined as the act of administrating increasing amounts of allergen extracts to an allergic patient, in order to minimize the symptoms related to aller‐ gen exposure. Immunotherapy is not an option for patients that do not produce IgE against allergens with clinical relevance [87]. As a result, 50 to 100% of dogs submitted to immuno‐ therapy may show improvement in clinical signs after four months of treatment, and some of these animals remain with no clinical sign for long periods [88]. Despite broadly applied in the treatment of human atopic dermatitis, there are just a few studies of the topical corti‐ costeroids use in veterinary medicine. Triamcinolone 0.015% spray was used with good re‐ sults during a month with minimal side effects [89]. Recently, hydrocortisone aceponate 0.0584% spray applied once daily in affected areas, in the dose of two sprays for each 100 cm2, during 84 days was markedly effective, showing results similar to those obtained with cyclosporine therapy, although with no adverse effects [90]. Frequently used oral glucocorti‐ coids are prednisone, prednisolone and methylprednisolone, considered efficient and with rapid anti-allergic effect, at 0.5 to 1.0 mg/kg once or twice daily. Once desired effect is ach‐ ieved, the dose may be decreased maintaining efficiency. Often observed adverse effects are related to dose and duration of the treatment [89]. Calcineurin inhibitors as tacrolimus 0.1% ointment applied twice daily on localized lesions is efficient in CAD treatment [91]. Good efficacy can also be observed with oral cyclosporine 5 mg/kg once daily during four to six weeks. When using cyclosporine the dose is usually reduced to half after obtaining improve‐ ment in clinical signs, maintaining the efficiency. Side effects as vomiting and transient diar‐ rhea can occur. Oral anti-histamines has been used for decades in CAD treatment, however, there is no study showing conclusive evidences of its efficacy. Adverse effects of anti-hista‐ mines may be sedation and lethargy but this signs are uncommon. Omega 3 and 6 essential fatty acids from vegetal or from fishes source has been used for longer than 20 years in the treatment of atopic dermatitis. They are commercially available in capsules, liquid or en‐ riched diet. The benefits of fatty acids therapy for CAD, is not clear despite the many studies in the area, however, they might reduce the corticosteroids dose, after 8 weeks of concurrent therapy [89].

## **8. Immunologic skin diseases**

#### **8.1. Pemphigus complex**

condition is known as "canine atopic like dermatitis" [80]. Other dermatosis might share similar signs with canine atopic dermatitis or could be concurrent diseases and it is why they should be eliminated or controlled before the conclusive diagnose of CAD. Differential diagnoses for CAD are FAD, CAFR, pruritic parasitic diseases, bacterial pyoderma, *Malasse‐ zia* dermatitis and other allergic diseases [82, 83]. Allergy tests goals are to determinate aller‐ gens, to contact avoidance and to be included in the allergy specific immunotherapy [83]. No allergy test is completely sensitive or specific, therefore, clinically normal animals can have positive responses and animals with negative results can have clinical characteristics of the disease [82]. Allergens generally related to CAD pathogenesis are domestic dust mites,

Atopic dermatitis is one of the most common skin diseases in dogs, however, its pathogene‐ sis is not completely understood, so there is no curative therapy available yet [85]. Diverse topical and systemic therapies are currently available for CAD treatment such as specific al‐ lergy immunotherapy, corticosteroids, calcineurin inhibitors, anti-histamines and essential fatty acids. Unfortunately, these therapies are not effective in all cases or show adverse ef‐ fects with long term use [86]. For most dogs with atopic dermatitis, the elimination or pre‐ vention of the contact with allergens is extremely difficult and drug therapy is not always satisfactory, in these cases the possibility to modulate the immune response to allergens is a good option. Specific allergy immunotherapy, also known as hyposensitization, desensitiza‐ tion or allergy vaccines [85], is defined as the act of administrating increasing amounts of allergen extracts to an allergic patient, in order to minimize the symptoms related to aller‐ gen exposure. Immunotherapy is not an option for patients that do not produce IgE against allergens with clinical relevance [87]. As a result, 50 to 100% of dogs submitted to immuno‐ therapy may show improvement in clinical signs after four months of treatment, and some of these animals remain with no clinical sign for long periods [88]. Despite broadly applied in the treatment of human atopic dermatitis, there are just a few studies of the topical corti‐ costeroids use in veterinary medicine. Triamcinolone 0.015% spray was used with good re‐ sults during a month with minimal side effects [89]. Recently, hydrocortisone aceponate 0.0584% spray applied once daily in affected areas, in the dose of two sprays for each 100 cm2, during 84 days was markedly effective, showing results similar to those obtained with cyclosporine therapy, although with no adverse effects [90]. Frequently used oral glucocorti‐ coids are prednisone, prednisolone and methylprednisolone, considered efficient and with rapid anti-allergic effect, at 0.5 to 1.0 mg/kg once or twice daily. Once desired effect is ach‐ ieved, the dose may be decreased maintaining efficiency. Often observed adverse effects are related to dose and duration of the treatment [89]. Calcineurin inhibitors as tacrolimus 0.1% ointment applied twice daily on localized lesions is efficient in CAD treatment [91]. Good efficacy can also be observed with oral cyclosporine 5 mg/kg once daily during four to six weeks. When using cyclosporine the dose is usually reduced to half after obtaining improve‐ ment in clinical signs, maintaining the efficiency. Side effects as vomiting and transient diar‐ rhea can occur. Oral anti-histamines has been used for decades in CAD treatment, however, there is no study showing conclusive evidences of its efficacy. Adverse effects of anti-hista‐ mines may be sedation and lethargy but this signs are uncommon. Omega 3 and 6 essential fatty acids from vegetal or from fishes source has been used for longer than 20 years in the

mold spores, trees and grasses pollens, antigens from insects and epidermis [84].

14 Insights from Veterinary Medicine

Pemphigus complex diseases result from the production of autoantibodies directed against epidermal intercellular content, resulting in cell separation with intraepidermal bullae for‐ mation. The deposition of antibodies promotes the physical separation of the cells; more‐ over, it is believed that the release of cell proteases occur, which digests the intercellular substance, further aggravating the condition. Among the forms observed in dogs and cats are pemphigus erythematosus, pemphigus vulgaris, pemphigus vegetans and pemphigus foliaceus, the latter being the most common autoimmune skin disorder in these species [92], which will be discussed in this chapter.

Pemphigus foliaceus is observed more frequently in middle-aged animals, especially in do‐ mestic short haired cats and in dogs of some predisposed breeds such as Bearded Collies, Japanese Akitas, Chow Chows, Doberman Pinschers and Newfoundlands [93, 94]. It is a vesiculobullous, erosive disorder, which produces evident footpad hyperkeratosis. The face, trunk and abdomen are also frequently affected [95, 96]. Pustules, crusted lesions, erythema, alopecia and secondary pyoderma may also be present [94, 96]. Cachexia and sepsis secon‐ dary to infection may be observed in severe cases [97].

When present, pustular lesions can be evaluated cytologically and can reveal non degen‐ erated granulocytes and acanthocytes [94]. Histologically intraepidermal and intrafollicu‐ lar pustules are observed, with the presence of superficial perivascular dermatitis and acanthocytes [92, 93, 98]. The histologic presence of acantholysis is the Hallmark of the pemphigus complex [98].

Therapy requires the use of immunosuppressant or immunomodulators. Prednisone, pre‐ dnisolone and methylprednisolone are commonly used in the treatment of pemphigus. Ini‐ tially it is recommended a dose of 2.2 to 4.4 mg/kg, every 24 hours, for all three drugs. If therapeutic response is seen in 14 days, the dosage should be reduced gradually over 30 to 40 days. After this, an alternate day dose should be implemented, reaching a final protocol of 1 mg/kg every 48 hours or less [98]. In resistant cases of canine pemphigus, the use of pre‐ dnisolone can be combined with azathioprine (2.2 mg/kg q12h or q24h) or cyclophospha‐ mide (50 mg/m2 q48h). Feline pemphigus normally responds well to prednisolone, although resistant cases may benefit from a combination of prednisolone and chlorambucil (0.1-0.2 mg/kg every 24 hours or every other day) [94]. Topical glucocorticoids can be used as a sin‐ gle therapy, in localized forms of pemphigus or used for persistent lesions, as adjunctive therapy to the systemic treatment [98]. The occurrence of deaths in cases of pemphigus folia‐ ceus are mainly from side effects of the drugs, secondary infections or in many cases, the pet owner requests euthanasia [97].

coids administration, or spontaneously, as a consequence of pituitary or adrenal gland pri‐ mary hyper function. In dogs and cats, 80 to 85% of spontaneous hyperadrenocorticism are hypophysis-dependent [112]. Poodles, Dachshunds, Boston Terriers and Boxers are the more

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 17

Symptoms as polyuria, polydipsia, polyphagia, hepatomegaly and abdominal swelling (potbellied appearance) are commonly observed. Dermatological signs include hypotrichosis, alopecia along the back tending to be symmetrical and bilateral, comedones, pyoderma and seborrhea [112]. Hyperpigmentation, thinner skin and calcinosis cutis occurs less frequently [114]. Clinical signs associated to laboratorial evaluation and diagnostic imaging findings, leads to the diagnosis. Plasma ACTH, low dexamethasone dose suppression test, ACTH stimulation test [113, 115] and urinary cortisol/creatinin ratio [116] confirms hyperadreno‐ corticism. It is also important to obtain the history of glucocorticoids use to eliminate iatro‐

Hyperadrenocorticism therapy depends on etiology, severity degree, tumor malignancy and availability of treatment options [118]. In hypophysis-dependent cases of adrenal dysfunc‐ tion, surgical resection of the pituitary gland can be performed with low mortality rate. Adrenalectomy is recommended in cases of adrenal neoplasms. Drug therapy to control hy‐ percortisolism involves the oral use of mitotane (o, p'-DDD), 50 mg/kg once a day [113] or oral trilostane, 2 to 3 mg/kg once a day. Some dogs may show a transient worsening of der‐ matological signs, especially with mitotane use. Complete resolution of dermatological signs

Canine pattern baldness is a common condition that affects short coated dog breeds with the development of acquired alopecia in specific body regions as, post-auricular, ventral cervi‐ cal, chest, abdomen and caudomedial aspect of the thighs [119] (Figure 5). Canine pattern baldness is an aesthetic problem, with no consequences to patients health however, treat‐ ment may be required by the owners. Treatment may be tried with oral melatonin 5 mg (one capsule), once daily during 30 days to promote hair growth in alopecic regions [119,120].

Follicular dysplasia is an inherited condition characterized by an abnormal development of the hair follicle, leading to hair structural abnormalities and alopecia [121]. Neck, back and hind limbs can be affected and secondary pyoderma and comedones are common clinical findings [122]. The diagnosis confirmation through histopathology revel a great number of dysplastic follicles, dysplastic hair shafts and melanin aggregates within the hairs [123]. Pa‐ tients can experiment spontaneous remission of follicle dysplasia, however, oral administra‐

tion of 3 mg melatonin twice daily may help hair regrowth [124].

often affected dog breeds, and it rarely occurs in cats [113].

genic cause [117].

usually takes months to be achieved [118].

**10. Acquired alopecia**

**10.1. Canine pattern baldness**

**10.2. Canine follicular dysplasia**

#### **8.2. Discoid lupus erythematosus**

Discoid lupus erythematosus (DLE) is an immune-mediated disease in which the lesions are similar to those of systemic lupus erythematosus (SLE), although, without the presence of the fluorescent antinuclear antibody and without the involvement of other body systems, as occurs in SLE [99]. The etiology is unknown, however, sunlight can precipitate or exacerbate the lesions. The most commonly affected regions of the body are the face and ears. Initially, there are depigmentation, erythema and desquamation. The lesions progress to scaly and swollen plaques. Definitive diagnosis is made by characteristic histopathology or immuno‐ fluorescence. Affected animals usually have a good response to systemic therapy with glu‐ cocorticoids, but topical presentations are also an option to the treatment [100]. Topical tacrolimus 0.1% can be an alternative therapy to the treatment with glucocorticoids, and was effective and safe for the treatment of DLE in dogs [101].

## **9. Metabolic and endocrine diseases**

#### **9.1. Hypothyroidism**

Hypothyroidism is an endocrine disorder associated with a reduced production of T4 and T3 hormones by the thyroid gland [102]. Rarely affects cats [103], however, is the most com‐ mon endocrinopathy in dogs [104]. The primary destruction of the thyroid gland occurs in more than 95% of adult dogs with hypothyroidism [104,105]. Thyroid hormones are ex‐ tremely important in the maintenance of normal skin function, and dermatologic conditions are reported in 60 to 80% of hypothyroidism cases [106,107]. Usual findings are changes in coat quality and color, alopecia (in most cases, bilaterally symmetric and sparing head and limbs), superficial pyoderma, dry and desquamated skin, dry or oily seborrhea, brittle and easily pulled hairs, hyperkeratosis, hyperpigmentation, comedones, otitis, deficient healing of wounds, pruritus, myxedema and obesity [108].

Diagnosis is based on clinical signs, and on serum total TSH and free T4 [109]. Histopatholo‐ gy may show highly suggestive signs of hypothyroidism as follicle atrophy, hyperkeratosis, epidermal melanosis, many follicles in telogen phase [110], hypertrophy and vacuolization of arrector pili muscles, increased dermal mucine and thickened dermis [111].

Oral sodium levothyroxine (T4), 20 µg/kg twice daily, is the drug of choice for hypothyroid‐ ism [108]. The patient should receive appropriate treatment for dermatologic conditions pre‐ sented, although they tend to disappear with the sodium levothyroxine administration.

#### **9.2. Hyperadrenocorticism**

Hyperadrenocorticism, also known as Cushing syndrome, results from chronic excessive cortisol secretion by the adrenal glands. It occurs iatrogenically, after synthetic glucocorti‐ coids administration, or spontaneously, as a consequence of pituitary or adrenal gland pri‐ mary hyper function. In dogs and cats, 80 to 85% of spontaneous hyperadrenocorticism are hypophysis-dependent [112]. Poodles, Dachshunds, Boston Terriers and Boxers are the more often affected dog breeds, and it rarely occurs in cats [113].

Symptoms as polyuria, polydipsia, polyphagia, hepatomegaly and abdominal swelling (potbellied appearance) are commonly observed. Dermatological signs include hypotrichosis, alopecia along the back tending to be symmetrical and bilateral, comedones, pyoderma and seborrhea [112]. Hyperpigmentation, thinner skin and calcinosis cutis occurs less frequently [114]. Clinical signs associated to laboratorial evaluation and diagnostic imaging findings, leads to the diagnosis. Plasma ACTH, low dexamethasone dose suppression test, ACTH stimulation test [113, 115] and urinary cortisol/creatinin ratio [116] confirms hyperadreno‐ corticism. It is also important to obtain the history of glucocorticoids use to eliminate iatro‐ genic cause [117].

Hyperadrenocorticism therapy depends on etiology, severity degree, tumor malignancy and availability of treatment options [118]. In hypophysis-dependent cases of adrenal dysfunc‐ tion, surgical resection of the pituitary gland can be performed with low mortality rate. Adrenalectomy is recommended in cases of adrenal neoplasms. Drug therapy to control hy‐ percortisolism involves the oral use of mitotane (o, p'-DDD), 50 mg/kg once a day [113] or oral trilostane, 2 to 3 mg/kg once a day. Some dogs may show a transient worsening of der‐ matological signs, especially with mitotane use. Complete resolution of dermatological signs usually takes months to be achieved [118].

## **10. Acquired alopecia**

ceus are mainly from side effects of the drugs, secondary infections or in many cases, the pet

Discoid lupus erythematosus (DLE) is an immune-mediated disease in which the lesions are similar to those of systemic lupus erythematosus (SLE), although, without the presence of the fluorescent antinuclear antibody and without the involvement of other body systems, as occurs in SLE [99]. The etiology is unknown, however, sunlight can precipitate or exacerbate the lesions. The most commonly affected regions of the body are the face and ears. Initially, there are depigmentation, erythema and desquamation. The lesions progress to scaly and swollen plaques. Definitive diagnosis is made by characteristic histopathology or immuno‐ fluorescence. Affected animals usually have a good response to systemic therapy with glu‐ cocorticoids, but topical presentations are also an option to the treatment [100]. Topical tacrolimus 0.1% can be an alternative therapy to the treatment with glucocorticoids, and was

Hypothyroidism is an endocrine disorder associated with a reduced production of T4 and T3 hormones by the thyroid gland [102]. Rarely affects cats [103], however, is the most com‐ mon endocrinopathy in dogs [104]. The primary destruction of the thyroid gland occurs in more than 95% of adult dogs with hypothyroidism [104,105]. Thyroid hormones are ex‐ tremely important in the maintenance of normal skin function, and dermatologic conditions are reported in 60 to 80% of hypothyroidism cases [106,107]. Usual findings are changes in coat quality and color, alopecia (in most cases, bilaterally symmetric and sparing head and limbs), superficial pyoderma, dry and desquamated skin, dry or oily seborrhea, brittle and easily pulled hairs, hyperkeratosis, hyperpigmentation, comedones, otitis, deficient healing

Diagnosis is based on clinical signs, and on serum total TSH and free T4 [109]. Histopatholo‐ gy may show highly suggestive signs of hypothyroidism as follicle atrophy, hyperkeratosis, epidermal melanosis, many follicles in telogen phase [110], hypertrophy and vacuolization

Oral sodium levothyroxine (T4), 20 µg/kg twice daily, is the drug of choice for hypothyroid‐ ism [108]. The patient should receive appropriate treatment for dermatologic conditions pre‐ sented, although they tend to disappear with the sodium levothyroxine administration.

Hyperadrenocorticism, also known as Cushing syndrome, results from chronic excessive cortisol secretion by the adrenal glands. It occurs iatrogenically, after synthetic glucocorti‐

of arrector pili muscles, increased dermal mucine and thickened dermis [111].

owner requests euthanasia [97].

16 Insights from Veterinary Medicine

**8.2. Discoid lupus erythematosus**

effective and safe for the treatment of DLE in dogs [101].

**9. Metabolic and endocrine diseases**

of wounds, pruritus, myxedema and obesity [108].

**9.1. Hypothyroidism**

**9.2. Hyperadrenocorticism**

#### **10.1. Canine pattern baldness**

Canine pattern baldness is a common condition that affects short coated dog breeds with the development of acquired alopecia in specific body regions as, post-auricular, ventral cervi‐ cal, chest, abdomen and caudomedial aspect of the thighs [119] (Figure 5). Canine pattern baldness is an aesthetic problem, with no consequences to patients health however, treat‐ ment may be required by the owners. Treatment may be tried with oral melatonin 5 mg (one capsule), once daily during 30 days to promote hair growth in alopecic regions [119,120].

#### **10.2. Canine follicular dysplasia**

Follicular dysplasia is an inherited condition characterized by an abnormal development of the hair follicle, leading to hair structural abnormalities and alopecia [121]. Neck, back and hind limbs can be affected and secondary pyoderma and comedones are common clinical findings [122]. The diagnosis confirmation through histopathology revel a great number of dysplastic follicles, dysplastic hair shafts and melanin aggregates within the hairs [123]. Pa‐ tients can experiment spontaneous remission of follicle dysplasia, however, oral administra‐ tion of 3 mg melatonin twice daily may help hair regrowth [124].

**11. Keratinization disorders**

that causes excessive skin desquamation [131,132].

and perivascular superficial dermatitis [130].

flammation and hyperkeratosis plaques formation [131,132].

tion disorders demand lifelong control and treatment [130].

Seborrheic dermatitis is a skin keratinization disorder which can be primary or secondary. Primary keratinization disorders are inherited and exhibits breed predisposition. Usually, clinical signs appear before two years of age [130]. About 90% of the cases are secondary to an underlying disease [131,132] such as metabolic, hereditary or nutritional disorders [133]

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 19

Seborrhea is classified in seborrhea sicca or oleosa according to hair and skin appearance [131,132]. In seborrhea sicca the coat is opaque and dry, containing aggregates of white to greyish scales, and in seborrhea oleosa there are adhesions of yellowish to brown lipid mate‐ rial, with greasy appearance [130]. Animals often exhibit pruritus, folliculitis, pyoderma, in‐

Diagnosis is based in history, clinical signs, physical examination findings and complemen‐ tary exams to eliminate differential diagnosis or to determinate the primary disease. Fre‐ quently, it is necessary to perform skin scrapings, fungal and bacterial cultures, allergy tests, endocrine function evaluation, skin biopsy and evaluation of therapy response to close the diagnosis. Histopathology reveals abnormal keratinization of the epidermis and hair folli‐ cles, orthokeratotic and parakeratotic hyperkeratosis, follicular hyperkeratosis, dyskeratosis

Seborrhea treatment goals are scales and crusts removal and oil, pruritus and inflammation reduction [131,132]. Cases of primary idiopathic seborrhea could be treated with oral Vita‐ min A (600 to 800 UI/kg, twice daily) or with retinoids as isotretinoin and etretin (1 a 2 mg/kg a q12h) [134]. Salicylic acid and sulfur shampoos are recommended and might have positive results in moderate cases. Tar shampoos are recommended to severe cases of sebor‐ rhea oleosa in dogs, however, they are contraindicated in cats [135]. Secondary cases have an excellent prognosis when the underlying disease is eliminated, while primary keratiniza‐

Acne is a common disorder in cats and it may result from an idiopathic keratinization defect or a secondary reaction pattern to another disorder [136]. The most common skin changes are found in the chin and lip margin, and they include comedones, crusts, papules, erythe‐ ma, alopecia and variable pruritus. Usually this disorder courses with secondary bacterial pyoderma and in some cases, with *M. pachydermatis* infections [137]. Papules, pustules, fu‐ runculosis and cellulitis might be signs of a secondary infection. Changes in hair follicle cy‐ cle, immunosuppression, stress, deficient grooming and concurrent viral infections are possible causes for this disorder [136]. Histology shows periductal linfoplasmocitary inflam‐ mation, sebaceous gland duct dilatation and follicular keratosis with obstruction and dilata‐ tion of the follicles [137]. Mild cases can be treated with anti-seborrheic shampoo; however, severe cases require association with systemic antimicrobial therapy or systemic corticoids

**11.1. Seborrhea**

**11.2. Acne**

**Figure 5.** Canine pattern baldness. A Doberman pinscher with alopecia of the chest, which is characteristic of pattern alopecia (Veterinary Hospital of the Federal University of Viçosa).

#### **10.3. Post clipping alopecia**

Post clipping alopecia or post clipping hair follicles arrest [125] is a common but poorly un‐ derstood syndrome in dogs, characterized by the absence of hair in consequence to a defi‐ cient growth in a previously clipped area [126]. In some cases the hair may take years to grow back. On clinical exam, the skin is normal with well delimited alopecic areas. Diagno‐ sis is closed by the history of alopecia after clipping with no other dermatologic sign. If any systemic sign or other skin disorders are present, it is recommended to perform appropriate laboratory procedures to search for the endocrinopathies [125]. Usually no treatment is re‐ quired [122]

#### **10.4. Alopecia X**

Alopecia X is a disease previously known as adrenal congenital hyperplasia, Cushing-like disease, dermatosis responsive to castration and adult-onset hyposomatotropism [127]. It is a condition associated with abnormalities on the hair cycle, affecting mainly the German Spitz breed. Affected animals display hairs in telogen phase what prevents new hair growth [128]. These animals are clinically healthy and show progressive hair loss, symmetric and non-pruritic, with variable degree of hyperpigmentation [129]. Histopathology findings are similar to those found in endocrinopathies, such as comedones, superficial and infundibu‐ lum hyperkeratinization, however, the presence of catagenization with flame follicle forma‐ tion, confirms alopecia X diagnosis [127].

Therapies with sex hormones, growth hormone, mitotane and castration usually do not pro‐ duce consistent results. Trilostane 10.5 mg/kg once a day, or twice a day, may produce com‐ plete hair growth in affected animals after four to eight weeks of treatment [129].

## **11. Keratinization disorders**

#### **11.1. Seborrhea**

Seborrheic dermatitis is a skin keratinization disorder which can be primary or secondary. Primary keratinization disorders are inherited and exhibits breed predisposition. Usually, clinical signs appear before two years of age [130]. About 90% of the cases are secondary to an underlying disease [131,132] such as metabolic, hereditary or nutritional disorders [133] that causes excessive skin desquamation [131,132].

Seborrhea is classified in seborrhea sicca or oleosa according to hair and skin appearance [131,132]. In seborrhea sicca the coat is opaque and dry, containing aggregates of white to greyish scales, and in seborrhea oleosa there are adhesions of yellowish to brown lipid mate‐ rial, with greasy appearance [130]. Animals often exhibit pruritus, folliculitis, pyoderma, in‐ flammation and hyperkeratosis plaques formation [131,132].

Diagnosis is based in history, clinical signs, physical examination findings and complemen‐ tary exams to eliminate differential diagnosis or to determinate the primary disease. Fre‐ quently, it is necessary to perform skin scrapings, fungal and bacterial cultures, allergy tests, endocrine function evaluation, skin biopsy and evaluation of therapy response to close the diagnosis. Histopathology reveals abnormal keratinization of the epidermis and hair folli‐ cles, orthokeratotic and parakeratotic hyperkeratosis, follicular hyperkeratosis, dyskeratosis and perivascular superficial dermatitis [130].

Seborrhea treatment goals are scales and crusts removal and oil, pruritus and inflammation reduction [131,132]. Cases of primary idiopathic seborrhea could be treated with oral Vita‐ min A (600 to 800 UI/kg, twice daily) or with retinoids as isotretinoin and etretin (1 a 2 mg/kg a q12h) [134]. Salicylic acid and sulfur shampoos are recommended and might have positive results in moderate cases. Tar shampoos are recommended to severe cases of sebor‐ rhea oleosa in dogs, however, they are contraindicated in cats [135]. Secondary cases have an excellent prognosis when the underlying disease is eliminated, while primary keratiniza‐ tion disorders demand lifelong control and treatment [130].

#### **11.2. Acne**

**Figure 5.** Canine pattern baldness. A Doberman pinscher with alopecia of the chest, which is characteristic of pattern

Post clipping alopecia or post clipping hair follicles arrest [125] is a common but poorly un‐ derstood syndrome in dogs, characterized by the absence of hair in consequence to a defi‐ cient growth in a previously clipped area [126]. In some cases the hair may take years to grow back. On clinical exam, the skin is normal with well delimited alopecic areas. Diagno‐ sis is closed by the history of alopecia after clipping with no other dermatologic sign. If any systemic sign or other skin disorders are present, it is recommended to perform appropriate laboratory procedures to search for the endocrinopathies [125]. Usually no treatment is re‐

Alopecia X is a disease previously known as adrenal congenital hyperplasia, Cushing-like disease, dermatosis responsive to castration and adult-onset hyposomatotropism [127]. It is a condition associated with abnormalities on the hair cycle, affecting mainly the German Spitz breed. Affected animals display hairs in telogen phase what prevents new hair growth [128]. These animals are clinically healthy and show progressive hair loss, symmetric and non-pruritic, with variable degree of hyperpigmentation [129]. Histopathology findings are similar to those found in endocrinopathies, such as comedones, superficial and infundibu‐ lum hyperkeratinization, however, the presence of catagenization with flame follicle forma‐

Therapies with sex hormones, growth hormone, mitotane and castration usually do not pro‐ duce consistent results. Trilostane 10.5 mg/kg once a day, or twice a day, may produce com‐

plete hair growth in affected animals after four to eight weeks of treatment [129].

alopecia (Veterinary Hospital of the Federal University of Viçosa).

**10.3. Post clipping alopecia**

18 Insights from Veterinary Medicine

quired [122]

**10.4. Alopecia X**

tion, confirms alopecia X diagnosis [127].

Acne is a common disorder in cats and it may result from an idiopathic keratinization defect or a secondary reaction pattern to another disorder [136]. The most common skin changes are found in the chin and lip margin, and they include comedones, crusts, papules, erythe‐ ma, alopecia and variable pruritus. Usually this disorder courses with secondary bacterial pyoderma and in some cases, with *M. pachydermatis* infections [137]. Papules, pustules, fu‐ runculosis and cellulitis might be signs of a secondary infection. Changes in hair follicle cy‐ cle, immunosuppression, stress, deficient grooming and concurrent viral infections are possible causes for this disorder [136]. Histology shows periductal linfoplasmocitary inflam‐ mation, sebaceous gland duct dilatation and follicular keratosis with obstruction and dilata‐ tion of the follicles [137]. Mild cases can be treated with anti-seborrheic shampoo; however, severe cases require association with systemic antimicrobial therapy or systemic corticoids [136]. Mupirocin 2% ointment is efficient when applied on lesions twice daily [138]. Topical tretinoin 0.01 to 0.025% (lotion or ointment) improves the clinical signs in chronic cases [139,140]. Proper recognition and treatment of secondary infections contribute to a success‐ ful therapy [137]. Feline acne has a good prognosis, however, symptomatic lifelong treat‐ ment is often necessary to control the condition [140].

**13. Nutritional skin diseases**

**13.2. Zinc responsive dermatosis**

**13.1. Vitamin A responsive dermatosis**

ment. Lifelong treatment is usually necessary [144].

or with excessive calcium phytate [144,146].

**13.3. External ear diseases**

Vitamin A is essential to the maintenance of epithelial tissue integrity and is especially im‐ portant in the keratinization process [144]. Vitamin A responsive dermatosis is an uncom‐ mon condition, observed mainly in properly fed Cocker Spaniels [145] that may exhibiting skin desquamation, pruritus, oily skin, characteristic hyperkeratotic plaques and secondary pyoderma [144]. Histopathology reveals follicular oriented exuberant hyperkeratosis [145].

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 21

Lesions do not improve with anti-seborrheic therapy, but oral supplementation of vitamin A (retinol), at 10.000 UI once a day, provides clinical improvement in 5 to 8 weeks of treat‐

Zinc is essential in the cellular metabolism and also in hair and skin health maintenance [144]. Zinc responsive dermatosis is an unusual disorder in dogs and it is characterized by

Two syndromes are recognized in dogs. Syndrome I affect young adult dogs fed with bal‐ anced diets, especially Siberian Huskies and Alaskan Malamutes, however, it was reported in other breeds [146,147]. Affected animals have a diminished ability to absorb zinc from the intestinal tract, due to some subclinical disease or to genetic factors [144]. Syndrome II is ob‐ served in rapidly growing puppies, especially of giant breeds, fed with zinc deficient diets

The diagnosis is based on history, clinical signs, characteristics lesions, breed, skin biopsy and response to zinc supplementation. The histopathological abnormalities found are papil‐ lary epidermal hyperplasia, confluent spongiotic parakeratosis and suppurative crusts [146].

Syndrome I control requires lifelong oral zinc supplementation with zinc methionine 1.7 mg/kg once a day, zinc gluconate 5 mg/kg once a day or zinc sulfate 10 mg/kg once a day [14]. Refractory cases to oral supplementation could receive intravenous administration of zinc sulfate at 10 to 15 mg/kg once a week, initially during four weeks and later each one, to six months [144]. Prognosis is good in most cases, although, lifelong supplementation may

Syndrome II has an excellent prognosis and only requires food balancing to supply the zinc deficiency, however, signs remission occur faster with oral zinc supplementation [144].

The external ear is divided in three parts: inner, middle and outer ear. External or outer ear comprises the pinna, vertical canal and horizontal canal, formed by auricular and annular cartilages. The diameter of the external ear canal varies according to the age, breed and size of the animal and it is separated from the middle ear by the tympanum, a thin semitranspar‐

scaling, focal erythema, crusts and alopecia, mainly over the head [146].

be required. Affected animals should be removed from breeding [147].

## **12. Psychogenic diseases**

### **12.1. Acral lick dermatitis**

Acral lick dermatitis is characterized by ulcerated, proliferative, firm and alopecic plaques, derived from compulsive licking of the distal portion of the limbs [141] (Figure 6). Secon‐ dary bacterial infection is frequent and contributes to the sorely pruritic nature of the lesions [142]. This condition may originate from psychogenic (fear, and/or anxiety-based condi‐ tions), dermatologic (secondary to hypersensitivity, demodicosis, neoplasms and fungal or bacterial infections), traumatic, neuropathies or articular diseases [141,142].

Diagnosis requires complete clinical evaluation (anamnesis, physical, neurological and der‐ matological exams), complete blood cell count, serum biochemical profile and urinalysis. Only after the elimination of possible organic causes for acral lick dermatitis, it can be con‐ sidered a behavioral disorder. In this case, the animal behavior should be evaluated, with the observation of its environmental and social stimuli and their motivational status. Treat‐ ing acral lick dermatitis is notoriously challenging. The animal's environment should be modified to eliminate or minimize their exposure to stress factors [142], and the use of antidepressives as fluoxetine 20 mg/day may help significantly in compulsion control which im‐ proves the lesions [143].

**Figure 6.** Acral lick dermatitis. Ulcerated plaques with tissue necrosis in the distal potion of a dog's forelimb.

## **13. Nutritional skin diseases**

[136]. Mupirocin 2% ointment is efficient when applied on lesions twice daily [138]. Topical tretinoin 0.01 to 0.025% (lotion or ointment) improves the clinical signs in chronic cases [139,140]. Proper recognition and treatment of secondary infections contribute to a success‐ ful therapy [137]. Feline acne has a good prognosis, however, symptomatic lifelong treat‐

Acral lick dermatitis is characterized by ulcerated, proliferative, firm and alopecic plaques, derived from compulsive licking of the distal portion of the limbs [141] (Figure 6). Secon‐ dary bacterial infection is frequent and contributes to the sorely pruritic nature of the lesions [142]. This condition may originate from psychogenic (fear, and/or anxiety-based condi‐ tions), dermatologic (secondary to hypersensitivity, demodicosis, neoplasms and fungal or

Diagnosis requires complete clinical evaluation (anamnesis, physical, neurological and der‐ matological exams), complete blood cell count, serum biochemical profile and urinalysis. Only after the elimination of possible organic causes for acral lick dermatitis, it can be con‐ sidered a behavioral disorder. In this case, the animal behavior should be evaluated, with the observation of its environmental and social stimuli and their motivational status. Treat‐ ing acral lick dermatitis is notoriously challenging. The animal's environment should be modified to eliminate or minimize their exposure to stress factors [142], and the use of antidepressives as fluoxetine 20 mg/day may help significantly in compulsion control which im‐

**Figure 6.** Acral lick dermatitis. Ulcerated plaques with tissue necrosis in the distal potion of a dog's forelimb.

bacterial infections), traumatic, neuropathies or articular diseases [141,142].

ment is often necessary to control the condition [140].

**12. Psychogenic diseases**

**12.1. Acral lick dermatitis**

20 Insights from Veterinary Medicine

proves the lesions [143].

### **13.1. Vitamin A responsive dermatosis**

Vitamin A is essential to the maintenance of epithelial tissue integrity and is especially im‐ portant in the keratinization process [144]. Vitamin A responsive dermatosis is an uncom‐ mon condition, observed mainly in properly fed Cocker Spaniels [145] that may exhibiting skin desquamation, pruritus, oily skin, characteristic hyperkeratotic plaques and secondary pyoderma [144]. Histopathology reveals follicular oriented exuberant hyperkeratosis [145].

Lesions do not improve with anti-seborrheic therapy, but oral supplementation of vitamin A (retinol), at 10.000 UI once a day, provides clinical improvement in 5 to 8 weeks of treat‐ ment. Lifelong treatment is usually necessary [144].

#### **13.2. Zinc responsive dermatosis**

Zinc is essential in the cellular metabolism and also in hair and skin health maintenance [144]. Zinc responsive dermatosis is an unusual disorder in dogs and it is characterized by scaling, focal erythema, crusts and alopecia, mainly over the head [146].

Two syndromes are recognized in dogs. Syndrome I affect young adult dogs fed with bal‐ anced diets, especially Siberian Huskies and Alaskan Malamutes, however, it was reported in other breeds [146,147]. Affected animals have a diminished ability to absorb zinc from the intestinal tract, due to some subclinical disease or to genetic factors [144]. Syndrome II is ob‐ served in rapidly growing puppies, especially of giant breeds, fed with zinc deficient diets or with excessive calcium phytate [144,146].

The diagnosis is based on history, clinical signs, characteristics lesions, breed, skin biopsy and response to zinc supplementation. The histopathological abnormalities found are papil‐ lary epidermal hyperplasia, confluent spongiotic parakeratosis and suppurative crusts [146].

Syndrome I control requires lifelong oral zinc supplementation with zinc methionine 1.7 mg/kg once a day, zinc gluconate 5 mg/kg once a day or zinc sulfate 10 mg/kg once a day [14]. Refractory cases to oral supplementation could receive intravenous administration of zinc sulfate at 10 to 15 mg/kg once a week, initially during four weeks and later each one, to six months [144]. Prognosis is good in most cases, although, lifelong supplementation may be required. Affected animals should be removed from breeding [147].

Syndrome II has an excellent prognosis and only requires food balancing to supply the zinc deficiency, however, signs remission occur faster with oral zinc supplementation [144].

#### **13.3. External ear diseases**

The external ear is divided in three parts: inner, middle and outer ear. External or outer ear comprises the pinna, vertical canal and horizontal canal, formed by auricular and annular cartilages. The diameter of the external ear canal varies according to the age, breed and size of the animal and it is separated from the middle ear by the tympanum, a thin semitranspar‐ ent membrane with an elliptic shape [148]. External ear diseases are particularly important in veterinary dermatology, since the outer ear is formed in the embryo life through a skin invagination, being susceptive to a number of dermatologic conditions [149]. Otitis externa is the most frequent disorder of the outer ear canal in dogs and cats, consisting in the inflam‐ mation of its epithelium [150,151].

on ear canal; cleaning the pinna, vertical and horizontal canal; eliminating debris, exudates and cerumen. It is also extremely important to instruct the owner about cleaning techniques

Gram positive bacterial infections can be treated with topical steroidal antibiotics (fusidic acid) and topical fluoroquinolones (marbofloxacin and orbifloxacin). Gram negative infec‐ tions could also be treated with fluoroquinolones or with topical cationic polipeptides (poly‐ myxin B) and aminoglycosides (neomycin, framycetin, gentamicin) [154]. Aminoglycosides are contraindicated in cases of tympanic membrane rupture due to their ototoxicity [150]. *Malassezia* species are usually susceptible to azoles (clotrimazol and myconazol) and poly‐

The use of topical drugs such as moxidectin 1% and imidacloprid 10% (0.1 mg/kg, two ap‐ plications with two weeks interval) is effective for *Otodectis cynotis* infestation in cats [157].

Animals with acute otitis should be treated twice daily during 7 to 14 days. Chronic or re‐ current cases should be treated for, at least 4 weeks, and systemic anti-inflammatory drugs are recommended in association to the topical therapy (prednisone or prednisolone 0.5 mg/kg q12h) [158]. Glucocorticoids efficiently control the inflammation and may prevent or reverse tissue hyperplasia and canal stenosis [154].Therapy should be discontinued only when cytology is negative for microorganisms, if there is no ear canal edema and if the epi‐

, Tássia Sell Ferreira3

and

Dermatology in Dogs and Cats http://dx.doi.org/10.5772/53660 23

and administration methods for topical medications [150].

ene macrolides (nistatin) [154].

thelium has a normal appearance [158].

3 General Practitioner, Juiz de For, Brazil

, Luciana Diegues Guimarães2

2 Veterinary Department, Federal University of Viçosa, Viçosa, Brazil

1 Veterinary Department, Pontifical Catholic University of Minas Gerais, Betim, Brazil

[1] Thomsett LR. Structure of Canine Skin. The British Veterinary Journal 1986; 142 (2)

[2] Favarato ES, Conceição LG. Hair Cycle in Dogs with Different Hair Types in a Tropi‐

cal Region of Brazil. Veterinary Dermatology 2008; 19(1) 15-20.

\*Address all correspondence to: dermatovet@gmail.com

**Author details**

Elisa Bourguignon1

**References**

116-23.

Evandro Silva Favarato2

In order to achieve the correct diagnosis and a successful therapy, it is essential to recognize and understand the primary predisposing and perpetuating causes. Primary causes are fac‐ tors or processes that trigger the inflammation in the outer canal [152]. The most frequent trigger factors are parasitic infestation (*Octodetes cynotis, Demodex canis, Sarcoptes scabiei, No‐ troedes cati*), foreign body, allergic diseases and hypersensitivities (atopic dermatitis, food al‐ lergy, contact allergic dermatitis), keratinization disorders (seborrhea, sebaceous adenitis), hormonal disorders and autoimmune diseases (pemphigus, lupus erythematous). Predis‐ posing causes are factors and process that increases the risk of otitis development, such as the ear conformation (narrow ear canal, excessive hair in the ear canal, long and pendulous ears), excessive humidity (baths, swimming habit), iatrogenic factors (use of cotton swab to clean the ear, use of irritant cleaning solutions) and the occurrence of obstructive ear diseas‐ es (polyps and neoplasms) [151].

Factors that help to perpetuate otitis are bacterial infection (*Staphylococcus pseudinterme‐ dius, Pseudomonas aeruginosa*), yeast infection (*M. pachydermatis*) and complications of oti‐ tis media, as ear canal narrowness due to hyperplasia and calcification of the cartilage structures [151,153,154].

The initial sign of otitis externa is erythema of the pinna and outer ear canal. Worsening of symptoms can lead to head shake, ear pruritus, malodorous purulent or ceruminous dis‐ charge, edema, pain and evidences of self-inflicted trauma as aural hematoma and acute moist dermatitis in the base of ears. Chronic or recurrent otitis can lead to soft tissue hyper‐ plasia, ear canal stenosis and occlusion [151].

Diagnosis requires physical and dermatological examination, knowledge of the dermatolog‐ ical history and clinical signs development. Otoscopic examination is the first choice techni‐ que to evaluate the outer ear canal [155] and it should be performed in every patient with signs of otitis [156]. It is important to notice that a healthy ear canal might have small amounts of yellowish or brown cerumen [150]. Through otoscopy the clinician can access the presence of inflammation, exudate, hyperplasia, stenosis, foreign body, neoplasms and evaluate the tympanic membrane. Depending on the degree of pain, inflammation and stenosis, it might be necessary to use topical or systemic corticoids for two to three weeks before performing an otoscopic examination [156].

Cytological analysis is required for the diagnosis, and samples should be obtained from both pinna and the outer vertical and horizontal canal. Cytology can provide early infor‐ mation about the inflammatory response and microbial organisms or parasites involved in the process [150].

A successful therapy is based on: removing or controlling the primary cause and predispos‐ ing factors; eliminating bacterial and yeast infection, parasite infestation and foreign bodies on ear canal; cleaning the pinna, vertical and horizontal canal; eliminating debris, exudates and cerumen. It is also extremely important to instruct the owner about cleaning techniques and administration methods for topical medications [150].

Gram positive bacterial infections can be treated with topical steroidal antibiotics (fusidic acid) and topical fluoroquinolones (marbofloxacin and orbifloxacin). Gram negative infec‐ tions could also be treated with fluoroquinolones or with topical cationic polipeptides (poly‐ myxin B) and aminoglycosides (neomycin, framycetin, gentamicin) [154]. Aminoglycosides are contraindicated in cases of tympanic membrane rupture due to their ototoxicity [150]. *Malassezia* species are usually susceptible to azoles (clotrimazol and myconazol) and poly‐ ene macrolides (nistatin) [154].

The use of topical drugs such as moxidectin 1% and imidacloprid 10% (0.1 mg/kg, two ap‐ plications with two weeks interval) is effective for *Otodectis cynotis* infestation in cats [157].

Animals with acute otitis should be treated twice daily during 7 to 14 days. Chronic or re‐ current cases should be treated for, at least 4 weeks, and systemic anti-inflammatory drugs are recommended in association to the topical therapy (prednisone or prednisolone 0.5 mg/kg q12h) [158]. Glucocorticoids efficiently control the inflammation and may prevent or reverse tissue hyperplasia and canal stenosis [154].Therapy should be discontinued only when cytology is negative for microorganisms, if there is no ear canal edema and if the epi‐ thelium has a normal appearance [158].

## **Author details**

ent membrane with an elliptic shape [148]. External ear diseases are particularly important in veterinary dermatology, since the outer ear is formed in the embryo life through a skin invagination, being susceptive to a number of dermatologic conditions [149]. Otitis externa is the most frequent disorder of the outer ear canal in dogs and cats, consisting in the inflam‐

In order to achieve the correct diagnosis and a successful therapy, it is essential to recognize and understand the primary predisposing and perpetuating causes. Primary causes are fac‐ tors or processes that trigger the inflammation in the outer canal [152]. The most frequent trigger factors are parasitic infestation (*Octodetes cynotis, Demodex canis, Sarcoptes scabiei, No‐ troedes cati*), foreign body, allergic diseases and hypersensitivities (atopic dermatitis, food al‐ lergy, contact allergic dermatitis), keratinization disorders (seborrhea, sebaceous adenitis), hormonal disorders and autoimmune diseases (pemphigus, lupus erythematous). Predis‐ posing causes are factors and process that increases the risk of otitis development, such as the ear conformation (narrow ear canal, excessive hair in the ear canal, long and pendulous ears), excessive humidity (baths, swimming habit), iatrogenic factors (use of cotton swab to clean the ear, use of irritant cleaning solutions) and the occurrence of obstructive ear diseas‐

Factors that help to perpetuate otitis are bacterial infection (*Staphylococcus pseudinterme‐ dius, Pseudomonas aeruginosa*), yeast infection (*M. pachydermatis*) and complications of oti‐ tis media, as ear canal narrowness due to hyperplasia and calcification of the cartilage

The initial sign of otitis externa is erythema of the pinna and outer ear canal. Worsening of symptoms can lead to head shake, ear pruritus, malodorous purulent or ceruminous dis‐ charge, edema, pain and evidences of self-inflicted trauma as aural hematoma and acute moist dermatitis in the base of ears. Chronic or recurrent otitis can lead to soft tissue hyper‐

Diagnosis requires physical and dermatological examination, knowledge of the dermatolog‐ ical history and clinical signs development. Otoscopic examination is the first choice techni‐ que to evaluate the outer ear canal [155] and it should be performed in every patient with signs of otitis [156]. It is important to notice that a healthy ear canal might have small amounts of yellowish or brown cerumen [150]. Through otoscopy the clinician can access the presence of inflammation, exudate, hyperplasia, stenosis, foreign body, neoplasms and evaluate the tympanic membrane. Depending on the degree of pain, inflammation and stenosis, it might be necessary to use topical or systemic corticoids for two to three weeks

Cytological analysis is required for the diagnosis, and samples should be obtained from both pinna and the outer vertical and horizontal canal. Cytology can provide early infor‐ mation about the inflammatory response and microbial organisms or parasites involved

A successful therapy is based on: removing or controlling the primary cause and predispos‐ ing factors; eliminating bacterial and yeast infection, parasite infestation and foreign bodies

mation of its epithelium [150,151].

22 Insights from Veterinary Medicine

es (polyps and neoplasms) [151].

plasia, ear canal stenosis and occlusion [151].

before performing an otoscopic examination [156].

structures [151,153,154].

in the process [150].

Elisa Bourguignon1 , Luciana Diegues Guimarães2 , Tássia Sell Ferreira3 and Evandro Silva Favarato2


## **References**


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**Chapter 2**

**Immunohistochemical Analysis of**

Additional information is available at the end of the chapter

frequency of metastasis and progressive disease [1].

and inflammatory mammary carcinoma [9,11-13].

Anna M. Badowska-Kozakiewicz

http://dx.doi.org/10.5772/54472

**1. Introduction**

**Progesterone Receptor and Proliferating Cell Nuclear**

**Antigen in Canine Inflammatory Mammary Carcinoma**

Cancer constitutes a major problem in animal pathology and is a subject of intensive research. Canine mammary tumors are common neoplasms and have been reported to account for up to half of all tumors in female dog [1]. All malignant canine mammary tumors have the potential to metastasize and in general, metastasis tends to occur via the lymphatic system to the inguinal lymph nodes or hematogenously to the lungs or to more distant body sites including the liver, spleen, heart, bone [2]. Canine mammary tumors share many similarities with breast cancer in human beings, including the high prevalence of adenocarcinomas,

Inflammatory mammary carcinoma is a special type of locally advanced mammary cancer that is associated with particularly aggressive behaviour and poor prognosis in women - in which case it is termed *inflammatory breast carcinoma* (IBC), and in the dog - in which case it is termed inflammatory mammary carcinoma (IMC) [3-8]. In both species, this uncommon type of tumors corresponds to a locally invasive mammary cancer that can be clinically misdiagnosed as a dermatitis or mastitis owing to its special inflammatory phenotype [9,10]. Although the pathogenesis of the disease remains obscure, some special clinical, genetic, biologic and hormonal characteristics have been found to be specific for inflammatory breast carcinoma

Inflammatory mammary carcinoma is a very specific type of rare, very aggressive and highly metastatic mammary cancer in dogs [7,8]. Clinical features include the presence of pain, erythema, edema and ulceration in the skin of the mammary gland region. These features are similar to symptoms of inflammatory diseases such as mastitis and dermatitis [2,7,8]. Histo‐

> © 2013 Badowska-Kozakiewicz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Badowska-Kozakiewicz; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.


**Chapter 2**

## **Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine Inflammatory Mammary Carcinoma**

Anna M. Badowska-Kozakiewicz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54472

## **1. Introduction**

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34 Insights from Veterinary Medicine

Cancer constitutes a major problem in animal pathology and is a subject of intensive research. Canine mammary tumors are common neoplasms and have been reported to account for up to half of all tumors in female dog [1]. All malignant canine mammary tumors have the potential to metastasize and in general, metastasis tends to occur via the lymphatic system to the inguinal lymph nodes or hematogenously to the lungs or to more distant body sites including the liver, spleen, heart, bone [2]. Canine mammary tumors share many similarities with breast cancer in human beings, including the high prevalence of adenocarcinomas, frequency of metastasis and progressive disease [1].

Inflammatory mammary carcinoma is a special type of locally advanced mammary cancer that is associated with particularly aggressive behaviour and poor prognosis in women - in which case it is termed *inflammatory breast carcinoma* (IBC), and in the dog - in which case it is termed inflammatory mammary carcinoma (IMC) [3-8]. In both species, this uncommon type of tumors corresponds to a locally invasive mammary cancer that can be clinically misdiagnosed as a dermatitis or mastitis owing to its special inflammatory phenotype [9,10]. Although the pathogenesis of the disease remains obscure, some special clinical, genetic, biologic and hormonal characteristics have been found to be specific for inflammatory breast carcinoma and inflammatory mammary carcinoma [9,11-13].

Inflammatory mammary carcinoma is a very specific type of rare, very aggressive and highly metastatic mammary cancer in dogs [7,8]. Clinical features include the presence of pain, erythema, edema and ulceration in the skin of the mammary gland region. These features are similar to symptoms of inflammatory diseases such as mastitis and dermatitis [2,7,8]. Histo‐

© 2013 Badowska-Kozakiewicz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Badowska-Kozakiewicz; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

logically there is evidence of a poorly differentiated carcinoma with extensive evidence of both mononuclear and polymorphonuclear cellular infiltrates and often edema. Dermal lymphatic invasion also can be seen histologically. Clinically, these neoplasms grow and metastasize extremely rapidly and invade lymphatics in the skin, resulting in marked edema and inflam‐ mation [14].

Inflammatory mammary carcinoma occurs most often in female dogs, but it can strike male dogs too. Most dogs that develop this form of reproductive cancer are females who have never been spayed, or who were spayed after they were two years old. Hormone therapy can also increase your female dog's risk of developing mammary cancer. Spay‐ ing your dog before she undergoes her first reproductive cycle at six months of age is the best way to prevent inflammatory carcinoma. Rarely, this type of cancer occurs in male dogs, which also have mammary glands. Dogs who develop a mammary carcino‐ ma will have one or more tumors in their mammary glands. More than half of dogs with this type of cancer develop tumors in more than one mammary gland. Malignant tumors grow quickly, are often irregular in shape, may attach themselves to the sur‐ rounding skin or tissue, and may cause painful inflammation and even ulceration of the

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 37

Inflammatory mammary carcinoma in dogs often causes pain and swelling in the affect‐ ed area. The tumors may be hard or soft. You will be able to feel them under the skin, and often they may be visible to the naked eye as well. The area will be warm and ten‐ der to the touch. It is advised to take tissue biopsies of the tumors to determine if they are cancerous and if they are inflammatory carcinoma. Blood tests and urinalysis can help the practitioner to determine if the cancer has spread, and how it may be affecting

Surgery is sometimes used to remove inflammatory carcinoma tumors, though this is not always advisable in dogs. If the cancer has not yet spread, or if ulceration and infection has occurred, it may be indicated to remove the tumor and affected mammary gland surgically. If the dog has not yet been spayed, your vet may want to perform this procedure as well. Inflammatory mammary cancer in dogs is an aggressive disease that spreads rapidly, and

The clinician may recommend chemotherapy and radiation therapy to treat your dog's cancer, even if the cancer has not yet spread. The prognosis for inflammatory carcinoma in dogs will depend upon the size of your dog's tumors at the time of diagnosis, and whether or not the cancer has already spread to other parts of the body. Survival time after treatment can range from nine months to two years. Prevent inflammatory carcinoma by having your dog spayed before she is six months old. Dogs who are obese, or who eat diets high in beef and pork, are

In the clinical evaluation of inflammatory mammary carcinoma observed a strong resemblance to the inflammatory process, so it is often confused with dermatitis [23-25]. Construction of histological inflammatory mammary carcinoma is not uniform - they can be all forms of cancer (Figure 2). Often these are tumors of low maturity. The lymphatic vessels of the skin congestion

The purpose of this study was to evaluate of proliferating cell nuclear antigen (PCNA), cytokeratin 19 (CK19), and progesterone receptor (PgR) in two cases of canine inflamma‐ tory mammary carcinoma and whether or not these markers might be useful in tumor

surgery alone often does little to slow or stop its progression [21,22].

at an increased risk of developing inflammatory carcinoma [22].

states of cancer cells. The prognosis is bad.

identification or prognosis.

affected area [8,12,13,20,21].

other body organs [22].

Two clinical forms of inflammatory mammary carcinoma have been described in women and dogs [8,9,15-17]: primary and secondary. Primary inflammatory mammary carcinoma occurs suddenly in dogs without previous detection of lesions of mammary tumors while the secondary inflammatory mammary carcinoma accompanies mammary tumors [8]. Secondary inflammatory mammary carcinoma is further classified into two types: postsurgical whenever it develops after surgical excision of a previous mammary tumors, and non-postsurgical when developing from a previous mammary tumor not surgically treated that leaded to inflamma‐ tory mammary carcinoma [9,18,19].

**Figure 1.** *Inflammatory mammary carcinoma* (IMC), Hematoxylin-eosin (H&E); Bar = 100µm.

Inflammatory mammary carcinoma occurs most often in female dogs, but it can strike male dogs too. Most dogs that develop this form of reproductive cancer are females who have never been spayed, or who were spayed after they were two years old. Hormone therapy can also increase your female dog's risk of developing mammary cancer. Spay‐ ing your dog before she undergoes her first reproductive cycle at six months of age is the best way to prevent inflammatory carcinoma. Rarely, this type of cancer occurs in male dogs, which also have mammary glands. Dogs who develop a mammary carcino‐ ma will have one or more tumors in their mammary glands. More than half of dogs with this type of cancer develop tumors in more than one mammary gland. Malignant tumors grow quickly, are often irregular in shape, may attach themselves to the sur‐ rounding skin or tissue, and may cause painful inflammation and even ulceration of the affected area [8,12,13,20,21].

logically there is evidence of a poorly differentiated carcinoma with extensive evidence of both mononuclear and polymorphonuclear cellular infiltrates and often edema. Dermal lymphatic invasion also can be seen histologically. Clinically, these neoplasms grow and metastasize extremely rapidly and invade lymphatics in the skin, resulting in marked edema and inflam‐

Two clinical forms of inflammatory mammary carcinoma have been described in women and dogs [8,9,15-17]: primary and secondary. Primary inflammatory mammary carcinoma occurs suddenly in dogs without previous detection of lesions of mammary tumors while the secondary inflammatory mammary carcinoma accompanies mammary tumors [8]. Secondary inflammatory mammary carcinoma is further classified into two types: postsurgical whenever it develops after surgical excision of a previous mammary tumors, and non-postsurgical when developing from a previous mammary tumor not surgically treated that leaded to inflamma‐

**Figure 1.** *Inflammatory mammary carcinoma* (IMC), Hematoxylin-eosin (H&E); Bar = 100µm.

mation [14].

36 Insights from Veterinary Medicine

tory mammary carcinoma [9,18,19].

Inflammatory mammary carcinoma in dogs often causes pain and swelling in the affect‐ ed area. The tumors may be hard or soft. You will be able to feel them under the skin, and often they may be visible to the naked eye as well. The area will be warm and ten‐ der to the touch. It is advised to take tissue biopsies of the tumors to determine if they are cancerous and if they are inflammatory carcinoma. Blood tests and urinalysis can help the practitioner to determine if the cancer has spread, and how it may be affecting other body organs [22].

Surgery is sometimes used to remove inflammatory carcinoma tumors, though this is not always advisable in dogs. If the cancer has not yet spread, or if ulceration and infection has occurred, it may be indicated to remove the tumor and affected mammary gland surgically. If the dog has not yet been spayed, your vet may want to perform this procedure as well. Inflammatory mammary cancer in dogs is an aggressive disease that spreads rapidly, and surgery alone often does little to slow or stop its progression [21,22].

The clinician may recommend chemotherapy and radiation therapy to treat your dog's cancer, even if the cancer has not yet spread. The prognosis for inflammatory carcinoma in dogs will depend upon the size of your dog's tumors at the time of diagnosis, and whether or not the cancer has already spread to other parts of the body. Survival time after treatment can range from nine months to two years. Prevent inflammatory carcinoma by having your dog spayed before she is six months old. Dogs who are obese, or who eat diets high in beef and pork, are at an increased risk of developing inflammatory carcinoma [22].

In the clinical evaluation of inflammatory mammary carcinoma observed a strong resemblance to the inflammatory process, so it is often confused with dermatitis [23-25]. Construction of histological inflammatory mammary carcinoma is not uniform - they can be all forms of cancer (Figure 2). Often these are tumors of low maturity. The lymphatic vessels of the skin congestion states of cancer cells. The prognosis is bad.

The purpose of this study was to evaluate of proliferating cell nuclear antigen (PCNA), cytokeratin 19 (CK19), and progesterone receptor (PgR) in two cases of canine inflamma‐ tory mammary carcinoma and whether or not these markers might be useful in tumor identification or prognosis.

tained 14 adenomas, 66 complex carcinomas (adenocarcinomas), 47 simple carcinomas (adenocarcinomas), 6 solid carcinomas and 2 inflammatory mammary carcinoma (IMC). The number of cancers with a defined grade amounted to, respectively, 1st grade – 48, 2nd grade – 39 and 3rd grade – 34. Mammary gland neoplasms were excised from fe‐ male dogs belonging to 9 breeds in the ages between 3 and 16 years. There were 106 mixed-breed dogs, 10 German Shepherd Dogs, 5 Boxers, 3, Rottweilers, 2 Beagles, 6 Yorkshire Terrier and 1 each of the following breed: English Springer Spaniel, Labra‐

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

Specimens were fixed in 8% buffered formalin, dehydrated and embedded in paraffin. 4µm thick sections were cut from each mammary neoplasm. The sections were mounted onto slides coated with 3-amino-propyltrietoxysilane (Sigma), deparaffinized in xylene and rehydrated in graded ethanol concentrations. Antigen retrieval was performed in 10mM citrate buffer in a microwave oven at 600W for 5 minutes and 300W for 10 minutes. The slides were left to cool at room temperature immersed in the buffer. Thereafter they were washed for 10 minutes in running tap water and rinsing in distilled water. Endogenous peroxidase was quenched by immersion in a solution of 30% hydrogen peroxide and methanol (50 ml of H202 and 50 ml of methanol) for 10 minutes. The slides were washed in distilled water then with TRIS (pH 7.4) for 10 minutes, and then incubated with the primary monoclonal antibodies in humid chamber for 1 hour at room temperature. Primary antibody clones and dilutions used for progesterone receptor (PgR), proliferating cell nuclear antigen (PCNA), and for cytokeratin 19 (CK19)

**Retrieval**

Mab \* 1:50 HTAR \*\* 1 hr, room temp DakoCytomation

Mab \* 1:50 HTAR \*\* 1 hr, room temp Novocastra

Mab \* 1:50 HTAR \*\* 1 hr, room temp DakoCytomation

HTAR – high-temperature antigen retrieval (with 10 mM citrate buffer, pH

**Incubation Source**

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 39

dor Retriever, Doberman Pinscher.

**2.2. Methods**

presented (table 1).

Cytokeratin 19 (CK19)

Progesterone Receptor (PgR)

Proliferating cell nuclear antigen (PCNA)

Mab - mouse monoclonal antibody; (

**Table 1.** The antibodies used for immunohistochemistry

( \*)

6.0).

**Antibody Type Dilution Antigen**

\*\*)

**Figure 2.** *Inflammatory mammary carcinoma* (IMC), Hematoxylin-eosin (H&E); Bar = 100µm.

## **2. Materials and methods**

#### **2.1. Materials**

A total of 135 samples from canine mammary tumours were used in this study, from which 80 were parafine-embeded archive samples from a period running from 2006 to 2008, and 55 were fresh samples obtained from mastectomy surgery, performed at Warsaw Veterinary Clinics and Small Animal Clinic of the Department of Clinical Sci‐ ences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW. Through three years period, only two cases of inflammatory mammary carcinoma (IMC) were diagnosed, which were further confirmed by clinical signs and histopathol‐ ogy were selected for further immunohistochemical studies.Investigated material con‐ tained 14 adenomas, 66 complex carcinomas (adenocarcinomas), 47 simple carcinomas (adenocarcinomas), 6 solid carcinomas and 2 inflammatory mammary carcinoma (IMC). The number of cancers with a defined grade amounted to, respectively, 1st grade – 48, 2nd grade – 39 and 3rd grade – 34. Mammary gland neoplasms were excised from fe‐ male dogs belonging to 9 breeds in the ages between 3 and 16 years. There were 106 mixed-breed dogs, 10 German Shepherd Dogs, 5 Boxers, 3, Rottweilers, 2 Beagles, 6 Yorkshire Terrier and 1 each of the following breed: English Springer Spaniel, Labra‐ dor Retriever, Doberman Pinscher.

#### **2.2. Methods**

**Figure 2.** *Inflammatory mammary carcinoma* (IMC), Hematoxylin-eosin (H&E); Bar = 100µm.

A total of 135 samples from canine mammary tumours were used in this study, from which 80 were parafine-embeded archive samples from a period running from 2006 to 2008, and 55 were fresh samples obtained from mastectomy surgery, performed at Warsaw Veterinary Clinics and Small Animal Clinic of the Department of Clinical Sci‐ ences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW. Through three years period, only two cases of inflammatory mammary carcinoma (IMC) were diagnosed, which were further confirmed by clinical signs and histopathol‐ ogy were selected for further immunohistochemical studies.Investigated material con‐

**2. Materials and methods**

38 Insights from Veterinary Medicine

**2.1. Materials**

Specimens were fixed in 8% buffered formalin, dehydrated and embedded in paraffin. 4µm thick sections were cut from each mammary neoplasm. The sections were mounted onto slides coated with 3-amino-propyltrietoxysilane (Sigma), deparaffinized in xylene and rehydrated in graded ethanol concentrations. Antigen retrieval was performed in 10mM citrate buffer in a microwave oven at 600W for 5 minutes and 300W for 10 minutes. The slides were left to cool at room temperature immersed in the buffer. Thereafter they were washed for 10 minutes in running tap water and rinsing in distilled water. Endogenous peroxidase was quenched by immersion in a solution of 30% hydrogen peroxide and methanol (50 ml of H202 and 50 ml of methanol) for 10 minutes. The slides were washed in distilled water then with TRIS (pH 7.4) for 10 minutes, and then incubated with the primary monoclonal antibodies in humid chamber for 1 hour at room temperature. Primary antibody clones and dilutions used for progesterone receptor (PgR), proliferating cell nuclear antigen (PCNA), and for cytokeratin 19 (CK19) presented (table 1).


( \*) Mab - mouse monoclonal antibody; ( \*\*) HTAR – high-temperature antigen retrieval (with 10 mM citrate buffer, pH 6.0).

**Table 1.** The antibodies used for immunohistochemistry

The slides were washed in TRIS for 10 minutes. The EnVision +TM System (DakoCytomation) was used for visualization. Visualization was achieved with 3,3'diaminobenzidine tetrahy‐ drocloride (DAB – DakoCytomation) in Tris-HCl buffer, and after rinsed slides were counter‐ stained with hematoxylin Ehrlicha, dehydrated in graded alcohol concentrations and xylene and closed in DPX mounting medium (Gurr®).For progesterone receptor (PgR), proliferating cell nuclear antigen (PCNA), and cytokeratin 19 (CK19), a canine mammary adenocarcinoma was used as positive control. Computer image analysis and Lucia v. 4.21 software were used for interpretation of the results of PCNA, CK19, PgR, expression; using those facilities we could count the number of neoplastic cells featuring stained cytoplasm and nucleus per 1,000 neoplastic cells. Results were analyzed using the SPSS 12.0 program. To determine whether differences for a few independent traits were significant, Kruskal-Wallis test was used. This test is an equivalent to the test of variance for traits without normal distribution. Two-sided correlations were performed using Spearman correlation test. The differences were deemed statistically significant at *p* ≤ 0.05.

**Year of bitches**

of the hind limbs.

**Adenoma**

**Carcinoma solidum**

**Adenocarcinoma simplex**

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

<8 lat 5 (21,7%) 1 (4,3%) 4 (17,4%) 13 (56,5%) 0 23

8-12 lat 7 (7,8%) 4 (4,4%) 32 (35,6%) 47 (52,2%) 2 (2.2%) 92

"/>12 lat 2 (10,0%) 1 (5,0%) 11 (55,0%) 6 (30,0%) 0 20

Total 14 (10,5%) 6 (4,5%) 47 (35,3%) 66 (49,6%) 2 (1.5%) 135

In our study, two inflammatory mammary carcinoma came from the dogs between the ages of 8 and 9 years (case 1 - 8 year, case 2 - 9 year) (Table 2). These dogs had a clinically diagnosis of IMC at the time of initial examination. The most important findings during physical examination included: erythema and warmth, generalized induration of the involved mam‐ mary glands, cutaneous nodules affecting the overlying skin, edema of the proximal portion

The value of mitotic index differed significantly between types of tumors. The lowest prolif‐ erative activity was observed in adenomas, the highest in simple carcinoma, solid carcinoma and inflammatory mammary carcinoma. The highest proliferative activity was found in tumors in the 3rd grade of malignancy, the lowest in the tumors in the 1st grade. Expression of PCNA was observed in the nuclei of neoplastic cells that have undergone division (Figure 3). Statistical analysis shows significant differences between particular types of tumors. The lowest number of cells exhibiting PCNA expression was observed in adenomas, the highest in the solid carcinoma, simple carcinoma, inflammatory mammary carcinoma and in tumors

In the two cases canine inflammatory mammary carcinoma on the basis of H&E histopathology found a high mitotic index (3.4 in the first case in the second case 4.1).PCNA index was similarly and markedly elevated in the two animals: in case 1 inflammatory mammary carcinoma was

**Table 3.** Occurrence of individual types of epithelial neoplasms in different age groups in bitches

**3.2. Results of proliferative activity in inflammatory mammary carcinoma**

82%, in case 2 the inflammatory mammary carcinoma was 85% (Figure 3).

with the highest histological grade of malignancy (3rd).

**Types of tumors Total**

**Canine inflammatory mammary carcinoma (IMC)**

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 41

**Adenocarcinoma complex**

### **3. Results and discussion**

#### **3.1. Relationship between age of bitches and the grade of malignancy and histological type of tumor of epithelial origin**

Dogs were divided into three age groups: <8 years, 8 -12 years and >12 years. In the group of bitches below the age of 8, majority (61.1%) consisted of tumors with the low‐ est histological grade of malignancy (1st). In the oldest group, 1st and 2nd grade tumors in the 1st and 2nd accounted for 77.8%. In the entire pool of studied tumors in all age groups, the largest share consisted of tumors with the lowest degree of histological ma‐ lignancy (40.4%) (Table 2). Assessment of the contribution of individual types of tumors at different ages showed that in bitches younger than 8 years the most common findings were adenomas (21.7%) and complex carcinomas (56.5%) and in those over 12 years sim‐ ple carcinomas occurred most often (55.0%) (Table 3).


**Table 2.** Incidence of malignancies of various grades in bitches in different age groups

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 41


**Table 3.** Occurrence of individual types of epithelial neoplasms in different age groups in bitches

The slides were washed in TRIS for 10 minutes. The EnVision +TM System (DakoCytomation) was used for visualization. Visualization was achieved with 3,3'diaminobenzidine tetrahy‐ drocloride (DAB – DakoCytomation) in Tris-HCl buffer, and after rinsed slides were counter‐ stained with hematoxylin Ehrlicha, dehydrated in graded alcohol concentrations and xylene and closed in DPX mounting medium (Gurr®).For progesterone receptor (PgR), proliferating cell nuclear antigen (PCNA), and cytokeratin 19 (CK19), a canine mammary adenocarcinoma was used as positive control. Computer image analysis and Lucia v. 4.21 software were used for interpretation of the results of PCNA, CK19, PgR, expression; using those facilities we could count the number of neoplastic cells featuring stained cytoplasm and nucleus per 1,000 neoplastic cells. Results were analyzed using the SPSS 12.0 program. To determine whether differences for a few independent traits were significant, Kruskal-Wallis test was used. This test is an equivalent to the test of variance for traits without normal distribution. Two-sided correlations were performed using Spearman correlation test. The differences were deemed

**3.1. Relationship between age of bitches and the grade of malignancy and histological type**

Dogs were divided into three age groups: <8 years, 8 -12 years and >12 years. In the group of bitches below the age of 8, majority (61.1%) consisted of tumors with the low‐ est histological grade of malignancy (1st). In the oldest group, 1st and 2nd grade tumors in the 1st and 2nd accounted for 77.8%. In the entire pool of studied tumors in all age groups, the largest share consisted of tumors with the lowest degree of histological ma‐ lignancy (40.4%) (Table 2). Assessment of the contribution of individual types of tumors at different ages showed that in bitches younger than 8 years the most common findings were adenomas (21.7%) and complex carcinomas (56.5%) and in those over 12 years sim‐

**<sup>o</sup> IIo IIIo**

**Tumor grade Total**

statistically significant at *p* ≤ 0.05.

40 Insights from Veterinary Medicine

**3. Results and discussion**

**of tumor of epithelial origin**

**Year of bitches**

ple carcinomas occurred most often (55.0%) (Table 3).

**I**

**Table 2.** Incidence of malignancies of various grades in bitches in different age groups

<8 lat (n=18) 11 (61.1%) 3 (16.7%) 4 (22.2%) 18

8-12 lat (n=85) 30 (36.0%) 29 (35.0%) 26 (30.5%) 85

"/>12 (n=18) 7 (38.9%) 7 (38.9%) 4 (22.2%) 18

total (n=121) 48 (40.4%) 39 (32.8%) 34 (28.1%) 121

In our study, two inflammatory mammary carcinoma came from the dogs between the ages of 8 and 9 years (case 1 - 8 year, case 2 - 9 year) (Table 2). These dogs had a clinically diagnosis of IMC at the time of initial examination. The most important findings during physical examination included: erythema and warmth, generalized induration of the involved mam‐ mary glands, cutaneous nodules affecting the overlying skin, edema of the proximal portion of the hind limbs.

#### **3.2. Results of proliferative activity in inflammatory mammary carcinoma**

The value of mitotic index differed significantly between types of tumors. The lowest prolif‐ erative activity was observed in adenomas, the highest in simple carcinoma, solid carcinoma and inflammatory mammary carcinoma. The highest proliferative activity was found in tumors in the 3rd grade of malignancy, the lowest in the tumors in the 1st grade. Expression of PCNA was observed in the nuclei of neoplastic cells that have undergone division (Figure 3). Statistical analysis shows significant differences between particular types of tumors. The lowest number of cells exhibiting PCNA expression was observed in adenomas, the highest in the solid carcinoma, simple carcinoma, inflammatory mammary carcinoma and in tumors with the highest histological grade of malignancy (3rd).

In the two cases canine inflammatory mammary carcinoma on the basis of H&E histopathology found a high mitotic index (3.4 in the first case in the second case 4.1).PCNA index was similarly and markedly elevated in the two animals: in case 1 inflammatory mammary carcinoma was 82%, in case 2 the inflammatory mammary carcinoma was 85% (Figure 3).

**Figure 4.** *Inflammatory mammary carcinoma* – ICM, immunostaining for cytokeratin 19 (CK19) in a case of IMC;

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 43

**Figure 5.** *Inflammatory mammary carcinoma* – ICM, immunostaining for cytokeratin (CK19) in a case of IMC; Bar=50 µm

Bar=100 µm

**Figure 3.** *Inflammatory mammary carcinoma* – ICM, immunostaining for PCNA in a case of IMC; Bar=100µm

#### **3.3. Results of cytokeratin 19 expression in inflammatory mammary carcinoma**

Expression of cytokeratin 19 was observed in the neoplastic cell cytoplasm. Positive CK19 reaction was found in all tumors of epithelial origin. The high expression of CK19 was found also in inflammatory mammary carcinoma (case 1 – intensity level of expression CK19 – 92%; case 2 – intensity level of expression CK19 – 98%) (Figure 4 and 5).

#### **3.4. Results of progesterone receptor expression in inflammatory mammary carcinoma**

Progesterone receptor expression was detected in the nuclei of tumor cells, but it was also seen in the cytoplasm. Cytoplasmic reaction was considered to be nonspecific. Among all tumors of epithelial origin, expression of progesterone receptors was found in 56 (41.4%), and no reaction was noted in 79 (58.5%). Expression of progesterone receptors was most commonly found in complex cancers (43.9%), simple cancers (42.6%), adenomas (28.6%), while solid cancers rarely expressed them (16.7%). The high expression of progesterone receptors was Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 43

**Figure 4.** *Inflammatory mammary carcinoma* – ICM, immunostaining for cytokeratin 19 (CK19) in a case of IMC; Bar=100 µm

**Figure 3.** *Inflammatory mammary carcinoma* – ICM, immunostaining for PCNA in a case of IMC; Bar=100µm

Expression of cytokeratin 19 was observed in the neoplastic cell cytoplasm. Positive CK19 reaction was found in all tumors of epithelial origin. The high expression of CK19 was found also in inflammatory mammary carcinoma (case 1 – intensity level of expression CK19 – 92%;

**3.4. Results of progesterone receptor expression in inflammatory mammary carcinoma**

Progesterone receptor expression was detected in the nuclei of tumor cells, but it was also seen in the cytoplasm. Cytoplasmic reaction was considered to be nonspecific. Among all tumors of epithelial origin, expression of progesterone receptors was found in 56 (41.4%), and no reaction was noted in 79 (58.5%). Expression of progesterone receptors was most commonly found in complex cancers (43.9%), simple cancers (42.6%), adenomas (28.6%), while solid cancers rarely expressed them (16.7%). The high expression of progesterone receptors was

**3.3. Results of cytokeratin 19 expression in inflammatory mammary carcinoma**

case 2 – intensity level of expression CK19 – 98%) (Figure 4 and 5).

42 Insights from Veterinary Medicine

**Figure 5.** *Inflammatory mammary carcinoma* – ICM, immunostaining for cytokeratin (CK19) in a case of IMC; Bar=50 µm

found also in inflammatory mammary carcinoma 2 (100%) (Figure 6 and 7). Analysis of the average number of cells showing positive expression of progesterone receptors reveals that the level of expression increases with the histological grade of malignancy. It was also found that most tumors expressing progesterone receptors came from dogs younger than 8 years. A positive correlation was found between mitotic index and expression of progesterone receptors in specific types of cancer and statistically significant differences between tumor characteristics were demonstrated (*p*=0.042).

**Figure 7.** *Inflammatory mammary carcinoma* – ICM, nuclear expression of progesterone receptor (PgR); Bar=100 µm

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 45

In our studies among all tumors of epithelial origin, expression of progesterone receptors was found in 41.4%. Amorim *et al.* (2008) in their study found no expression of PgR in 9 cases inflammatory mammary carcinoma [26]. However, Pen *et al*. (2003) found in their study high positive immune reaction to PgR in inflammatory mammary carcinoma [12]. The high positive immune reaction to PgR in canine inflammatory mammary carcinoma suggests,the possible involvement of special endocrine mechanisms in inflammatory mammary carcinoma devel‐

During the three years in the research material collected was diagnosed only two cases of canine inflammatory mammary carcinoma, therefore it can be said that canine inflammatory mammary carcinoma is a rare cancer in dogs [8]. Perez *et al.* showed that inflammatory mammary carcinoma is rare in dogs (17,7% of all cases within 4 years) [8]. Yet Susaneck *et al.* [13] claimed that the incidence of canine inflammatory mammary carcinoma has doubled in the past 15 years [7]. Pena *et al.* [12,13] in their research work diagnosed 33 of canine inflam‐

All these authors described this type of cancer as a rare, unusual and different. Authors investigated the relationship between clinical and histopathological characteristics of canine inflammatory mammary carcinoma specifying nuclear antigen expression of PCNA, and

matory mammary carcinoma cases in a 5 years period.

expression of progesterone receptor [7,8,12,13].

opment.

**Figure 6.** *Inflammatory mammary carcinoma* – ICM, nuclear expression of progesterone receptor (PgR); Bar=50 µm

Clinical signs and histopathological findings are necessary for the accurate diagnosis of inflammatory mammary carcinoma. Our studies have suggested that inflammatory mammary carcinoma is an aggressive malignancy because in our studies, the percentage of PCNA positive cells within the tumor was high in the two cases. An important marker of malignancy is the proliferative activity. Proper evaluation of proliferative activity of tumor cells is crucial for the evaluation of its biological activity and is used in determining the treatment of cancer. In our study, proliferative activity depended on both, the type of tumor and the degree of histological malignancy. The highest values of mitotic index were recorded in simple carci‐ nomas, solid carcinomas and in two cases of inflammatory mammary carcinoma and in tumors with the highest histological grade of malignancy. Similar results were reported for the expression of PCNA. The highest expression of PCNA was seen in solid carcinomas, simple carcinomas and in inflammatory mammary carcinoma and in 3rd grade tumors.

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 45

found also in inflammatory mammary carcinoma 2 (100%) (Figure 6 and 7). Analysis of the average number of cells showing positive expression of progesterone receptors reveals that the level of expression increases with the histological grade of malignancy. It was also found that most tumors expressing progesterone receptors came from dogs younger than 8 years. A positive correlation was found between mitotic index and expression of progesterone receptors in specific types of cancer and statistically significant differences between tumor characteristics

**Figure 6.** *Inflammatory mammary carcinoma* – ICM, nuclear expression of progesterone receptor (PgR); Bar=50 µm

carcinomas and in inflammatory mammary carcinoma and in 3rd grade tumors.

Clinical signs and histopathological findings are necessary for the accurate diagnosis of inflammatory mammary carcinoma. Our studies have suggested that inflammatory mammary carcinoma is an aggressive malignancy because in our studies, the percentage of PCNA positive cells within the tumor was high in the two cases. An important marker of malignancy is the proliferative activity. Proper evaluation of proliferative activity of tumor cells is crucial for the evaluation of its biological activity and is used in determining the treatment of cancer. In our study, proliferative activity depended on both, the type of tumor and the degree of histological malignancy. The highest values of mitotic index were recorded in simple carci‐ nomas, solid carcinomas and in two cases of inflammatory mammary carcinoma and in tumors with the highest histological grade of malignancy. Similar results were reported for the expression of PCNA. The highest expression of PCNA was seen in solid carcinomas, simple

were demonstrated (*p*=0.042).

44 Insights from Veterinary Medicine

**Figure 7.** *Inflammatory mammary carcinoma* – ICM, nuclear expression of progesterone receptor (PgR); Bar=100 µm

In our studies among all tumors of epithelial origin, expression of progesterone receptors was found in 41.4%. Amorim *et al.* (2008) in their study found no expression of PgR in 9 cases inflammatory mammary carcinoma [26]. However, Pen *et al*. (2003) found in their study high positive immune reaction to PgR in inflammatory mammary carcinoma [12]. The high positive immune reaction to PgR in canine inflammatory mammary carcinoma suggests,the possible involvement of special endocrine mechanisms in inflammatory mammary carcinoma devel‐ opment.

During the three years in the research material collected was diagnosed only two cases of canine inflammatory mammary carcinoma, therefore it can be said that canine inflammatory mammary carcinoma is a rare cancer in dogs [8]. Perez *et al.* showed that inflammatory mammary carcinoma is rare in dogs (17,7% of all cases within 4 years) [8]. Yet Susaneck *et al.* [13] claimed that the incidence of canine inflammatory mammary carcinoma has doubled in the past 15 years [7]. Pena *et al.* [12,13] in their research work diagnosed 33 of canine inflam‐ matory mammary carcinoma cases in a 5 years period.

All these authors described this type of cancer as a rare, unusual and different. Authors investigated the relationship between clinical and histopathological characteristics of canine inflammatory mammary carcinoma specifying nuclear antigen expression of PCNA, and expression of progesterone receptor [7,8,12,13].

## **4. Conclusion**

Based on the literature and our own experience it can be concluded that canine inflammatory mammary carcinoma is a rare tumors with poor prognosis. Our research suggests that inflammatory mammary carcinoma is an aggressive malignancy, with a tendency to metasta‐ size at an early stage.

[5] Giordano SH, Hortobagyi GN. Inflammatory breast cancer: clinical progress and the main problems that must be addressed. Breast Cancer Research 2003; 5:284–288. [6] Kleer CG, van Golen KL, Merajver SD. Molecular biology of breast cancer metastasis. Inflammatory breast cancer: clinical syndrome and molecular determinants. Breast

Immunohistochemical Analysis of Progesterone Receptor and Proliferating Cell Nuclear Antigen in Canine

Inflammatory Mammary Carcinoma http://dx.doi.org/10.5772/54472 47

[7] Susaneck SJ, Allen TA, Hoopes J, Withrow SJ, Macy DW. Inflammatory mammary carcinoma in the dog. Journal of the American Animal Hospital Association 1983; 9:

[8] Perez-Alenza MD, Tabanera E, Pena L. Inflammatory mammary carcinoma in dogs: 33 cases (1995–1999). Journal of the American Veterinary Medical Association 2001; 219:

[9] Clemente M, Rerez-Alenza MD, Illera JC, Pena L. Histological, Immunohistochemical, and Ultrastructural Description of Vasculogenic Mimicry in canine Mammary Cancer.

[10] Jaiyesimi IA, Buzdar AU, Hortobagyi G. Inflammatory breast cancer: a review. Journal

[11] Illera JC, Perez-Alenza MD, Nieto A, Jimenez MA, Silvan G, Dunner S, Pena L. Steroids

[12] Pena L, Perez-Alenza MD, Rodriguez-Bertos A, Nieto A. Canine inflammatory mammary carcinoma: histopathology, immunohistochemistry and clinical implica‐

[13] Pene L, Perez-Alenza MD, Silvan G, Lopes C, Nieto A. Steroid hormone profile of canine inflammatory mammary carcinoma: a preliminary study. Journal of Steroid

[14] Marconato L, Romanelli G, Stefanello D, et al. Prognostic factors for dogs with mam‐ mary inflammatory carcinoma:43 cases (2003-2008). Journal of the American Veterinary

[15] Attia-Sobol J, Ferriere JP, Cure H, Kwiatkowski F, Achard JL, Verrelle P, Feillel V, De Latour M, Lafaye C, Deloche C. Treatment results, survival and prognostic factors in 109 inflammatory breast cancers: univariate and multivariate analysis. European

[16] Resetkova E. Pathologic aspects of inflammatory breast carcinoma: part 1. Histomor‐ phology and differential diagnosis. Seminars in Oncology 2008; 35: 25-32.

[17] Taylor G, Meltzer A. Inflammatory carcinoma of the breast. American Journal of Cancer

[18] Kim T, Lau J, Erban J. Lack of uniform diagnostic criteria for inflammatory breast cancer limits interpretation of treatment outcomes: a systematic review. Clinical Breast Cancer

tions of 21 cases. Breast Cancer Research and Treatment 2003; 78: 141-148.

and receptors in canine mammary cancer. Steroids 2006; 71: 541-548.

Cancer Research 2000; 2: 423–429.

Veterinary Pathology 2012; 47(2): 265-274.

Biochemistry&Molecular Biology 2003; 84: 211-216.

Medical Association 2009; 235: 967-972.

Journal Cancer 1993; 29A: 1081-1088.

1938; 33: 33049.

2006; 7: 386-395.

Clinical Oncology 1992; 10: 1014-1024.

971–976.

1110–1114.

## **Acknowledgements**

Study was carried out at the Institute of Pathology, Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw School of Life Sciences, 159C Nowour‐ synowska Street, 02-766 Warsaw. Research was conducted as a part of a doctoral dissertation, partially funded by a grant from the Ministry of Science and Information Technology, No N30800632/0667.

## **Author details**

Anna M. Badowska-Kozakiewicz

Address all correspondence to: abadowska@op.pl

Department of Biophysics and Human Physiology, Medical University of Warsaw, Warsaw, Poland

## **References**


[5] Giordano SH, Hortobagyi GN. Inflammatory breast cancer: clinical progress and the main problems that must be addressed. Breast Cancer Research 2003; 5:284–288.

**4. Conclusion**

46 Insights from Veterinary Medicine

size at an early stage.

N30800632/0667.

**Author details**

Poland

**References**

455-477.

1079-1082.

Anna M. Badowska-Kozakiewicz

Address all correspondence to: abadowska@op.pl

**Acknowledgements**

Based on the literature and our own experience it can be concluded that canine inflammatory mammary carcinoma is a rare tumors with poor prognosis. Our research suggests that inflammatory mammary carcinoma is an aggressive malignancy, with a tendency to metasta‐

Study was carried out at the Institute of Pathology, Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw School of Life Sciences, 159C Nowour‐ synowska Street, 02-766 Warsaw. Research was conducted as a part of a doctoral dissertation, partially funded by a grant from the Ministry of Science and Information Technology, No

Department of Biophysics and Human Physiology, Medical University of Warsaw, Warsaw,

[1] Rutteman GR, Withrow SJ, MacEwen EG. Tumors of the mammary gland. W: Small Animal Clinicae Oncology Withrow SJ, MacEwen EG. (edit), 3rd ed., Philadelphia 2001;

[2] Jong-Hyuk K, Keum-Soon I, Na-Hyun K, Seung-Ki C, Alan RD, Jung-Hyang S.. Inflammatory mammary carcinoma with metastasis to the brain and distant organs in a spayed Shih Tzu dog. Journal of Veterinary Diagnostic Investigation 2011; 23(5):

[3] Perez-Alenza MD, Jimenez A, Nieto AI, Pena L. First description of feline inflammatory mammary carcinoma: Clinicopathological and immunohistochemical characteristics of

[4] Tavassoli FA. Infiltrating carcinoma: special types. Inflammatory carcinoma. In Pathology of the Breast 2nd edition. New York: McGraw-Hill 1999; 538–541.

three casus. Breast Cancer Research 2004; 6: 300-307.


[19] Kumar V, Abbas AK, Fausto N, ed.: Robbins and Cotran Pathologic Basis of Disease, 7th ed. Elsevier Saunders, Philadelphia, PA, 2005.

**Chapter 3**

**Indicators of Poor Welfare in**

James Nguhiu-Mwangi, Joshua W. Aleri, Eddy G. M. Mogoa and Peter M. F. Mbithi

http://dx.doi.org/10.5772/53678

**1. Introduction**

mal's state [10].

Additional information is available at the end of the chapter

**the Peri-Urban Areas of Nairobi, Kenya**

**Dairy Cows Within Smallholder Zero-Grazing Units in**

Animal welfare lacks a good universal definition and a satisfactory distinction from the term "well being". However, a consensual definition is essential for practical, legislative and sci‐ entific purposes. Without a clear definition, animal welfare cannot be effectively studied or conclusively assessed to provide remedial measures to its violation [1-3]. Animal welfare is therefore defined as the ability of an animal to interact or cope comfortably with its environ‐ ment, resulting in satisfaction of both its physical and mental state [4-6]. This satisfaction en‐

In the context of welfare, "environment" refers to internal factors (within the animal) and external factors (in the animal's physical environment) to which the animal responds with its physiological and psychological systems [6,9]. In contrast, animal "well being" is de‐ fined as the animal's perception of its state in trying to cope with its environment [1,5]. Concisely, animal "well-being" refers to the current state of the animal, but animal wel‐ fare is a more general term referring to past, present and future implications of the ani‐

The assessment of animal welfare is base on the provisions of five freedoms, which include: **a.** Freedom from hunger and thirst, availed through provision of ready access to water

**b.** Freedom from pain, injury and disease, availed through disease prevention and treatment,

© 2013 Nguhiu-Mwangi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Nguhiu-Mwangi et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

hances expression of normal behavioural patterns by the animal [7,8].

and a diet to maintain health and vigour,


## **Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi, Kenya**

James Nguhiu-Mwangi, Joshua W. Aleri, Eddy G. M. Mogoa and Peter M. F. Mbithi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53678

## **1. Introduction**

[19] Kumar V, Abbas AK, Fausto N, ed.: Robbins and Cotran Pathologic Basis of Disease,

[20] Allen SW, Prasse KW, Mahaffey EA. Cytologic differentiationn of bening from malignant canine mammary tumors. Veterinary Pathology 1986; 23: 649-655.

[21] Ferguson RH. Canine mammary gland tumors. Veterinary Clinics of North America

[22] Hellmen E. The pathogenesis of canine mammary tumors. Cancer Journal 1996; 9:

[23] Sarli G, Prezioso R, Benazzi C, Castellani G, Marcato PS. Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors. Journal of

[24] Alpaugh ML, Tomlinson JS, Shao ZM, Barsky SH. A novel human xenograft model of

[25] Ellis DL, Teitelbaum SL. Inflammatory carcinoma of the breast: a pathologic definition.

[26] Amorim RL, Souza CHM, Bandarra EP, Sanches OC, Piza ET. Immunohistochemical study of estrogen and progesterone reptors and cell proliferative indexes in canine inflammatory mammary carcinoma: 9 cases. Brazilian Journal of Veterinary Pathology

inflammatory breast cancer. Cancer Research 1999; 59: 5079-5084.

7th ed. Elsevier Saunders, Philadelphia, PA, 2005.

Veterinary Diagnostic Investigation 2002; 14: 25-34.

Small Animal Practice 1985; 15: 501-511.

Cancer 1974; 33: 1045-1047.

2008; 1(1): 16-20.

282-286.

48 Insights from Veterinary Medicine

Animal welfare lacks a good universal definition and a satisfactory distinction from the term "well being". However, a consensual definition is essential for practical, legislative and sci‐ entific purposes. Without a clear definition, animal welfare cannot be effectively studied or conclusively assessed to provide remedial measures to its violation [1-3]. Animal welfare is therefore defined as the ability of an animal to interact or cope comfortably with its environ‐ ment, resulting in satisfaction of both its physical and mental state [4-6]. This satisfaction en‐ hances expression of normal behavioural patterns by the animal [7,8].

In the context of welfare, "environment" refers to internal factors (within the animal) and external factors (in the animal's physical environment) to which the animal responds with its physiological and psychological systems [6,9]. In contrast, animal "well being" is de‐ fined as the animal's perception of its state in trying to cope with its environment [1,5]. Concisely, animal "well-being" refers to the current state of the animal, but animal wel‐ fare is a more general term referring to past, present and future implications of the ani‐ mal's state [10].

The assessment of animal welfare is base on the provisions of five freedoms, which include:


© 2013 Nguhiu-Mwangi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Nguhiu-Mwangi et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**c.** Freedom from fear and distress, availed through avoidance of conditions that cause mental suffering,

residents [13,18]. The high and rapid population growth in developing countries has led to a reduction of agricultural lands that support the livelihood of the people. This has triggered a shift from fewer large-scale farms to numerous intensified smallholder production units in an endeavor to maximize economic profits [22]. The resulting low income following land subdivision to smallholder enterprises, affects the livelihood of majority of the citizens in the involved countries [16,21]. The low income poses financial challenges that make it difficult to afford adequate dairy farming facilities, hence the progressively deteriorating husbandry standards that precipitate stressful conditions, which further exacerbate poor welfare of the dairy cattle in these smallholder units. These interacting multiple factors, cause a vicious cir‐ cle of events that eventually have negative effects on physiology, behavior, disease suscepti‐ bility and productivity of the dairy cows [23,24]. The welfare of food animals has become a major concern to consumers of animal products in many parts of the world. Consumers of products such as meat and meat products, milk and eggs are demanding to know how the animals from which these products have been obtained are handled with respect to animal

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

Kenya

51

http://dx.doi.org/10.5772/53678

Dairy cattle housing should provide the animal with protection from harsh environmental extremes [27]. Good housing systems are those that are well designed for ease of manage‐ ment and maintenance at all times [27-29]. It is proposed that all confinement for animals should be constructed and operated to meet the legal requirements for protection of the ani‐ mal as well as maintain high quality animal products [30]. Good animal housing systems are those that enhance provision of all the five freedoms that an animal should have to satisfy its welfare [28,31]. If these basic needs cannot be met in the animal house, then health, wel‐ fare and production of the animal will be compromised. These concerns are particularly crit‐ ical in the smallholder zero-grazing systems, in which dairy cows are confined throughout their growth and production life. Naturally, cattle are grazing animals and therefore pas‐ ture-grazing is a more welfare-friendly system because it allows free expression of normal animal behavior compared to the restricted indoor zero-grazing systems. Conversely, high yielding dairy cows may not get all their nutritional demands from grazing only, and this may compromise their welfare with regard to nutrition. This means that both zero-grazing and pasture-grazing systems have positive and negative effects on the welfare of dairy cattle [32]. However, zero-grazing systems demand more articulate precision in design, construc‐ tion and management because they have a higher inclination to compromising welfare of the housed dairy cattle. Although pasture-grazing allows free expression of normal cattle behavior and provides sufficient comfortable lying space, the pasture forage has lower nu‐ tritional value than the high plane feeding of the zero-grazing units and therefore cattle in pastures may spent long hours grazing depending on the quality and amount of forage in the pasture, hence less time resting, which influences the resting aspect of welfare negative‐ ly [33]. In comparison, indoor housing systems provide high level feeding and increase in‐ take rates, thus fulfilling nutritional requirements faster, reducing eating times, leaving more time for cattle to rest and ruminate [34]. However, indoor housing systems have limit‐ ed space allowance, which increases competitive aggressive behavior within the herd [35], restriction of natural foraging behavior and opportunity to feed selectively [36], negative ef‐ fects on the cow comfort [33], and high incidence of diseases such as lameness and mastitis

welfare ethics [25,26].


Knowledge of animal physiology, animal behavior and animal needs based on the five freedoms is paramount in assessing as well as enforcing animal welfare. Animals need to be provided with amble comfort related to these five freedoms. They should be kept in housing or environments that will minimize adverse climatic variations or exposures to extremes of cold or heat, rain, strong continuous winds and direct solar exposures. Ap‐ propriate conditions minimizing trauma, development of lesions and disease outbreaks are essential. Continuous availability of water and provision of adequate wholesome feeds, which consist of balanced constituent rations supplying specific nutritional needs to the body, is required. Animals should be provided with housing conditions and environ‐ ments that allow them to display natural behavior such as unhindered movement, free ex‐ pression of oestrus or heat symptoms necessary for mating or insemination in order to have continued sustainable reproduction, social relationships that include animal-to-ani‐ mal and animal-to-human cordial interactions; and finally minimizing or preventing any causes of suffering as much as possible [11].

Smallholder dairy farming occupies a vast proportion of agricultural production and the main livelihood of the people in most developing (third world) countries particularly in Af‐ rica, Asia and South America. In Kenya, smallholder zero-grazing dairy units contribute about 80% of the national commercial dairy herd [12] and over 70% of all the marketed milk [13-16]. Each of the Kenyan smallholder zero-grazing dairy units has 2 to 10 milking cows most of which are exotic breeds (Friesian, Ayrshire, Guernsey, Jersey or crosses of these exotic breeds). Some smallholder farmers, who have better financial resources, manage to have up to 20 or more cows. The cows are raised on small plots of land measuring between 0.25 to 2 acres. Only few smallholder farmers would have land measuring a maximum of 5 acres. The Kenyan smallholder zero-grazing dairy units are unique because they have var‐ ied designs and management practices. They vary in housing designs, nutritional and man‐ agement protocol from unit to unit to the extent that they can correctly be referred to as zero-grazing "subunits" that are devoid of a consistent production system. The nutritional regimes and management practices not only vary from unit to unit, but also within the same unit from time to time [17]. The cows in these units are invariably zero-grazed [13,18] and have sub-optimal production [14,18,19], which is attributed to a number of constraints such as inadequate feeding, poor nutrition, substandard animal husbandry, lack of proper dairy farming facilities that include inadequate space to move and interact freely. All these factors predispose the cows to diseases and other stressful conditions [14,20,21].

A high number of smallholder zero-grazing dairy units are concentrated in the peri-urban areas owing to availability of ready market for milk and milk products among city and town residents [13,18]. The high and rapid population growth in developing countries has led to a reduction of agricultural lands that support the livelihood of the people. This has triggered a shift from fewer large-scale farms to numerous intensified smallholder production units in an endeavor to maximize economic profits [22]. The resulting low income following land subdivision to smallholder enterprises, affects the livelihood of majority of the citizens in the involved countries [16,21]. The low income poses financial challenges that make it difficult to afford adequate dairy farming facilities, hence the progressively deteriorating husbandry standards that precipitate stressful conditions, which further exacerbate poor welfare of the dairy cattle in these smallholder units. These interacting multiple factors, cause a vicious cir‐ cle of events that eventually have negative effects on physiology, behavior, disease suscepti‐ bility and productivity of the dairy cows [23,24]. The welfare of food animals has become a major concern to consumers of animal products in many parts of the world. Consumers of products such as meat and meat products, milk and eggs are demanding to know how the animals from which these products have been obtained are handled with respect to animal welfare ethics [25,26].

**c.** Freedom from fear and distress, availed through avoidance of conditions that cause

**d.** Freedom to have normal behaviour patterns, availed through provision of sufficient

**e.** Freedom from thermal or physical discomfort, availed through provision of a comforta‐

Knowledge of animal physiology, animal behavior and animal needs based on the five freedoms is paramount in assessing as well as enforcing animal welfare. Animals need to be provided with amble comfort related to these five freedoms. They should be kept in housing or environments that will minimize adverse climatic variations or exposures to extremes of cold or heat, rain, strong continuous winds and direct solar exposures. Ap‐ propriate conditions minimizing trauma, development of lesions and disease outbreaks are essential. Continuous availability of water and provision of adequate wholesome feeds, which consist of balanced constituent rations supplying specific nutritional needs to the body, is required. Animals should be provided with housing conditions and environ‐ ments that allow them to display natural behavior such as unhindered movement, free ex‐ pression of oestrus or heat symptoms necessary for mating or insemination in order to have continued sustainable reproduction, social relationships that include animal-to-ani‐ mal and animal-to-human cordial interactions; and finally minimizing or preventing any

Smallholder dairy farming occupies a vast proportion of agricultural production and the main livelihood of the people in most developing (third world) countries particularly in Af‐ rica, Asia and South America. In Kenya, smallholder zero-grazing dairy units contribute about 80% of the national commercial dairy herd [12] and over 70% of all the marketed milk [13-16]. Each of the Kenyan smallholder zero-grazing dairy units has 2 to 10 milking cows most of which are exotic breeds (Friesian, Ayrshire, Guernsey, Jersey or crosses of these exotic breeds). Some smallholder farmers, who have better financial resources, manage to have up to 20 or more cows. The cows are raised on small plots of land measuring between 0.25 to 2 acres. Only few smallholder farmers would have land measuring a maximum of 5 acres. The Kenyan smallholder zero-grazing dairy units are unique because they have var‐ ied designs and management practices. They vary in housing designs, nutritional and man‐ agement protocol from unit to unit to the extent that they can correctly be referred to as zero-grazing "subunits" that are devoid of a consistent production system. The nutritional regimes and management practices not only vary from unit to unit, but also within the same unit from time to time [17]. The cows in these units are invariably zero-grazed [13,18] and have sub-optimal production [14,18,19], which is attributed to a number of constraints such as inadequate feeding, poor nutrition, substandard animal husbandry, lack of proper dairy farming facilities that include inadequate space to move and interact freely. All these factors

predispose the cows to diseases and other stressful conditions [14,20,21].

A high number of smallholder zero-grazing dairy units are concentrated in the peri-urban areas owing to availability of ready market for milk and milk products among city and town

mental suffering,

50 Insights from Veterinary Medicine

ble environment.

space and appropriate physical structures,

causes of suffering as much as possible [11].

Dairy cattle housing should provide the animal with protection from harsh environmental extremes [27]. Good housing systems are those that are well designed for ease of manage‐ ment and maintenance at all times [27-29]. It is proposed that all confinement for animals should be constructed and operated to meet the legal requirements for protection of the ani‐ mal as well as maintain high quality animal products [30]. Good animal housing systems are those that enhance provision of all the five freedoms that an animal should have to satisfy its welfare [28,31]. If these basic needs cannot be met in the animal house, then health, wel‐ fare and production of the animal will be compromised. These concerns are particularly crit‐ ical in the smallholder zero-grazing systems, in which dairy cows are confined throughout their growth and production life. Naturally, cattle are grazing animals and therefore pas‐ ture-grazing is a more welfare-friendly system because it allows free expression of normal animal behavior compared to the restricted indoor zero-grazing systems. Conversely, high yielding dairy cows may not get all their nutritional demands from grazing only, and this may compromise their welfare with regard to nutrition. This means that both zero-grazing and pasture-grazing systems have positive and negative effects on the welfare of dairy cattle [32]. However, zero-grazing systems demand more articulate precision in design, construc‐ tion and management because they have a higher inclination to compromising welfare of the housed dairy cattle. Although pasture-grazing allows free expression of normal cattle behavior and provides sufficient comfortable lying space, the pasture forage has lower nu‐ tritional value than the high plane feeding of the zero-grazing units and therefore cattle in pastures may spent long hours grazing depending on the quality and amount of forage in the pasture, hence less time resting, which influences the resting aspect of welfare negative‐ ly [33]. In comparison, indoor housing systems provide high level feeding and increase in‐ take rates, thus fulfilling nutritional requirements faster, reducing eating times, leaving more time for cattle to rest and ruminate [34]. However, indoor housing systems have limit‐ ed space allowance, which increases competitive aggressive behavior within the herd [35], restriction of natural foraging behavior and opportunity to feed selectively [36], negative ef‐ fects on the cow comfort [33], and high incidence of diseases such as lameness and mastitis [37,38]. All these factors in the indoor housing have adverse effects on the welfare of cattle. In Kenya, the practice of zero-grazing dairy production is inevitable owing to the reduced land sizes. Hence, the importance of drawing reliable direct indicators of poor welfare exist‐ ing in these zero-grazing systems in order to introduce corrective remedial measures, partic‐ ularly in relation to designing of the construction of welfare-acceptable and cowcomfortable zero-grazing units no matter how simple or cheap.

welfare problems of lameness in cattle and this would even be more prevalent in zero-graz‐ ing systems in developing countries, is the lack of valid and reliable lameness diagnostic methods. There is generally lack of sensitive methods of recognizing early change in the gait of lame cattle [44,47,48]. The most reliable and sensitive way of detecting early changes in gait for diagnosis of lameness is the use of automated gait-scoring computer aided systems, which are very scarcely used all over the world [49]. Moreover, these automated facilities are expensively unaffordable to the poor smallholder farmers in developing countries such as Kenya. Claw disorders particularly those related to laminitis are highly prevalent in smallholder zero-grazing dairy units and subunits in the peri-urban areas of Nairobi, Kenya and probably in other parts of Kenya with similar production systems [50]. These have been found to be highly associated with housing and management factors within the zero-graz‐ ing units [17,50]. This high prevalence of claw lesions together with a high prevalence of in‐ juries or signs of injuries in specific parts of the body as well as soiling and body condition scores of dairy cows in the smallholder zero-grazing units in the peri-urban areas of Nairobi, Kenya [51,52] was thought to be reliable indicators of the state of welfare of dairy cattle par‐

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

Kenya

53

http://dx.doi.org/10.5772/53678

Parameters used to assess animal welfare should be able to inform us about the state of welfare. Three requirements are essential for parameters or indicators used to assess ani‐ mal welfare. These include: "validity", which asks the question, "what does the parameter in consideration tell us about the animal's welfare state?"; "reliability", which considers inter-observer reliability and asks the question, "do different observers see the same thing?" and the third requirement is "feasibility", which considers the practical aspects of doing the recordings, asking the questions, "how easy is it to record the parameter?, how long does it take to assess the parameter?, and what equipment is needed for measuring

There is a high likelihood among farmers with zero-grazed dairy cows to focus more on whatever it takes to cause their cows produce as much milk as possible at the expense of the health and welfare considerations of the animal. High milk yielding cows often develop a compromise of energy-balance deficits, which infringes on their welfare. As a result of ener‐ gy deficit stress, these dairy cows become easily susceptible to metabolic and reproductive problems [53]. The uniqueness of the zero-grazing systems in Kenya which consists of subu‐ nits that are inconsistently varied in designs, in feeding regimes in relation to feed types, quality and quantity, as well as substandard management practices makes them a rich source of information on management of welfare of cattle. Information acquired from stud‐ ies in these smallholder zero-grazing subunits will serve to demonstrate how animal-level parameters can be useful in indicating the welfare state of the dairy cattle and how these in‐ dicators are associated with the housing design, feeding and management practices in these varied and substandard zero-grazing units and generally suggest possible remedial welfare

The intent of this paper is to present the results from two studies carried out at different times with collection of data from some of the zero-grazing units in the same area but look‐ ing at separate objectives. These studies dealt with assessment of the state of welfare of dai‐

ticularly when correlated with the prevailing zero-grazing conditions.

the parameter?" [39].

improvement measures.

Improvements of animal welfare may be achieved through (a) assessment of animal welfare, (b) identification of risk factors potentially leading to welfare problems and (c), interven‐ tions in response to the risk factors. Improvements can be enhanced by directly dealing with the risk factors of animal welfare within the farming unit. Therefore, there must be good re‐ liable way of measuring or assessing whether or not poor animal welfare exists within the practiced farming systems. In this process the animal based parameters help us to identify the animal's response to the system, and therefore indicating the negative impact of the po‐ tential risk factors existing within the farming system [39]. Traditionally, farm animal wel‐ fare assessment has focused on the measurement of resources provided to the animal such as housing-and-housing design criteria [40,41]. Although such indirect resource-based wel‐ fare assessment criteria are quick, easy and have some degree of reliability, basing the wel‐ fare verdict solely on their findings may not necessarily mean that the welfare of the animals is good or poor. Other husbandry aspects that affect animal welfare are management practi‐ ces and the human-animal relationship, but their measurement may be more difficult. How‐ ever, the provision of good management and environmental resources does not necessarily result in a high standard of animal welfare. Direct animal-level parameters such as health or behavior can be taken as indicators of the animals' feelings and a measure of bodily state of the animal. These are more reliable because they indicate how the animal has been affected by some factors existing within the proximate environment or housing system of the animal and how it has responded to these factors. Welfare assessment should therefore be based primarily on such animal-related parameters. In practice, resource or management-based parameters should also be included in an on-farm assessment protocol when closely corre‐ lated to animal-associated measurements and because they can form the basis for the identi‐ fication of causes of welfare problems [39]. It is however challenging to select and develop reliable and at the same time feasible measurements for on-farm assessment protocols. At‐ tempts to create an operational welfare assessment protocol primarily relying on animal-re‐ lated parameters have mainly been made with regard to dairy cows [42-45].

Animal-level indices for on-farm welfare assessment can be divided into ethological or be‐ havioural and pathological or health parameters; physiological indicators are mostly un‐ available for feasibility reasons. Ethological parameters include individual animal behavior, animal-to-animal interaction, human-animal interaction, agonistic behavior and other ab‐ normal behavior. The commonest animal health indicators of cattle welfare are lameness, ex‐ ternal body injuries, disease incidence, body condition score and body cleanliness. The main welfare health problem in cattle is lameness, particularly caused by lesions resulting from disruptions of the horn of the claw predisposed by factors such as concrete floors, zero-graz‐ ing systems and uncomfortable stalls [45,46]. One of the main shortcomings that exacerbates welfare problems of lameness in cattle and this would even be more prevalent in zero-graz‐ ing systems in developing countries, is the lack of valid and reliable lameness diagnostic methods. There is generally lack of sensitive methods of recognizing early change in the gait of lame cattle [44,47,48]. The most reliable and sensitive way of detecting early changes in gait for diagnosis of lameness is the use of automated gait-scoring computer aided systems, which are very scarcely used all over the world [49]. Moreover, these automated facilities are expensively unaffordable to the poor smallholder farmers in developing countries such as Kenya. Claw disorders particularly those related to laminitis are highly prevalent in smallholder zero-grazing dairy units and subunits in the peri-urban areas of Nairobi, Kenya and probably in other parts of Kenya with similar production systems [50]. These have been found to be highly associated with housing and management factors within the zero-graz‐ ing units [17,50]. This high prevalence of claw lesions together with a high prevalence of in‐ juries or signs of injuries in specific parts of the body as well as soiling and body condition scores of dairy cows in the smallholder zero-grazing units in the peri-urban areas of Nairobi, Kenya [51,52] was thought to be reliable indicators of the state of welfare of dairy cattle par‐ ticularly when correlated with the prevailing zero-grazing conditions.

[37,38]. All these factors in the indoor housing have adverse effects on the welfare of cattle. In Kenya, the practice of zero-grazing dairy production is inevitable owing to the reduced land sizes. Hence, the importance of drawing reliable direct indicators of poor welfare exist‐ ing in these zero-grazing systems in order to introduce corrective remedial measures, partic‐ ularly in relation to designing of the construction of welfare-acceptable and cow-

Improvements of animal welfare may be achieved through (a) assessment of animal welfare, (b) identification of risk factors potentially leading to welfare problems and (c), interven‐ tions in response to the risk factors. Improvements can be enhanced by directly dealing with the risk factors of animal welfare within the farming unit. Therefore, there must be good re‐ liable way of measuring or assessing whether or not poor animal welfare exists within the practiced farming systems. In this process the animal based parameters help us to identify the animal's response to the system, and therefore indicating the negative impact of the po‐ tential risk factors existing within the farming system [39]. Traditionally, farm animal wel‐ fare assessment has focused on the measurement of resources provided to the animal such as housing-and-housing design criteria [40,41]. Although such indirect resource-based wel‐ fare assessment criteria are quick, easy and have some degree of reliability, basing the wel‐ fare verdict solely on their findings may not necessarily mean that the welfare of the animals is good or poor. Other husbandry aspects that affect animal welfare are management practi‐ ces and the human-animal relationship, but their measurement may be more difficult. How‐ ever, the provision of good management and environmental resources does not necessarily result in a high standard of animal welfare. Direct animal-level parameters such as health or behavior can be taken as indicators of the animals' feelings and a measure of bodily state of the animal. These are more reliable because they indicate how the animal has been affected by some factors existing within the proximate environment or housing system of the animal and how it has responded to these factors. Welfare assessment should therefore be based primarily on such animal-related parameters. In practice, resource or management-based parameters should also be included in an on-farm assessment protocol when closely corre‐ lated to animal-associated measurements and because they can form the basis for the identi‐ fication of causes of welfare problems [39]. It is however challenging to select and develop reliable and at the same time feasible measurements for on-farm assessment protocols. At‐ tempts to create an operational welfare assessment protocol primarily relying on animal-re‐

comfortable zero-grazing units no matter how simple or cheap.

52 Insights from Veterinary Medicine

lated parameters have mainly been made with regard to dairy cows [42-45].

Animal-level indices for on-farm welfare assessment can be divided into ethological or be‐ havioural and pathological or health parameters; physiological indicators are mostly un‐ available for feasibility reasons. Ethological parameters include individual animal behavior, animal-to-animal interaction, human-animal interaction, agonistic behavior and other ab‐ normal behavior. The commonest animal health indicators of cattle welfare are lameness, ex‐ ternal body injuries, disease incidence, body condition score and body cleanliness. The main welfare health problem in cattle is lameness, particularly caused by lesions resulting from disruptions of the horn of the claw predisposed by factors such as concrete floors, zero-graz‐ ing systems and uncomfortable stalls [45,46]. One of the main shortcomings that exacerbates

Parameters used to assess animal welfare should be able to inform us about the state of welfare. Three requirements are essential for parameters or indicators used to assess ani‐ mal welfare. These include: "validity", which asks the question, "what does the parameter in consideration tell us about the animal's welfare state?"; "reliability", which considers inter-observer reliability and asks the question, "do different observers see the same thing?" and the third requirement is "feasibility", which considers the practical aspects of doing the recordings, asking the questions, "how easy is it to record the parameter?, how long does it take to assess the parameter?, and what equipment is needed for measuring the parameter?" [39].

There is a high likelihood among farmers with zero-grazed dairy cows to focus more on whatever it takes to cause their cows produce as much milk as possible at the expense of the health and welfare considerations of the animal. High milk yielding cows often develop a compromise of energy-balance deficits, which infringes on their welfare. As a result of ener‐ gy deficit stress, these dairy cows become easily susceptible to metabolic and reproductive problems [53]. The uniqueness of the zero-grazing systems in Kenya which consists of subu‐ nits that are inconsistently varied in designs, in feeding regimes in relation to feed types, quality and quantity, as well as substandard management practices makes them a rich source of information on management of welfare of cattle. Information acquired from stud‐ ies in these smallholder zero-grazing subunits will serve to demonstrate how animal-level parameters can be useful in indicating the welfare state of the dairy cattle and how these in‐ dicators are associated with the housing design, feeding and management practices in these varied and substandard zero-grazing units and generally suggest possible remedial welfare improvement measures.

The intent of this paper is to present the results from two studies carried out at different times with collection of data from some of the zero-grazing units in the same area but look‐ ing at separate objectives. These studies dealt with assessment of the state of welfare of dai‐ ry cattle in those units and the prevalent risk factors for poor welfare. In particular, it was planned 1) to determine the role of claw lesions in predicting the welfare of zero-grazed dai‐ ry cows with respect to housing designs, floor type, feeding and management practices in the peri-urban areas of Nairobi Kenya; 2) and to determine the role of body injuries, body soiling and body condition scores in predicting the welfare of zero-grazed dairy cows with respect to housing designs, floor type, feeding and management practices in the peri-urban areas of Nairobi Kenya.

and the suppression of functions are the main physiological responses to short-term welfare problems [5]. Adrenocorticotropic hormone (ACTH) challenge technique and the immune response provide measurement of long-term welfare problems. In bovines, the heart rate has been found to be a suitable parameter for studying dairy cow response to stress [67,68].

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

Kenya

55

http://dx.doi.org/10.5772/53678

Pathologic signs are widely accepted as indicators of poor welfare because they are a mani‐ festation of current suffering of the animal [3]. Reduction in health could be a reflection of compromised welfare; hence animal-level parameters are likely to be the best welfare indi‐ cators [42,44]. Clinical signs of disease and injuries are the animal-level parameters associat‐ ed with reduced health that may be useful indicators of poor animal welfare [5,44]. Lameness, skin injuries and measurement of immune function are the most commonly used pathological indicators of poor welfare in dairy cattle [3,44]. However, absence of injury and disease is not sufficient proof of good animal welfare [69]. Therefore, pre-pathological state of the animal which includes suppressed immunity (hence increasing vulnerability to dis‐ eases), reduced ability to reproduce and cessation of normal growth, tend to suggest that the animal is already suffering and these factors could be used as indicators of poor welfare [70,71]. Assessment of pre-pathological immunity state is based on white cell counts in blood or milk [72]. However, results obtained from such studies have been inconsistent [63]. Some of the short comings of these studies are that pre-pathological conditions do not neces‐ sary lead to adverse effects on animals [3] and also animal welfare may be impaired at the

Behaviour is an important indicator of animal welfare. It can be measured and recorded with minimal animal disturbance [73]. However, the main difficulty is the understanding of animal's normal, natural or ideal behavior in order to quantify abnormal behaviour [74]. Behavioural indicators of poor welfare include the inability of the animal to carry out normal behaviour and the exhibition of a persistent undesirable action by a minority of the population that could be termed as abnormal behaviour [3,61]. Abnormal animal be‐ haviour is classified into five categories which include: detrimental behaviour that causes injury, sham behaviours that are performed in the absence of adequate substrate or envi‐ ronmental stimuli, apathetic behaviour that is a reduced attentiveness towards external stimuli, escape behavior that manifests as a desire to leave the confined environment and redirected behaviour that may ritualize into stereotypes [75]. Abnormal behaviour is dam‐ aging to the animals [76]. Expression of abnormal behaviour is a sign that an animal has problems adapting to its environment [3]. It may be an expression of the level of distress

Cattle are referred to as group animals because they express synchronized behaviour within the herd [73]. On daily basis, cows confined and housed spend 5-6 hours eating, 4-9 hours

*2.1.3. Pathological indicators*

time of pre-pathological assessment [66].

that the animal is experiencing [6].

*2.1.5. Bovine ethology*

*2.1.4. Ethological indicators*

## **2. Material and methods**

#### **2.1. Assessment of animal welfare**

Assessment of animal welfare can be done using both animal-based and environmental-re‐ lated parameters (which includes housing factors and management factors) [40,44,54]. These parameters can be evaluated using indicators that show the state of the animal such as pro‐ duction performance, physiological, pathological, ethological and integrated factors [3,55].

#### *2.1.1. Production performance as an indicator of welfare*

The production performance indicators of animal welfare are growth rate, productivity, re‐ productive output and duration of productive life of an animal [1,56]. Many researchers have stated that if the welfare of an animal is good, then production will be optimal [1,57,58]. However, high productivity may not necessarily be an indicator of good welfare, nor low productivity an indicator of poor welfare [46]. For example, dairy cows with high milk production are likely to be predisposed to increased lameness, mastitis, damaged ud‐ der ligaments, infertility and problems at parturition [59,60]. It has been suggested that milk yield can be used as an on-farm indicator of animal welfare [44].

#### *2.1.2. Physiological indicators*

The main physiological indicators of welfare are hormone levels from the pituitary and adrenal glands and the changes induced on target organs by these hormones such as tachy‐ cardia, blood pressure, hyperglycaemia, lymphocytosis and eosinopaenia [3]. The advantag‐ es of physiological indicators of animal welfare are that their measurements use reliable analytical methods [61,62] that are less invasive within the body [6]. Cortisol levels indicate the degree of stress experienced by an animal [63]. However, other normal activities such as mating, can lead to an increase in stress hormone levels. Moreover, results of different stud‐ ies on stress hormones have been inconsistent and hence their reliability as indicators of ani‐ mal welfare is doubtful [64]. In spite of these arguments, the use of stress hormone response as a welfare indicator has gained credibility because it can easily be measured [65].

Methods used as welfare indicators should not be generalized to all species but rather con‐ sidered within species, and the search for more reliable methods should be intensified [66]. It has been shown that heart rate, adrenal function, brain biochemistry, regulatory responses and the suppression of functions are the main physiological responses to short-term welfare problems [5]. Adrenocorticotropic hormone (ACTH) challenge technique and the immune response provide measurement of long-term welfare problems. In bovines, the heart rate has been found to be a suitable parameter for studying dairy cow response to stress [67,68].

#### *2.1.3. Pathological indicators*

ry cattle in those units and the prevalent risk factors for poor welfare. In particular, it was planned 1) to determine the role of claw lesions in predicting the welfare of zero-grazed dai‐ ry cows with respect to housing designs, floor type, feeding and management practices in the peri-urban areas of Nairobi Kenya; 2) and to determine the role of body injuries, body soiling and body condition scores in predicting the welfare of zero-grazed dairy cows with respect to housing designs, floor type, feeding and management practices in the peri-urban

Assessment of animal welfare can be done using both animal-based and environmental-re‐ lated parameters (which includes housing factors and management factors) [40,44,54]. These parameters can be evaluated using indicators that show the state of the animal such as pro‐ duction performance, physiological, pathological, ethological and integrated factors [3,55].

The production performance indicators of animal welfare are growth rate, productivity, re‐ productive output and duration of productive life of an animal [1,56]. Many researchers have stated that if the welfare of an animal is good, then production will be optimal [1,57,58]. However, high productivity may not necessarily be an indicator of good welfare, nor low productivity an indicator of poor welfare [46]. For example, dairy cows with high milk production are likely to be predisposed to increased lameness, mastitis, damaged ud‐ der ligaments, infertility and problems at parturition [59,60]. It has been suggested that milk

The main physiological indicators of welfare are hormone levels from the pituitary and adrenal glands and the changes induced on target organs by these hormones such as tachy‐ cardia, blood pressure, hyperglycaemia, lymphocytosis and eosinopaenia [3]. The advantag‐ es of physiological indicators of animal welfare are that their measurements use reliable analytical methods [61,62] that are less invasive within the body [6]. Cortisol levels indicate the degree of stress experienced by an animal [63]. However, other normal activities such as mating, can lead to an increase in stress hormone levels. Moreover, results of different stud‐ ies on stress hormones have been inconsistent and hence their reliability as indicators of ani‐ mal welfare is doubtful [64]. In spite of these arguments, the use of stress hormone response

as a welfare indicator has gained credibility because it can easily be measured [65].

Methods used as welfare indicators should not be generalized to all species but rather con‐ sidered within species, and the search for more reliable methods should be intensified [66]. It has been shown that heart rate, adrenal function, brain biochemistry, regulatory responses

areas of Nairobi Kenya.

54 Insights from Veterinary Medicine

**2. Material and methods**

*2.1.2. Physiological indicators*

**2.1. Assessment of animal welfare**

*2.1.1. Production performance as an indicator of welfare*

yield can be used as an on-farm indicator of animal welfare [44].

Pathologic signs are widely accepted as indicators of poor welfare because they are a mani‐ festation of current suffering of the animal [3]. Reduction in health could be a reflection of compromised welfare; hence animal-level parameters are likely to be the best welfare indi‐ cators [42,44]. Clinical signs of disease and injuries are the animal-level parameters associat‐ ed with reduced health that may be useful indicators of poor animal welfare [5,44]. Lameness, skin injuries and measurement of immune function are the most commonly used pathological indicators of poor welfare in dairy cattle [3,44]. However, absence of injury and disease is not sufficient proof of good animal welfare [69]. Therefore, pre-pathological state of the animal which includes suppressed immunity (hence increasing vulnerability to dis‐ eases), reduced ability to reproduce and cessation of normal growth, tend to suggest that the animal is already suffering and these factors could be used as indicators of poor welfare [70,71]. Assessment of pre-pathological immunity state is based on white cell counts in blood or milk [72]. However, results obtained from such studies have been inconsistent [63]. Some of the short comings of these studies are that pre-pathological conditions do not neces‐ sary lead to adverse effects on animals [3] and also animal welfare may be impaired at the time of pre-pathological assessment [66].

#### *2.1.4. Ethological indicators*

Behaviour is an important indicator of animal welfare. It can be measured and recorded with minimal animal disturbance [73]. However, the main difficulty is the understanding of animal's normal, natural or ideal behavior in order to quantify abnormal behaviour [74]. Behavioural indicators of poor welfare include the inability of the animal to carry out normal behaviour and the exhibition of a persistent undesirable action by a minority of the population that could be termed as abnormal behaviour [3,61]. Abnormal animal be‐ haviour is classified into five categories which include: detrimental behaviour that causes injury, sham behaviours that are performed in the absence of adequate substrate or envi‐ ronmental stimuli, apathetic behaviour that is a reduced attentiveness towards external stimuli, escape behavior that manifests as a desire to leave the confined environment and redirected behaviour that may ritualize into stereotypes [75]. Abnormal behaviour is dam‐ aging to the animals [76]. Expression of abnormal behaviour is a sign that an animal has problems adapting to its environment [3]. It may be an expression of the level of distress that the animal is experiencing [6].

#### *2.1.5. Bovine ethology*

Cattle are referred to as group animals because they express synchronized behaviour within the herd [73]. On daily basis, cows confined and housed spend 5-6 hours eating, 4-9 hours ruminating and 11-11.5 hours lying down [3,77,78]. However, the behavioural patterns may vary according to the type of housing system in which they are [79,80]. Friesian cows under cubicle system were found to have lying time of 13.7 hours/day compared to 6.5 hours/day in open out-door systems [81]. Reduced lying time has been found to exacerbate the inci‐ dence of claw lesions [82]. Prolonged standing causes cows to expend more energy and ex‐ poses hooves to longer periods on slurry, which may increase incidence of lameness [17,83]. Eating behaviour is the most characteristic indication of the state of comfort in animals, that is, the degree to which the biological requirements of animals are met [84]. It has been ob‐ served that feeding cows with smooth quality fodder and high concentrates is very benefi‐ cial compared to rough fodder. The explanation here is that reduced eating time reduces standing time of the animals [84]. Increased milk yield has been observed in cows with lon‐ ger lying times. This is thought to be due to increased blood supply to the udder through the milk vein, increasing nutrient supply to the udder [86,87].

once, from which 40% were in their first and second parities and 60% in their third and fourth parities. Both lame and non-lame cows of any of the breeds were included in the study group. Selection of the cows meeting the inclusion criteria was performed as previ‐ ously described [50]. Briefly, in each smallholder unit, cows that met the selection criteria were isolated from the rest and serially numbered as 1, 2, 3, to S, where S was the last serial number depending on the total number of cows isolated in that unit. To avoid biased sam‐ pling, a farm worker numbered the isolated cows. From the serially numbered cows, the in‐ vestigator, starting with either serial number 1 or 2, systematically selected every second cow in the series. For example in the series S1, S2, S3, S4, S5, S6, S7, S8, S9, and S10, if the first cow selected was S1 the next one selected serially would be S3, S5, S7 and S9 respective‐ ly, thus all odd serial numbers. But if the first cow selected was S2, then the next ones select‐ ed serially would be S4, S6, S8 and S10 respectively, thus all even serial numbers. If the first cow selected in one smallholder unit was serial number S1, then in the next smallholder unit, the first cow selected would be serial number S2. This selection of the first cow was alternated between odd and even numbers from one smallholder unit to the other until in‐ vestigation in all the 32 units was completed. Therefore, the cows selected in any individual

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

Kenya

57

http://dx.doi.org/10.5772/53678

smallholder unit were either all with odd or all with even serial numbers.

was applied. The lesions found on each cow were recorded.

Data on claw disorders were collected by examining only the hind claws of each cow, due to poor restraint facilities that make it difficult to examine the fore limbs. General observation of gait for signs of lameness was done first. The floor state and small sizes of the units made the examination for lameness quite restrictive, and it needed an experienced veterinarian to conclude on whether a cow was lame or not, particularly when mildly lame. Each cow was restrained in a standing posture in the crush or the sleeping cubicle. Lifting of one hind limb at a time was done using a rope tied to an overhead pole or cross-bar. After washing with soap and water, claws were examined for any lesions, particularly on the weight-bearing surface. About 1-2 mm thickness of the horn of the sole was trimmed-off using a sharp quit‐ tor knife to expose any underlying lesions. Trimming did not reach the level of the corium and therefore was non-invasive and non-painful to the cows. In case of painful claw condi‐ tion, local analgesia using 2% lignocaine hydrochloride and a tourniquet at mid-metatarsus

Data on cow-level factors were collected by the first author (as interviewer) administering questionnaires either to farmers, or the stockmen managing the cows in the zero-grazing units (as respondent interviewees) before examination of the cows. The data which included breed, parity, milk yield per day, and lactation stage were pre-coded and recorded in the questionnaires. The questionnaires were structured simple "Yes" and "No" and "I do not know" responses to minimize variations and information bias from the respondents. Data on farm-level factors were collected during visitation to each of the 32 farms. Some data (housing and stall design, presence and number of cubicles, type of cubicle bedding and floor, presence or absence of a curb, and lunging space, and adequacy of feeding space) were collected through observation. Other data such as kerb height were collected through measurements, while the rest (frequency of concentrate feeding, mineral supplementation, type of fodder, and frequency of slurry removal from the walk-alleys were collected by the

#### **2.2. Study procedures**

#### *2.2.1. Study area*

The study was carried out in the peri-urban areas of Nairobi, Kenya. Nairobi is the capital city of Kenya with an area of 696 square kilometers and a population of over 2.1 million peo‐ ple. It is surrounded by a fertile peri-urban agricultural region lying between 01º 18´S and 36º 45´E, and 1798 meters above sea level. It has an annual rainfall estimated at 765 mm maximum and 36 mm minimum in two distinct seasons (March to June, and October to De‐ cember). The rest of the months of the year are moderately dry. The cold months are begin‐ ning of July to the end of August with temperatures ranging from 18º C to 21ºC at day time and 11ºC to 15ºC at night time. The North-Western side of Nairobi is the coolest with high humidity, while the Eastern side is the warmest with very low humidity. The region has a high concentration of zero-grazed smallholder dairy units owing to its ready market for milk and milk-related dairy products.

#### *2.2.2. Study design*

Study 1 – Can claw lesions be used for predicting welfare of zero-grazed dairy cows?

The study consisted of a cross-sectional study in which each zero-grazing unit was visited once and each cow included in the study was examined only once. Even when a zero-graz‐ ing unit was visited more than once, no cow was examined twice. Thirty-two smallholder zero-grazing dairy units were purposively selected from those with median cow number of 10 (ranging from 5 to 20 adult cows). It was difficult to get enough farmers allowing their cows to be used for the study, hence another major criteria for inclusion of the zero-grazing dairy units was the willing smallholder farmers. Selection of the zero-grazing dairy units was facilitated by local veterinarians and animal health technicians with whom the farmers were more acquainted. A total of 300 dairy cows that included Friesians 76% (n=228), Ayr‐ shires 20% (n=60) and 4% (n=12) being Guernsey and Jersey crosses were recruited from the 32 smallholder zero-grazing units. Cows that were included in the study had calved at least ruminating and 11-11.5 hours lying down [3,77,78]. However, the behavioural patterns may vary according to the type of housing system in which they are [79,80]. Friesian cows under cubicle system were found to have lying time of 13.7 hours/day compared to 6.5 hours/day in open out-door systems [81]. Reduced lying time has been found to exacerbate the inci‐ dence of claw lesions [82]. Prolonged standing causes cows to expend more energy and ex‐ poses hooves to longer periods on slurry, which may increase incidence of lameness [17,83]. Eating behaviour is the most characteristic indication of the state of comfort in animals, that is, the degree to which the biological requirements of animals are met [84]. It has been ob‐ served that feeding cows with smooth quality fodder and high concentrates is very benefi‐ cial compared to rough fodder. The explanation here is that reduced eating time reduces standing time of the animals [84]. Increased milk yield has been observed in cows with lon‐ ger lying times. This is thought to be due to increased blood supply to the udder through

The study was carried out in the peri-urban areas of Nairobi, Kenya. Nairobi is the capital city of Kenya with an area of 696 square kilometers and a population of over 2.1 million peo‐ ple. It is surrounded by a fertile peri-urban agricultural region lying between 01º 18´S and 36º 45´E, and 1798 meters above sea level. It has an annual rainfall estimated at 765 mm maximum and 36 mm minimum in two distinct seasons (March to June, and October to De‐ cember). The rest of the months of the year are moderately dry. The cold months are begin‐ ning of July to the end of August with temperatures ranging from 18º C to 21ºC at day time and 11ºC to 15ºC at night time. The North-Western side of Nairobi is the coolest with high humidity, while the Eastern side is the warmest with very low humidity. The region has a high concentration of zero-grazed smallholder dairy units owing to its ready market for

Study 1 – Can claw lesions be used for predicting welfare of zero-grazed dairy cows?

The study consisted of a cross-sectional study in which each zero-grazing unit was visited once and each cow included in the study was examined only once. Even when a zero-graz‐ ing unit was visited more than once, no cow was examined twice. Thirty-two smallholder zero-grazing dairy units were purposively selected from those with median cow number of 10 (ranging from 5 to 20 adult cows). It was difficult to get enough farmers allowing their cows to be used for the study, hence another major criteria for inclusion of the zero-grazing dairy units was the willing smallholder farmers. Selection of the zero-grazing dairy units was facilitated by local veterinarians and animal health technicians with whom the farmers were more acquainted. A total of 300 dairy cows that included Friesians 76% (n=228), Ayr‐ shires 20% (n=60) and 4% (n=12) being Guernsey and Jersey crosses were recruited from the 32 smallholder zero-grazing units. Cows that were included in the study had calved at least

the milk vein, increasing nutrient supply to the udder [86,87].

**2.2. Study procedures**

56 Insights from Veterinary Medicine

milk and milk-related dairy products.

*2.2.1. Study area*

*2.2.2. Study design*

once, from which 40% were in their first and second parities and 60% in their third and fourth parities. Both lame and non-lame cows of any of the breeds were included in the study group. Selection of the cows meeting the inclusion criteria was performed as previ‐ ously described [50]. Briefly, in each smallholder unit, cows that met the selection criteria were isolated from the rest and serially numbered as 1, 2, 3, to S, where S was the last serial number depending on the total number of cows isolated in that unit. To avoid biased sam‐ pling, a farm worker numbered the isolated cows. From the serially numbered cows, the in‐ vestigator, starting with either serial number 1 or 2, systematically selected every second cow in the series. For example in the series S1, S2, S3, S4, S5, S6, S7, S8, S9, and S10, if the first cow selected was S1 the next one selected serially would be S3, S5, S7 and S9 respective‐ ly, thus all odd serial numbers. But if the first cow selected was S2, then the next ones select‐ ed serially would be S4, S6, S8 and S10 respectively, thus all even serial numbers. If the first cow selected in one smallholder unit was serial number S1, then in the next smallholder unit, the first cow selected would be serial number S2. This selection of the first cow was alternated between odd and even numbers from one smallholder unit to the other until in‐ vestigation in all the 32 units was completed. Therefore, the cows selected in any individual smallholder unit were either all with odd or all with even serial numbers.

Data on claw disorders were collected by examining only the hind claws of each cow, due to poor restraint facilities that make it difficult to examine the fore limbs. General observation of gait for signs of lameness was done first. The floor state and small sizes of the units made the examination for lameness quite restrictive, and it needed an experienced veterinarian to conclude on whether a cow was lame or not, particularly when mildly lame. Each cow was restrained in a standing posture in the crush or the sleeping cubicle. Lifting of one hind limb at a time was done using a rope tied to an overhead pole or cross-bar. After washing with soap and water, claws were examined for any lesions, particularly on the weight-bearing surface. About 1-2 mm thickness of the horn of the sole was trimmed-off using a sharp quit‐ tor knife to expose any underlying lesions. Trimming did not reach the level of the corium and therefore was non-invasive and non-painful to the cows. In case of painful claw condi‐ tion, local analgesia using 2% lignocaine hydrochloride and a tourniquet at mid-metatarsus was applied. The lesions found on each cow were recorded.

Data on cow-level factors were collected by the first author (as interviewer) administering questionnaires either to farmers, or the stockmen managing the cows in the zero-grazing units (as respondent interviewees) before examination of the cows. The data which included breed, parity, milk yield per day, and lactation stage were pre-coded and recorded in the questionnaires. The questionnaires were structured simple "Yes" and "No" and "I do not know" responses to minimize variations and information bias from the respondents. Data on farm-level factors were collected during visitation to each of the 32 farms. Some data (housing and stall design, presence and number of cubicles, type of cubicle bedding and floor, presence or absence of a curb, and lunging space, and adequacy of feeding space) were collected through observation. Other data such as kerb height were collected through measurements, while the rest (frequency of concentrate feeding, mineral supplementation, type of fodder, and frequency of slurry removal from the walk-alleys were collected by the first author interviewing the farmer, or stockmen. All the information collected from the zero-grazing units by measurements and by interview on the questionnaires was recorded in data collection sheets in codes allocated for each parameter.

points that separated between the unit body condition scores. BCS 1 meant poor body con‐ dition, BCS 2 represented moderate body condition, BCS 3 represented good body condi‐ tion, BCS 4 meant a fat cow, and BCS 5 represented a very fat cow. The farmers' and stockmen's perspective or knowledge on animal welfare was evaluated through interview‐

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

The data representing each parameter information was coded with a specific numerical code for each parameter for the purposes of entry into Microsoft Office Excel sheets. The data were imported into SAS© 2002-2003 (SAS Institute Inc., Cary, NC, USA). Descriptive statis‐ tics were computed for cow-level and farm-level factors. From study 1, the prevalence rate of each claw disorder was calculated independent of other claw disorders. The prevalence of each claw disorder was calculated as the number of cows (CL) affected by the specific claw disorder divided by the total number of cows (300) examined, then multiplied by 100 to

factors and the claw lesions. An association was considered significant at the level of P<0.05. Multiple logistic regressions were done through a step-down regression in which the risk factors that made the least variation to the occurrence of the claw lesions were eliminated one at a time through consideration of their odds ratios. Only the factors that were found to influence the occurrence of claw lesions significantly were retained in the model. The effects of confounding the risk factors were dealt with in the analysis but they were minimal be‐

From study 2, prevalence of body injuries were calculated as simple percentages of occur‐ rences of lesions, injuries and the risk factors. By use of SAS (Statistical Analytical System) descriptive statistics were generated and tests of simple associations between zero-grazing

icance level. Chi Square values were determined using 2x2 contingency tables. In these asso‐ ciations, the Chi Square calculations were determined by evaluating each risk factor (variable) against each welfare predictor (outcome) on the animal. The degrees of freedom (df) in each case was standard, being calculated by [(rows-1)(columns-1), hence [(2-1) x (2-1)

A high prevalence of acquired claw disorders was encountered in the cows from both studies, but higher in the first than the second study. The difference can be attributed to

cause of some similarities of the management in the smallholder farms.

unit-level and animal-level factors were done using Chi Square (χ<sup>2</sup>

= 1]. Therefore df was 1 for each association test.

**3.1. Lameness and claw disorders as indicators of welfare**

**3. Results and discussion**

) statistics were used to determine unconditional associations between all risk

) statistics at p<0.05 signif‐

Kenya

59

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ing them as respondents. All the data were recorded in data collection sheets.

*2.2.4. Data management and analysis*

make it a percentage.

Chi-square (χ<sup>2</sup>

Prevalence (*%*) <sup>=</sup> *CL* ×100

300

Study 2 - Are body injuries, body soiling or body condition scores useful in predicting the welfare of zero-grazed dairy cows?

In this cross-sectional study each zero-grazing smallholder unit (defined as one with a mini‐ mum of 3 and a maximum of 16 adult dairy cows) was visited once for the whole study peri‐ od. A total of 80 smallholder zero-grazing dairy units were included in the study (It is important to note that apart from these zero-grazing units being in the same area as those in study 1, none of them was included in both studies). Selection of the 80 zero-grazing units to include in study 2 was performed as for study 1. Furthermore, for logistical reasons units were also chosen based on the farmers' willingness to co-operate and to allow their dairy units to be used in the study.

The animals included for examination were adult dairy cows, whether in milk or dry. In any smallholder unit that had 5 or less adult cows, all the adult females were selected for examina‐ tion. In those having more than 5 adult cows, only 5 were selected for examination. The five were selected using a simple systematic sampling method, similar to the one used in study 1. In all the 80 smallholder units, a total of 306 dairy cows were selected for examination.

In each unit, the selected cows were closely examined for signs of external body injuries. In‐ juries were recorded according the body regions on which they occurred. These body re‐ gions were mainly those that were prone to injury from housing structures and they included the neck, brisket, carpal joint area, rib-cage area, area over the tuber coxae, ischial area, hock joint area, teats and udder. The main signs that were considered as indicators of body injuries included external presence of raw wounds, ulcerations, swellings, scars, local‐ ized hair loss and skin hyperkeratosis/callus-like formation.

#### *2.2.3. Evaluation of housing and animal management*

Some of the factors of housing design and the quality of construction finishes were evaluat‐ ed only by visual observation while others were assessed by taking actual measurement of the dimensions. Those factors that were evaluated only by visual observation included types and state of roofing, walls, and floor (mainly at the walk alleys and cubicles), as well as types and adequacy of feed bunks/troughs, presence or absence of neck-bars over the feed bunks and presence or absence of cubicles. Presence and type of cubicle bedding was also observed. Those housing factors that were evaluated by measuring actual dimensions in‐ cluded height of neck-bar from the upper edge of the feed bunk, width of the walk alleys from the rear edge of the cubicles to the front (near edge) of the feed bunk as well as width and length of the cubicles. Besides the physical aspects of the facilities, other animal-related aspects were also evaluated. The stocking density was evaluated by calculating cows to cu‐ bicle ratios. Presence of slurry on the walk alleys and gross body soiling of the cows was noted. Frequency of slurry removal was obtained through questionnaires. The individual body condition score (BCS) was evaluated on a simple scale of 1 to 5, which included half points that separated between the unit body condition scores. BCS 1 meant poor body con‐ dition, BCS 2 represented moderate body condition, BCS 3 represented good body condi‐ tion, BCS 4 meant a fat cow, and BCS 5 represented a very fat cow. The farmers' and stockmen's perspective or knowledge on animal welfare was evaluated through interview‐ ing them as respondents. All the data were recorded in data collection sheets.

#### *2.2.4. Data management and analysis*

first author interviewing the farmer, or stockmen. All the information collected from the zero-grazing units by measurements and by interview on the questionnaires was recorded

Study 2 - Are body injuries, body soiling or body condition scores useful in predicting the

In this cross-sectional study each zero-grazing smallholder unit (defined as one with a mini‐ mum of 3 and a maximum of 16 adult dairy cows) was visited once for the whole study peri‐ od. A total of 80 smallholder zero-grazing dairy units were included in the study (It is important to note that apart from these zero-grazing units being in the same area as those in study 1, none of them was included in both studies). Selection of the 80 zero-grazing units to include in study 2 was performed as for study 1. Furthermore, for logistical reasons units were also chosen based on the farmers' willingness to co-operate and to allow their dairy

The animals included for examination were adult dairy cows, whether in milk or dry. In any smallholder unit that had 5 or less adult cows, all the adult females were selected for examina‐ tion. In those having more than 5 adult cows, only 5 were selected for examination. The five were selected using a simple systematic sampling method, similar to the one used in study 1. In

In each unit, the selected cows were closely examined for signs of external body injuries. In‐ juries were recorded according the body regions on which they occurred. These body re‐ gions were mainly those that were prone to injury from housing structures and they included the neck, brisket, carpal joint area, rib-cage area, area over the tuber coxae, ischial area, hock joint area, teats and udder. The main signs that were considered as indicators of body injuries included external presence of raw wounds, ulcerations, swellings, scars, local‐

Some of the factors of housing design and the quality of construction finishes were evaluat‐ ed only by visual observation while others were assessed by taking actual measurement of the dimensions. Those factors that were evaluated only by visual observation included types and state of roofing, walls, and floor (mainly at the walk alleys and cubicles), as well as types and adequacy of feed bunks/troughs, presence or absence of neck-bars over the feed bunks and presence or absence of cubicles. Presence and type of cubicle bedding was also observed. Those housing factors that were evaluated by measuring actual dimensions in‐ cluded height of neck-bar from the upper edge of the feed bunk, width of the walk alleys from the rear edge of the cubicles to the front (near edge) of the feed bunk as well as width and length of the cubicles. Besides the physical aspects of the facilities, other animal-related aspects were also evaluated. The stocking density was evaluated by calculating cows to cu‐ bicle ratios. Presence of slurry on the walk alleys and gross body soiling of the cows was noted. Frequency of slurry removal was obtained through questionnaires. The individual body condition score (BCS) was evaluated on a simple scale of 1 to 5, which included half

all the 80 smallholder units, a total of 306 dairy cows were selected for examination.

ized hair loss and skin hyperkeratosis/callus-like formation.

*2.2.3. Evaluation of housing and animal management*

in data collection sheets in codes allocated for each parameter.

welfare of zero-grazed dairy cows?

58 Insights from Veterinary Medicine

units to be used in the study.

The data representing each parameter information was coded with a specific numerical code for each parameter for the purposes of entry into Microsoft Office Excel sheets. The data were imported into SAS© 2002-2003 (SAS Institute Inc., Cary, NC, USA). Descriptive statis‐ tics were computed for cow-level and farm-level factors. From study 1, the prevalence rate of each claw disorder was calculated independent of other claw disorders. The prevalence of each claw disorder was calculated as the number of cows (CL) affected by the specific claw disorder divided by the total number of cows (300) examined, then multiplied by 100 to make it a percentage.

Prevalence (*%*) <sup>=</sup> *CL* ×100 300

Chi-square (χ<sup>2</sup> ) statistics were used to determine unconditional associations between all risk factors and the claw lesions. An association was considered significant at the level of P<0.05. Multiple logistic regressions were done through a step-down regression in which the risk factors that made the least variation to the occurrence of the claw lesions were eliminated one at a time through consideration of their odds ratios. Only the factors that were found to influence the occurrence of claw lesions significantly were retained in the model. The effects of confounding the risk factors were dealt with in the analysis but they were minimal be‐ cause of some similarities of the management in the smallholder farms.

From study 2, prevalence of body injuries were calculated as simple percentages of occur‐ rences of lesions, injuries and the risk factors. By use of SAS (Statistical Analytical System) descriptive statistics were generated and tests of simple associations between zero-grazing unit-level and animal-level factors were done using Chi Square (χ<sup>2</sup> ) statistics at p<0.05 signif‐ icance level. Chi Square values were determined using 2x2 contingency tables. In these asso‐ ciations, the Chi Square calculations were determined by evaluating each risk factor (variable) against each welfare predictor (outcome) on the animal. The degrees of freedom (df) in each case was standard, being calculated by [(rows-1)(columns-1), hence [(2-1) x (2-1) = 1]. Therefore df was 1 for each association test.

## **3. Results and discussion**

#### **3.1. Lameness and claw disorders as indicators of welfare**

A high prevalence of acquired claw disorders was encountered in the cows from both studies, but higher in the first than the second study. The difference can be attributed to the fact that in study 1 the claws were trimmed during evaluation, while in study 2 only observation for lameness was done without trimming for specific examination of the claws. In study 1, out of 300 cows the prevalence was 88% (n=264) of which 69% (n=182) were subclinical (the affected cows were not lame), the diagnoses being made through trimming of the claws; and 31% (n=82) were clinical (the affected cows were lame), with animals showing evidence of lameness. About 70% (n=211) of the cows had laminitis, which was either sublinical laminitis in 49% (n=148) diagnosed by presence of sole hae‐ morrhages seen after trimming a thin layer of the horn of the sole, or chronic laminitis in 21% (n=63) diagnosed by presence of extensive diffuse sole haemorrhages coupled with various degrees of claw deformities. The Pictorial description of the claw lesions and the associated predisposing causes was detailed in a previous publication [50]. In study 2, lame cows were encountered in 73% (n=58) of the 80 zero-grazing units, for which the to‐ tal prevalence was 35% (n=107) among the 306 cows examined. The lameness was caused by different claw disorders, which included various degrees of claw deformities ranging from moderate claw overgrowth to severe twisting of the claws. Lameness caused by le‐ sions in proximal parts of the limbs (proximal to the claws) had very low prevalence of less than 2% in both studies; most of these lesions did not cause any lameness.

the neck-bar during the many hours of feeding at the feed bunks. A neck-bar is fixed over the feed bunk to prevent cattle from wasting feeds and placing their forelimbs into the feed bunks (Figure 2). Similar effects of neck-bars have been previously described [88]. The neckbars over the feed bunks were present in 60% (n=48) of the zero-grazing units, and in 77.1% (n=37) of these units the neck-bars were fixed at less or up to 50cm of the top edge of the feed bunk, while in 22.9% (n=11) of the units they were more than 50 cm from the top edge of the feed bunk. When the level is too low, the dorsal surface of the neck would always scrap against the neck-bar as long as the animal is at the feed bunk feeding, and injuries are exacerbated by animals pushing one another and fighting at the feed bunks due to inade‐ quate feeding space or social dominance molestation. All the neck-bars in these zero-grazing units were made of timber, some of which had side-facing sharp edges that contact the dor‐ sal surface of the neck, precipitating the occurrence of injuries (Figure 2). Also, the width of some feed bunks was excessive that cows struggled to reach the feed on the far end and this

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predisposed them to more of the neck injuries.

**Zero-grazing units with cows showing**

**Body region n % n %**

Carpal joint 77 96 230 75.16

Hock joint 76 95 260 85.00

Rib cage area 76 95 228 74.51

Tuber coxae 72 91 204 66.70

Ischial area 61 76 124 40.52

Neck 52 65 186 60.78

Brisket 51 64 134 43.79

Teats / udder 50 63 89 29.10

infringes partly on freedom from hunger (one of the five freedoms of animal welfare).

**Table 1.** Distribution of injuries on various parts of the body surface as found among 306 cows examined in the 80 smallholder zero-grazed dairy units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

Inadequate feeding space per animal at the feed bunk was a common finding in these small‐ holder zero-grazing dairy cattle units. This led to increased competitiveness and aggressive behavior of the cows toward each other and particularly toward the subordinate cattle dur‐ ing feeding times. Such behavior is likely to result in physical injuries not only in the neck area but also in other regions of the body, and to reduce feeding time as well, a fact that also

**surface body injuries Cows with surface body injuries**

#### **3.2. Body injuries as indicators of welfare**

Injuries on body surface were found distributed in various body regions among the 306 cows that were examined from the 80 smallholder zero-grazed dairy units in study 2 (Ta‐ ble 1). These body regions included the neck, brisket, hock joint area, carpal joint area, tu‐ ber coxae, ischial and rib cage areas, teats and udder. These areas being protuberant were prone to be easily injuried by house structures. The protuberant areas of the body are the parts on which pressure is exerted the most when lying down, and therefore injuries in these areas indicate the comfort state of the lying places of the animal house, hence re‐ flecting good or poor animal welfare with respect to lying comfort. Injuries on the men‐ tioned body protuberances also serve as indicators of the traumatic tendencies of certain structural parts of the animal housing unit, and this in turn reflects good or poor animal welfare state of the housing unit.

In 65% (n=52) of the zero-grazed units, cows showed injuries in the dorsal part of the neck between the middle area and over the shoulders, which presented various signs such as hy‐ perkeratosis and callus-like skin tissue, large patched hair loss, raw wounds, and scars (Fig‐ ure 1). These affected 60.8% (n=186) of the 306 cows examined. Hyperkeratosis and calluslike skin tissue were the predominant lesions indicating chronic injuries to the skin and constituted 70% (n=130) of the cows with signs of neck injuries. Prevalence of hair loss and raw wounds or scars on the dorsal aspect of the neck as signs of injury was low, being 20% (n=37) and 10% (n=19) of the cows with signs of neck injuries respectively. Evaluation of the housing structures showed that only in 35% (n=28) of the zero-grazing units they were not the cause for trauma to the neck areas of the cows. The low level of neck-bars over the feed bunks was the main risk factor for injuries on the dorsal surface of the neck. Hyperkeratosis and callus-like skin in the dorsal surface of the neck are caused by constant friction against the neck-bar during the many hours of feeding at the feed bunks. A neck-bar is fixed over the feed bunk to prevent cattle from wasting feeds and placing their forelimbs into the feed bunks (Figure 2). Similar effects of neck-bars have been previously described [88]. The neckbars over the feed bunks were present in 60% (n=48) of the zero-grazing units, and in 77.1% (n=37) of these units the neck-bars were fixed at less or up to 50cm of the top edge of the feed bunk, while in 22.9% (n=11) of the units they were more than 50 cm from the top edge of the feed bunk. When the level is too low, the dorsal surface of the neck would always scrap against the neck-bar as long as the animal is at the feed bunk feeding, and injuries are exacerbated by animals pushing one another and fighting at the feed bunks due to inade‐ quate feeding space or social dominance molestation. All the neck-bars in these zero-grazing units were made of timber, some of which had side-facing sharp edges that contact the dor‐ sal surface of the neck, precipitating the occurrence of injuries (Figure 2). Also, the width of some feed bunks was excessive that cows struggled to reach the feed on the far end and this predisposed them to more of the neck injuries.

the fact that in study 1 the claws were trimmed during evaluation, while in study 2 only observation for lameness was done without trimming for specific examination of the claws. In study 1, out of 300 cows the prevalence was 88% (n=264) of which 69% (n=182) were subclinical (the affected cows were not lame), the diagnoses being made through trimming of the claws; and 31% (n=82) were clinical (the affected cows were lame), with animals showing evidence of lameness. About 70% (n=211) of the cows had laminitis, which was either sublinical laminitis in 49% (n=148) diagnosed by presence of sole hae‐ morrhages seen after trimming a thin layer of the horn of the sole, or chronic laminitis in 21% (n=63) diagnosed by presence of extensive diffuse sole haemorrhages coupled with various degrees of claw deformities. The Pictorial description of the claw lesions and the associated predisposing causes was detailed in a previous publication [50]. In study 2, lame cows were encountered in 73% (n=58) of the 80 zero-grazing units, for which the to‐ tal prevalence was 35% (n=107) among the 306 cows examined. The lameness was caused by different claw disorders, which included various degrees of claw deformities ranging from moderate claw overgrowth to severe twisting of the claws. Lameness caused by le‐ sions in proximal parts of the limbs (proximal to the claws) had very low prevalence of

less than 2% in both studies; most of these lesions did not cause any lameness.

Injuries on body surface were found distributed in various body regions among the 306 cows that were examined from the 80 smallholder zero-grazed dairy units in study 2 (Ta‐ ble 1). These body regions included the neck, brisket, hock joint area, carpal joint area, tu‐ ber coxae, ischial and rib cage areas, teats and udder. These areas being protuberant were prone to be easily injuried by house structures. The protuberant areas of the body are the parts on which pressure is exerted the most when lying down, and therefore injuries in these areas indicate the comfort state of the lying places of the animal house, hence re‐ flecting good or poor animal welfare with respect to lying comfort. Injuries on the men‐ tioned body protuberances also serve as indicators of the traumatic tendencies of certain structural parts of the animal housing unit, and this in turn reflects good or poor animal

In 65% (n=52) of the zero-grazed units, cows showed injuries in the dorsal part of the neck between the middle area and over the shoulders, which presented various signs such as hy‐ perkeratosis and callus-like skin tissue, large patched hair loss, raw wounds, and scars (Fig‐ ure 1). These affected 60.8% (n=186) of the 306 cows examined. Hyperkeratosis and calluslike skin tissue were the predominant lesions indicating chronic injuries to the skin and constituted 70% (n=130) of the cows with signs of neck injuries. Prevalence of hair loss and raw wounds or scars on the dorsal aspect of the neck as signs of injury was low, being 20% (n=37) and 10% (n=19) of the cows with signs of neck injuries respectively. Evaluation of the housing structures showed that only in 35% (n=28) of the zero-grazing units they were not the cause for trauma to the neck areas of the cows. The low level of neck-bars over the feed bunks was the main risk factor for injuries on the dorsal surface of the neck. Hyperkeratosis and callus-like skin in the dorsal surface of the neck are caused by constant friction against

**3.2. Body injuries as indicators of welfare**

60 Insights from Veterinary Medicine

welfare state of the housing unit.


**Table 1.** Distribution of injuries on various parts of the body surface as found among 306 cows examined in the 80 smallholder zero-grazed dairy units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

Inadequate feeding space per animal at the feed bunk was a common finding in these small‐ holder zero-grazing dairy cattle units. This led to increased competitiveness and aggressive behavior of the cows toward each other and particularly toward the subordinate cattle dur‐ ing feeding times. Such behavior is likely to result in physical injuries not only in the neck area but also in other regions of the body, and to reduce feeding time as well, a fact that also infringes partly on freedom from hunger (one of the five freedoms of animal welfare).

Signs of injuries on the cranial surface of carpal joint area of the cows were observed in 96% (n=77) of the 80 units evaluated. These included healing wounds and scars, soft tissue swel‐ lings, hardening of skin in callus-like appearance and various degrees of hair loss. Out of the 306 cows examined, 75.2% (n=230) had signs of injuries in the carpal joint area. The main signs of injury in this area of the body were healing wounds and scars found in 75% (n=173) of the 230 cows with carpal area injuries, but soft tissue swellings and hair loss alone were found in 15% (n=34) and 10% (n=23) of the cows respectively. The high prevalence of signs of injuries on the cranial surface of the carpal joint area served as indicators of the rough and abrasive state of the floors where the cows lie on. It also meant that the cubicles in which the cows lay had inadequate or no bedding at all (Figure 3). Cattle get up from the lying posture by first kneeling on the carpus before extending the hind limbs to support their weight and finally stand up. This behavior predisposes cattle to likelihood of injuries to the carpal area every time the animal kneels on bare abrasive floor or bare concrete cubicle surfaces. Con‐ crete or loose stone floors of the walk alley and cubicle lying surfaces were the commonest abrasive surfaces in these zero-grazing units. Repeated friction and contusion on such floors may cause injuries that will heal with time, leaving scars and hair loss. The repeated pro‐ longed friction on the cranial surface of the carpus might eventually extend deep and lead to contusion of the underlying subcutaneous connective tissue with subsequent development of false pre-carpal bursa that consequently results into carpal hygroma (Figure 4). Inade‐ quate lunge space and bob zone in the animal cubicle may exacerbate occurrence of injuries on the cranial surface of the carpus. All these traumatic signs indicate existence of poor ani‐

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In this study, floors were evaluated in the walk alleys where the cows spent most of their time standing during feeding times. In total, 28.8% (n=23) of the 80 studied units were earth‐ en floors in the walk alleys, while the remainder 71.3% (n=57) had concrete or stoned floors. The concrete floors were grossly worn-out and pot-holed in 41.3% (n=24) of the 57 units with concrete walk alleys, while 26.2% (n=15) were smooth and slippery and 32.5% (n=18) were good and non-slippery (32.50%). In 53.75% (n=43) of the units there was no bedding material in the cubicles or animal resting areas, these areas were bare earth in 53.5% (n=23) and bare concrete in 46.5% (n=20). The bedding materials used in the rest of the zero-grazing units were wheat straw, saw-dust, wood-shavings, plastic mats or bare wooden slabs. The grossly worn-out or pot-holed concrete floors and bare concreted cubicles were the main causes of injuries and discomfort on the cranial surface of the carpal joint area whenever the cows rose up from the lying position. Slippery concrete on the walk alley poses a risk by increas‐ ing chances of slipping and falling, particularly in the presence of slurry on the floor, mak‐ ing of it an increased risk for poor animal welfare [28]. Considering that cows spend an average of about 12 hours per day standing even when provided with soft lying area [77,89], it makes it necessary to have soft, non-slip, smooth washable floor systems with adequate slope for drainage in order to enhance claw hygiene and health [28,31]. Such materials on floors would promote good animal welfare. The types and conditions of the floors in these studies predisposed the cows to poor claw health, hence the high prevalence of claw lesions subsequently precipitating to lameness [90,91]. Provision of comfortable bedding in the cu‐ bicles and resting areas of the cow housing unit influences cow resting behavior positively,

mal welfare in the evaluated zero-grazing units.

**Figure 1.** Signs of injuries on the dorsal surface of the neck in some of the cows among the 80 farms evaluated for welfare of dairy cattle in the smallholder zero-grazing units in the peri-urban areas of Nairobi, Kenya. Picture A shows severe hyperkeratosis, callus-like skin with complete hair loss (arrow), Picture B shows moderate hyperkeratosis and a patch of hair loss (arrow), and Picture C shows beginning of hair loss with skin crust (arrow).

**Figure 2.** Position of the neck-bars in some of the zero-grazing units evaluated for dairy cattle welfare in the periurban areas of Nairobi, Kenya. Pictures A, C and D show low-level positioned neck-bars (arrow) that always rubs the dorsal surface of the cow neck whenever she feeds from the feed bunk; Picture A also shows excessively wide feed bunk (double-headed arrow) from which a cow struggles to reach feed in the far wide-end; Picture B shows a cow attempting to squeeze the head and the neck between a very low sharp-edged neck-bar (arrow) and a broken sharpedged under-bar (arrow head).

Signs of injuries on the cranial surface of carpal joint area of the cows were observed in 96% (n=77) of the 80 units evaluated. These included healing wounds and scars, soft tissue swel‐ lings, hardening of skin in callus-like appearance and various degrees of hair loss. Out of the 306 cows examined, 75.2% (n=230) had signs of injuries in the carpal joint area. The main signs of injury in this area of the body were healing wounds and scars found in 75% (n=173) of the 230 cows with carpal area injuries, but soft tissue swellings and hair loss alone were found in 15% (n=34) and 10% (n=23) of the cows respectively. The high prevalence of signs of injuries on the cranial surface of the carpal joint area served as indicators of the rough and abrasive state of the floors where the cows lie on. It also meant that the cubicles in which the cows lay had inadequate or no bedding at all (Figure 3). Cattle get up from the lying posture by first kneeling on the carpus before extending the hind limbs to support their weight and finally stand up. This behavior predisposes cattle to likelihood of injuries to the carpal area every time the animal kneels on bare abrasive floor or bare concrete cubicle surfaces. Con‐ crete or loose stone floors of the walk alley and cubicle lying surfaces were the commonest abrasive surfaces in these zero-grazing units. Repeated friction and contusion on such floors may cause injuries that will heal with time, leaving scars and hair loss. The repeated pro‐ longed friction on the cranial surface of the carpus might eventually extend deep and lead to contusion of the underlying subcutaneous connective tissue with subsequent development of false pre-carpal bursa that consequently results into carpal hygroma (Figure 4). Inade‐ quate lunge space and bob zone in the animal cubicle may exacerbate occurrence of injuries on the cranial surface of the carpus. All these traumatic signs indicate existence of poor ani‐ mal welfare in the evaluated zero-grazing units.

**Figure 1.** Signs of injuries on the dorsal surface of the neck in some of the cows among the 80 farms evaluated for welfare of dairy cattle in the smallholder zero-grazing units in the peri-urban areas of Nairobi, Kenya. Picture A shows severe hyperkeratosis, callus-like skin with complete hair loss (arrow), Picture B shows moderate hyperkeratosis and a

**Figure 2.** Position of the neck-bars in some of the zero-grazing units evaluated for dairy cattle welfare in the periurban areas of Nairobi, Kenya. Pictures A, C and D show low-level positioned neck-bars (arrow) that always rubs the dorsal surface of the cow neck whenever she feeds from the feed bunk; Picture A also shows excessively wide feed bunk (double-headed arrow) from which a cow struggles to reach feed in the far wide-end; Picture B shows a cow attempting to squeeze the head and the neck between a very low sharp-edged neck-bar (arrow) and a broken sharp-

edged under-bar (arrow head).

62 Insights from Veterinary Medicine

patch of hair loss (arrow), and Picture C shows beginning of hair loss with skin crust (arrow).

In this study, floors were evaluated in the walk alleys where the cows spent most of their time standing during feeding times. In total, 28.8% (n=23) of the 80 studied units were earth‐ en floors in the walk alleys, while the remainder 71.3% (n=57) had concrete or stoned floors. The concrete floors were grossly worn-out and pot-holed in 41.3% (n=24) of the 57 units with concrete walk alleys, while 26.2% (n=15) were smooth and slippery and 32.5% (n=18) were good and non-slippery (32.50%). In 53.75% (n=43) of the units there was no bedding material in the cubicles or animal resting areas, these areas were bare earth in 53.5% (n=23) and bare concrete in 46.5% (n=20). The bedding materials used in the rest of the zero-grazing units were wheat straw, saw-dust, wood-shavings, plastic mats or bare wooden slabs. The grossly worn-out or pot-holed concrete floors and bare concreted cubicles were the main causes of injuries and discomfort on the cranial surface of the carpal joint area whenever the cows rose up from the lying position. Slippery concrete on the walk alley poses a risk by increas‐ ing chances of slipping and falling, particularly in the presence of slurry on the floor, mak‐ ing of it an increased risk for poor animal welfare [28]. Considering that cows spend an average of about 12 hours per day standing even when provided with soft lying area [77,89], it makes it necessary to have soft, non-slip, smooth washable floor systems with adequate slope for drainage in order to enhance claw hygiene and health [28,31]. Such materials on floors would promote good animal welfare. The types and conditions of the floors in these studies predisposed the cows to poor claw health, hence the high prevalence of claw lesions subsequently precipitating to lameness [90,91]. Provision of comfortable bedding in the cu‐ bicles and resting areas of the cow housing unit influences cow resting behavior positively, by encouraging them to lie down frequently. Hence, by reducing the long hours of standing, which subsequently minimizes the risk of lameness from claw lesions [17,92], the cow wel‐ fare is enhanced. However, some of the bedding such as sawdust, which are used in these zero-grazing units owing to ease of their availability and cost, could be incriminated as risk factor for mastitis [93], but in the current studies, mastitis was not a problem.

**Figure 4.** Signs of injuries on the cranial surface of the carpus. Picture A shows early swelling developing on the carpus (ar‐ row); Picture B shows massively swollen carpal hygroma (arrow) as a result of prolonged repeated contusion of soft tissues cranial to the carpal joint subsequently forming a subcutaneous pre-carpal bursa. This swelling was full of viscous strawcoloured sterile fluid. Sometimes the carpal hygroma lesion could become infected and progress to joint ankylosis as in Pic‐ ture C (arrow), which impairs the animal ability to move and feed, hence originating loss of body condition, which is also a sign of poor welfare. Frequent lying down from standing discomfort in a lame cow may result to development of decubital wounds as seen on the caudo-lateral aspect of the thigh in Picture C (arrow) and these aggravate poor animal welfare.

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Cows in 64% (n=51) of the zero-grazing units had brisket injuries, which were evidenced by extensive patches of hair loss and/or scars on the brisket (Figure 5). Brisket injuries were found in 43.8% (n=134) of the 306 cows examined in this study. Injuries at the brisket area were caused by abrasive action of bare concrete in the cubicles and by high and sharp upper edge of the feed bunk on which the brisket rubbed continuously during feeding (Figure 6). A good feed bunk that takes into consideration animal welfare should be made of concrete, because it can be smoothened during construction to eliminate sharp edges that would in‐ jure cattle as they feed [28,31,89]. The feed bunk front side should not be too high but low enough for cattle to reach feeds without the brisket rubbing against the upper edge of the bunk. The few concrete feed bunks that were worn-out, and a high number of others made of iron sheets and timber, had sharpened edges that predisposed the cows to injuries of the mouth, head and neck regions. Nails and iron sheet pieces are likely to break from the iron sheet-lined feed bunks with time, and if ingested by the cows can lead to hardware disease apart from causing direct wounds on the body surface in the head, neck and brisket regions. Therefore, the state of feed bunks as found in this study, exposed cows to poor welfare.

**Figure 3.** Bare or damaged floors and cubicles without bedding, which predisposed the cows to injuries and poor wel‐ fare. Picture A shows a cow lying on bare floor of a cubicle that is fallen apart with wooden planks on the floor. Picture B shows a floor made of blocks of stone with gaps between them. Picture C shows cubicles with loose stones in them. Picture D shows a cow attempting to stand by kneeling, which injures the carpus if the cubicle or concrete floor is bare and lunge space small, note the neck is under a wooden cross-bar. Picture E is a damaged pot-holed concrete floor. Picture F shows cows lying on rubber mats.

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by encouraging them to lie down frequently. Hence, by reducing the long hours of standing, which subsequently minimizes the risk of lameness from claw lesions [17,92], the cow wel‐ fare is enhanced. However, some of the bedding such as sawdust, which are used in these zero-grazing units owing to ease of their availability and cost, could be incriminated as risk

**Figure 3.** Bare or damaged floors and cubicles without bedding, which predisposed the cows to injuries and poor wel‐ fare. Picture A shows a cow lying on bare floor of a cubicle that is fallen apart with wooden planks on the floor. Picture B shows a floor made of blocks of stone with gaps between them. Picture C shows cubicles with loose stones in them. Picture D shows a cow attempting to stand by kneeling, which injures the carpus if the cubicle or concrete floor is bare and lunge space small, note the neck is under a wooden cross-bar. Picture E is a damaged pot-holed concrete floor.

Picture F shows cows lying on rubber mats.

factor for mastitis [93], but in the current studies, mastitis was not a problem.

64 Insights from Veterinary Medicine

**Figure 4.** Signs of injuries on the cranial surface of the carpus. Picture A shows early swelling developing on the carpus (ar‐ row); Picture B shows massively swollen carpal hygroma (arrow) as a result of prolonged repeated contusion of soft tissues cranial to the carpal joint subsequently forming a subcutaneous pre-carpal bursa. This swelling was full of viscous strawcoloured sterile fluid. Sometimes the carpal hygroma lesion could become infected and progress to joint ankylosis as in Pic‐ ture C (arrow), which impairs the animal ability to move and feed, hence originating loss of body condition, which is also a sign of poor welfare. Frequent lying down from standing discomfort in a lame cow may result to development of decubital wounds as seen on the caudo-lateral aspect of the thigh in Picture C (arrow) and these aggravate poor animal welfare.

Cows in 64% (n=51) of the zero-grazing units had brisket injuries, which were evidenced by extensive patches of hair loss and/or scars on the brisket (Figure 5). Brisket injuries were found in 43.8% (n=134) of the 306 cows examined in this study. Injuries at the brisket area were caused by abrasive action of bare concrete in the cubicles and by high and sharp upper edge of the feed bunk on which the brisket rubbed continuously during feeding (Figure 6). A good feed bunk that takes into consideration animal welfare should be made of concrete, because it can be smoothened during construction to eliminate sharp edges that would in‐ jure cattle as they feed [28,31,89]. The feed bunk front side should not be too high but low enough for cattle to reach feeds without the brisket rubbing against the upper edge of the bunk. The few concrete feed bunks that were worn-out, and a high number of others made of iron sheets and timber, had sharpened edges that predisposed the cows to injuries of the mouth, head and neck regions. Nails and iron sheet pieces are likely to break from the iron sheet-lined feed bunks with time, and if ingested by the cows can lead to hardware disease apart from causing direct wounds on the body surface in the head, neck and brisket regions. Therefore, the state of feed bunks as found in this study, exposed cows to poor welfare.

Signs of injuries at the hock joint area were observed in 95% (n=76) of all the 80 zero-grazing units. These included healing wounds and scars, soft tissue swellings and hair loss (Figure 7). The lesions were found in 87% (n= 260) of the 306 cows examined. The high prevalence of injuries at the hock area was a good indicator of the uncomfortable state of the concrete floor and the bare concrete cubicles, which in turn were a definite reflection of the existing poor welfare of cattle in these zero-grazing units. Another region of the body with signs of inju‐ ries related to concrete floor and bare concrete cubicles was the ischial area. Injuries in this area were found in 76% (n=61) of the zero-grazing units and affected 41% (n=124) of the 306 cows examined. Cows tend to lie leaning more toward one side than the other, with the lat‐ eral aspect of the hock pressed against the floor (Figure 8). This explains the high prevalence

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**Figure 7.** Injuries on the hock. Picture A shows hyperaemic skin with hair loss (arrow), Picture B shows hygroma swel‐ ling at the initial developing stage (arrow), Picture C shows healing wound caudal on the hock area (arrow). Picture D

shows nodular scarring tumour-like swelling on the lateral aspect of the hock (arrow).

of these injuries.

**Figure 5.** Brisket injury consisting of swelling, scar tissue and hair loss (arrow) in one of the examined cows.

**Figure 6.** Various feed bunks with different designs and state of the upper front edge. Picture A shows concrete feed bunk with smooth upper front edge, which is at an acceptable low level off the brisket (arrow). Picture B shows low smoothedged concrete feed bunk (arrow), but addition of wooden bars above the upper front edge (arrow head) on which the brisket could rub and be injured with time. Picture C shows the edges and main part of feed bunk lined with sharp broken iron sheet pieces (arrow), which could injure not only the brisket but also the tongue as it scoops the feed.

Signs of injuries at the hock joint area were observed in 95% (n=76) of all the 80 zero-grazing units. These included healing wounds and scars, soft tissue swellings and hair loss (Figure 7). The lesions were found in 87% (n= 260) of the 306 cows examined. The high prevalence of injuries at the hock area was a good indicator of the uncomfortable state of the concrete floor and the bare concrete cubicles, which in turn were a definite reflection of the existing poor welfare of cattle in these zero-grazing units. Another region of the body with signs of inju‐ ries related to concrete floor and bare concrete cubicles was the ischial area. Injuries in this area were found in 76% (n=61) of the zero-grazing units and affected 41% (n=124) of the 306 cows examined. Cows tend to lie leaning more toward one side than the other, with the lat‐ eral aspect of the hock pressed against the floor (Figure 8). This explains the high prevalence of these injuries.

**Figure 5.** Brisket injury consisting of swelling, scar tissue and hair loss (arrow) in one of the examined cows.

66 Insights from Veterinary Medicine

**Figure 6.** Various feed bunks with different designs and state of the upper front edge. Picture A shows concrete feed bunk with smooth upper front edge, which is at an acceptable low level off the brisket (arrow). Picture B shows low smoothedged concrete feed bunk (arrow), but addition of wooden bars above the upper front edge (arrow head) on which the brisket could rub and be injured with time. Picture C shows the edges and main part of feed bunk lined with sharp broken

iron sheet pieces (arrow), which could injure not only the brisket but also the tongue as it scoops the feed.

**Figure 7.** Injuries on the hock. Picture A shows hyperaemic skin with hair loss (arrow), Picture B shows hygroma swel‐ ling at the initial developing stage (arrow), Picture C shows healing wound caudal on the hock area (arrow). Picture D shows nodular scarring tumour-like swelling on the lateral aspect of the hock (arrow).

**Figure 8.** Pictures showing that the lying position of a cow presses on lateral aspect of one hock joint area such that if there is no adequate bedding material or padding, the hock area is easily injured by repeated pressure on hard floor particularly bare concrete floor. Picture A shows a cow lying on lateral aspect of the right hock, and Picture B the cow is lying on lateral aspect of the left hock.

**Figure 9.** Scars sustained at the rib cage area (arrows) in one of the cows examined in the 80 zero-grazing units.

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**Figure 10.** Signs of wounds on the tuber coxae. Picture A: hair loss and skin abrasion (arrow). Picture B: beginning of

hair loss due to abrasion by housing structures (arrow)

Other signs of injuries found on the cows in some of the units evaluated were located on the rib cage area (Figure 9) and the tuber coxae (Figure 10). On the rib cage area they were found in 95% (n=76) of the units and they affected 75% (n=228) of the 306 cows examined; lesions on tuber coxae were found in 91% (n=72) of the units and they affected 67% (n= 204) of the 306 cows examined. In both areas the signs of injuries were mainly healing wounds or scars. Tuber coxae and rib cage injuries were associated with small cubicle space and pro‐ truding traumatic parts of the cattle housing structures, such as side dividing timber or wooden pieces, nails and iron sheets on the side walls. Some of the studied units had broken wooden sidewalls and collapsing roofing material that easily injure the animals (Figure 11). Small-sized cubicles, measuring 1.80 meters by 0.95 meters or less, were found in 74.6% (n=50) of the units evaluated in this study. Overstocking was found in more than half of the zero-grazing units. It caused squeezing and competition for space and feed among the ani‐ mals, which facilitates injury from the protruding traumatic objects and collapsed roofing material in the cattle housing units. Overstocking meant that there were more cows than the number of cubicles available to rest (Figure 12) and in some cases the feeding space was in‐ adequate for all the cows present. All these factors contributed to poor welfare of the cattle.

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**Figure 9.** Scars sustained at the rib cage area (arrows) in one of the cows examined in the 80 zero-grazing units.

**Figure 8.** Pictures showing that the lying position of a cow presses on lateral aspect of one hock joint area such that if there is no adequate bedding material or padding, the hock area is easily injured by repeated pressure on hard floor particularly bare concrete floor. Picture A shows a cow lying on lateral aspect of the right hock, and Picture B the cow

Other signs of injuries found on the cows in some of the units evaluated were located on the rib cage area (Figure 9) and the tuber coxae (Figure 10). On the rib cage area they were found in 95% (n=76) of the units and they affected 75% (n=228) of the 306 cows examined; lesions on tuber coxae were found in 91% (n=72) of the units and they affected 67% (n= 204) of the 306 cows examined. In both areas the signs of injuries were mainly healing wounds or scars. Tuber coxae and rib cage injuries were associated with small cubicle space and pro‐ truding traumatic parts of the cattle housing structures, such as side dividing timber or wooden pieces, nails and iron sheets on the side walls. Some of the studied units had broken wooden sidewalls and collapsing roofing material that easily injure the animals (Figure 11). Small-sized cubicles, measuring 1.80 meters by 0.95 meters or less, were found in 74.6% (n=50) of the units evaluated in this study. Overstocking was found in more than half of the zero-grazing units. It caused squeezing and competition for space and feed among the ani‐ mals, which facilitates injury from the protruding traumatic objects and collapsed roofing material in the cattle housing units. Overstocking meant that there were more cows than the number of cubicles available to rest (Figure 12) and in some cases the feeding space was in‐ adequate for all the cows present. All these factors contributed to poor welfare of the cattle.

is lying on lateral aspect of the left hock.

68 Insights from Veterinary Medicine

**Figure 10.** Signs of wounds on the tuber coxae. Picture A: hair loss and skin abrasion (arrow). Picture B: beginning of hair loss due to abrasion by housing structures (arrow)

**Figure 11.** Collapsed iron sheet roof and sides in one of the zero-grazing units evaluated. Both the roof and side tim‐ ber are broken and collapsing. Yet cows are still housed inside (arrow).

**Figure 13.** Pictures showing lateral limb injuries. Picture A shows a scarring bruised skin on the lateral side of the thigh (arrow); Picture B shows a healing longitudinal skin cut on the lateral aspect of the lower part of the limb (arrow).

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The skin injuries observed in the current study were a reflection of the housing type and size, as well as the structures used to construct cattle houses. Similar injuries have been described in other studies [44]. Skin injuries in any part of the body of an animal are indicators of the welfare status of the animal particularly in relation to its environment. These lesions are associated with pain and suffering [43]. An environment that allows free movement of the animal without risk of disease or injury is paramount [28,31]. The key predisposing factors to external body in‐ juries are the restrictiveness of housing types and structures that affect the cows' behavioral patterns [94]. The external injuries observed in the cows in this study were mainly located on body protuberances such as the hock and the tuber coxae. Others were in the areas of the body subjected to pressure during recumbency and feeding times such as the brisket, ischial region, udder, rib cage, and neck. These findings are in agreement with previous reports [94,95]. In this study, although injuries at different parts of the body were attributed to different risk factors,

Statistical analysis of simple associations between injuries and disease was carried out and several factors were associated. Injuries in the various body regions reported in the foregone pages were found to be associated with various factors within the zero-grazing units. The

**Injured body region Risk factor Chi-square value (χ2) P value**

Hock joint area Narrow walk alley 10.68 <0.0011 Ischial region Concrete floor 8.86 0.012

zero-grazing units evaluated for the welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

**Table 2.** Risk factors associated with the occurrence of body injuries in the 306 cows examined in the 80 smallholder

Presence of neck bar 20.25 <0.0001 Height of neck bar 22.93 <0.0001

Presence of neck bar 8.14 0.0043 Height of neck bar 7.37 0.025

Bare concrete floor 12.57 0.014 Quality of bedding 5.15 0.023

they still related to the nature of the housing environment.

factors with strong association are presented in table 2.

Dorsal surface of the neck

Brisket area

Teat /udder/thighs

**Figure 12.** One of the overstocked zero-grazing units with narrowed walk alley and the cows hardly having any room to turn or move. Animal interactions here and scrambling for space can cause them to press each other against the sharp wooden structures leading to injuries on the rib cage, tuber coxae and other protuberances.

Additional areas with lesions, but showing lower prevalence of injuries include the teats, udder, thighs and other areas of the limbs (Figure 13). These were mainly abrasions with hy‐ peraemia of skin and hair loss. Injuries in these regions were mainly associated with rough‐ ness and bareness of the concrete floor and the cubicles. Occasionally, the skin in the thigh areas can also be injured by protruding sharp edges or objects in the housing unit. There were also pin-point nodular lesions on the teats of some of the cows, which resembled poxlike lesions. Although these lesions could have been caused by microorganisms such as vi‐ ruses, poor environmental conditions would facilitate the entrance of such agents and persistence of infection.

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**Figure 13.** Pictures showing lateral limb injuries. Picture A shows a scarring bruised skin on the lateral side of the thigh (arrow); Picture B shows a healing longitudinal skin cut on the lateral aspect of the lower part of the limb (arrow).

**Figure 11.** Collapsed iron sheet roof and sides in one of the zero-grazing units evaluated. Both the roof and side tim‐

**Figure 12.** One of the overstocked zero-grazing units with narrowed walk alley and the cows hardly having any room to turn or move. Animal interactions here and scrambling for space can cause them to press each other against the

Additional areas with lesions, but showing lower prevalence of injuries include the teats, udder, thighs and other areas of the limbs (Figure 13). These were mainly abrasions with hy‐ peraemia of skin and hair loss. Injuries in these regions were mainly associated with rough‐ ness and bareness of the concrete floor and the cubicles. Occasionally, the skin in the thigh areas can also be injured by protruding sharp edges or objects in the housing unit. There were also pin-point nodular lesions on the teats of some of the cows, which resembled poxlike lesions. Although these lesions could have been caused by microorganisms such as vi‐ ruses, poor environmental conditions would facilitate the entrance of such agents and

sharp wooden structures leading to injuries on the rib cage, tuber coxae and other protuberances.

persistence of infection.

ber are broken and collapsing. Yet cows are still housed inside (arrow).

70 Insights from Veterinary Medicine

The skin injuries observed in the current study were a reflection of the housing type and size, as well as the structures used to construct cattle houses. Similar injuries have been described in other studies [44]. Skin injuries in any part of the body of an animal are indicators of the welfare status of the animal particularly in relation to its environment. These lesions are associated with pain and suffering [43]. An environment that allows free movement of the animal without risk of disease or injury is paramount [28,31]. The key predisposing factors to external body in‐ juries are the restrictiveness of housing types and structures that affect the cows' behavioral patterns [94]. The external injuries observed in the cows in this study were mainly located on body protuberances such as the hock and the tuber coxae. Others were in the areas of the body subjected to pressure during recumbency and feeding times such as the brisket, ischial region, udder, rib cage, and neck. These findings are in agreement with previous reports [94,95]. In this study, although injuries at different parts of the body were attributed to different risk factors, they still related to the nature of the housing environment.

Statistical analysis of simple associations between injuries and disease was carried out and several factors were associated. Injuries in the various body regions reported in the foregone pages were found to be associated with various factors within the zero-grazing units. The factors with strong association are presented in table 2.


**Table 2.** Risk factors associated with the occurrence of body injuries in the 306 cows examined in the 80 smallholder zero-grazing units evaluated for the welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

The presence of neck-bar over the feed bunk had a strong association with injuries on the dorsal surface of the neck (χ<sup>2</sup> =20.25; p<0.0001) and the surface of the brisket (χ<sup>2</sup> = 8.14; p=0.0043). The position of the height of the neck-bar from the top edge of the feed bunk was also found to influence presence or absence of injuries at the dorsal surface of the neck (χ<sup>2</sup> =22.93; p<0.0001) and the surface of the brisket (χ<sup>2</sup> =7.37; p=0.025. Injuries in the hock joint area were significantly influenced by narrow walk alleys (χ<sup>2</sup> =10.68; p<0.001), whilst injuries at the ischial area were significantly associated with poor quality (excessively rough and pot-holed) concrete floors (χ<sup>2</sup> =8.86; p=0.012). Teat, udder and thigh injuries were found to have a significant association with bare concrete-floored cubicles (χ<sup>2</sup> =12.57; p=0.014) and al‐ so with presence or absence of bedding and the quality of bedding (χ<sup>2</sup> =5.15; p=0.023). Lame‐ ness was found to be associated with excess slurry in the walk alley (χ<sup>2</sup> =29.58; p=0.042).

From the results, about 91% (n=277) of the 306 cows examined had fair body condition (BCS between 2 and 3.5), which indicated that the feeding practiced in these zero-grazing units was moderate. Only a few cows had poor body condition below 1.5 (6%; n=19). Similarly, good body condition above 3.5 was found in only 3% (n=10) of the cows examined, which shows that nutritional quality and feed quantity or feeding regime in these zero-grazing units falls below the optimal expectations of good feeding practices for dairy cows. The body condition score (BCS) was influenced by the presence, amount and frequency of con‐ centrate feeding, mineral supplementation and protein supplementation, as shown in Table 4. Body condition score 1 had significant association with occasional (irregular) feeding of

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

=14.77; p=0.022), absence of concentrate feeding (χ<sup>2</sup>

=13.29; p=0.021), variations in amounts of mineral supplementation (χ<sup>2</sup>

to have a significant association with regular protein supplementation (χ<sup>2</sup>

variation (number of times) in the frequency of concentrate feeding (χ<sup>2</sup>

=8.23; p=0.042). Body condition score 2 was found to have a significant association with

=12.02; p=0.035). Body condition score 3 was found to have a significant association with

**BCS Associated factor Chi-square value ( χ2) P value**

Occasional concentrate feeding 14.77 0.022 Absence of concentrate feeding 7.90 0.048 Occasional mineral supplementation 49.87 <0.0001 Absence of mineral supplementation 8.23 0.0415

Variation in frequency of concentrate feeding 22.69 0.012 Regular concentrate feeding 13.29 0.021 Regular mineral supplementation 12.02 0.035

High levels of concentrate feeding 35.65 0.017 Variations in amounts of mineral supplementation 29.08 0.016 Regular mineral supplementation 15.03 <0.01 Regular concentrate feeding 13.19 0.022 4 Regular protein supplementation 14.46 0.0023

**Table 4.** Factors associated with the body condition score (BCS) for the cows examined in 80 smallholder zero-grazing

Generally, the feeding of forages to cows in these zero-grazing units was more consistent than the feeding of concentrates. Forages included mainly grasses such as napier grass (Pen‐ nisetum purpurem), Kikuyu grass (pennisetum clandestum), Rhodes grass [Chloris gayana],

units evaluated for the welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

=7.90; p=0.048), occasional

http://dx.doi.org/10.5772/53678

= 22.69; p=0.012), reg‐

=14.46; p=0.023).

=29.08; p=0.016)

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73

=49.87; p<0.0001) as well as absence of mineral supplements

=13.29; p=0.021) and regular mineral supplementation

=35.65; p=0.017), regular (daily) concentrate feeding

=15.03; p<0.01). Body condition score 4 was found

concentrates (χ<sup>2</sup>

(χ<sup>2</sup>

(χ<sup>2</sup>

(χ<sup>2</sup>

1

2

3

mineral supplementation (χ<sup>2</sup>

ular (daily) concentrate feeding (χ<sup>2</sup>

high levels of concentrate feeding (χ<sup>2</sup>

and regular mineral supplementation (χ<sup>2</sup>

The housing systems in the smallholder zero-grazing units in this study greatly restricted the cows from freely expressing their normal behavior and enjoying free movement. The restrict‐ ing sizes of these animal units are normally due to the small pieces of land owned and the finan‐ cial constraints of these smallholder farmers, which makes it difficult for them to build cattle housing units with the recommended dimensions [29]. This means that it may be difficult to guarantee the freedom of expression of normal behavior and movement for the cows in such smallholder zero-grazing units. The restriction of movement is likely to predispose the cows to lameness [96]. The particularly small size of cubicles found in these units were contrary to what is recommended [97] and was incriminated as one of the factors that predisposed the cows to frequent injuries on the rib-cage, tuber coxae and ischial area, thus supporting previous find‐ ings [98]. All these housing factors predisposing the cows to body injuries and lameness are as‐ sociated with causing pain and suffering, hence poor welfare.

#### **3.3. Body condition score as indicators of welfare**

Body condition score (BCS) was also found to be a good indicator of the dairy cow welfare for these zero-grazing units. It reflects mainly on the feeding regime, nutritional value of the diet and the feed quantities supplied to the cows. The average body condition score of the cows evaluated in these 80 units was 2.20. Out of the 306 cows examined, the distribution of the body condition score was found to be as is presented in table 3.


**Table 3.** Distribution of the body condition scores among the 306 cows examined in the 80 smallholder zero-grazing units evaluated for welfare of dairy cattle in the peri-urabn areas of Nairobi, Kenya.

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The presence of neck-bar over the feed bunk had a strong association with injuries on the

p=0.0043). The position of the height of the neck-bar from the top edge of the feed bunk was also found to influence presence or absence of injuries at the dorsal surface of the neck

at the ischial area were significantly associated with poor quality (excessively rough and

have a significant association with bare concrete-floored cubicles (χ<sup>2</sup> =12.57; p=0.014) and al‐

The housing systems in the smallholder zero-grazing units in this study greatly restricted the cows from freely expressing their normal behavior and enjoying free movement. The restrict‐ ing sizes of these animal units are normally due to the small pieces of land owned and the finan‐ cial constraints of these smallholder farmers, which makes it difficult for them to build cattle housing units with the recommended dimensions [29]. This means that it may be difficult to guarantee the freedom of expression of normal behavior and movement for the cows in such smallholder zero-grazing units. The restriction of movement is likely to predispose the cows to lameness [96]. The particularly small size of cubicles found in these units were contrary to what is recommended [97] and was incriminated as one of the factors that predisposed the cows to frequent injuries on the rib-cage, tuber coxae and ischial area, thus supporting previous find‐ ings [98]. All these housing factors predisposing the cows to body injuries and lameness are as‐

Body condition score (BCS) was also found to be a good indicator of the dairy cow welfare for these zero-grazing units. It reflects mainly on the feeding regime, nutritional value of the diet and the feed quantities supplied to the cows. The average body condition score of the cows evaluated in these 80 units was 2.20. Out of the 306 cows examined, the distribution of

**BCS Number of cows Percentage of cows (%)**

1 – 1.5 19 6 2 – 2.5 177 58 3 – 3.5 100 33 4 – 4.5 10 3 5 0 0 Total 306 100

**Table 3.** Distribution of the body condition scores among the 306 cows examined in the 80 smallholder zero-grazing

=20.25; p<0.0001) and the surface of the brisket (χ<sup>2</sup>

=8.86; p=0.012). Teat, udder and thigh injuries were found to

=7.37; p=0.025. Injuries in the hock joint

=10.68; p<0.001), whilst injuries

=5.15; p=0.023). Lame‐

=29.58; p=0.042).

= 8.14;

dorsal surface of the neck (χ<sup>2</sup>

72 Insights from Veterinary Medicine

pot-holed) concrete floors (χ<sup>2</sup>

=22.93; p<0.0001) and the surface of the brisket (χ<sup>2</sup>

area were significantly influenced by narrow walk alleys (χ<sup>2</sup>

sociated with causing pain and suffering, hence poor welfare.

the body condition score was found to be as is presented in table 3.

units evaluated for welfare of dairy cattle in the peri-urabn areas of Nairobi, Kenya.

**3.3. Body condition score as indicators of welfare**

so with presence or absence of bedding and the quality of bedding (χ<sup>2</sup>

ness was found to be associated with excess slurry in the walk alley (χ<sup>2</sup>

(χ<sup>2</sup>

From the results, about 91% (n=277) of the 306 cows examined had fair body condition (BCS between 2 and 3.5), which indicated that the feeding practiced in these zero-grazing units was moderate. Only a few cows had poor body condition below 1.5 (6%; n=19). Similarly, good body condition above 3.5 was found in only 3% (n=10) of the cows examined, which shows that nutritional quality and feed quantity or feeding regime in these zero-grazing units falls below the optimal expectations of good feeding practices for dairy cows. The body condition score (BCS) was influenced by the presence, amount and frequency of con‐ centrate feeding, mineral supplementation and protein supplementation, as shown in Table 4. Body condition score 1 had significant association with occasional (irregular) feeding of concentrates (χ<sup>2</sup> =14.77; p=0.022), absence of concentrate feeding (χ<sup>2</sup> =7.90; p=0.048), occasional mineral supplementation (χ<sup>2</sup> =49.87; p<0.0001) as well as absence of mineral supplements (χ<sup>2</sup> =8.23; p=0.042). Body condition score 2 was found to have a significant association with variation (number of times) in the frequency of concentrate feeding (χ<sup>2</sup> = 22.69; p=0.012), reg‐ ular (daily) concentrate feeding (χ<sup>2</sup> =13.29; p=0.021) and regular mineral supplementation (χ<sup>2</sup> =12.02; p=0.035). Body condition score 3 was found to have a significant association with high levels of concentrate feeding (χ<sup>2</sup> =35.65; p=0.017), regular (daily) concentrate feeding (χ<sup>2</sup> =13.29; p=0.021), variations in amounts of mineral supplementation (χ<sup>2</sup> =29.08; p=0.016) and regular mineral supplementation (χ<sup>2</sup> =15.03; p<0.01). Body condition score 4 was found to have a significant association with regular protein supplementation (χ<sup>2</sup> =14.46; p=0.023).


**Table 4.** Factors associated with the body condition score (BCS) for the cows examined in 80 smallholder zero-grazing units evaluated for the welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

Generally, the feeding of forages to cows in these zero-grazing units was more consistent than the feeding of concentrates. Forages included mainly grasses such as napier grass (Pen‐ nisetum purpurem), Kikuyu grass (pennisetum clandestum), Rhodes grass [Chloris gayana], maize stover, and in few occasions banana plant stems. Forages were fed to all the cows in all the 80 zero-grazing units. Moreover, main variations in dairy cow feeding practices in these zero-grazing units were found on concentrate feeding. Concentrates were fed to cows only in 85% (n= 68) of the zero-grazing units evaluated in this study. In the remaining 15% (n=12) of the units, cows were not fed on concentrates at all. Of the zero-grazing units that provided concentrates, 98.5% (n=67) used commercially available concentrates, while 1.5% (n=1) used farm-made concentrate mixtures. The farm-made concentrate mixtures consisted of pollard, maize germ, wheat bran, yeast, cotton seed cake and minerals. The formulation ratios of the ingredients in the farm-made mixtures were not revealed to the investigator. In the farms that provided concentrates, 83.8% (n=57) fed it only to lactating cows while 16.2% (n=11) fed it to all cows. Concentrates were provided 2-3 times per day, intentionally coin‐ ciding with milking times. In the farms that provided concentrates, 32.4% (n=22) fed each cow on an average of 2-4 kilograms of concentrates per day, 29.4% (n=20) on an average of 5-7 kilograms per day, 23.5% (n=16) on an average of 8-10 kilograms per day and in 14.7% (n=10) on more than 10 kilograms per day (Table 5).

tion has been shown to be protective on occurrence of some claw conditions such as sole

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

Kenya

75

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Additional protein supplements such as cotton seed cake, sorghum, fish-meal and high protein forage (Alfafa/Lucerne-*Medicago sativa*) were provided in 36% (n=29) of the zerograzing units. These protein supplements were added to concentrates, but the high pro‐ tein forages such as Lucerne were mixed with fodder feeds. Protein supplements were added and fed to cattle only during early lactation. Protein supplementation had no in‐ fluence on occurrence of claw lesions, but on body condition score, which is discussed in

Concentrates are rich in proteins and carbohydrates and have some levels of minerals and vitamins, hence their usefulness in supplementing forages that generally have less of these nutrients. Apart from being essential for growth and for improved milk production [6,99], concentrates also make the diet of dairy cows more complete, thus contributing to their good welfare [99]. However, if fed in large quantities, carbohydrate feeds could lead to ruminal tympany, sub-acute ruminal acidosis and subsequent laminitis [100], which consequently results in lameness that negatively impacts on the welfare of the cow [31,44]. The inconsistencies of concentrate feeding observed in this study including total failure to feed the cows on any concentrate, irregular feeding frequencies and feed‐ ing irregular amounts, demonstrated the farmers' ignorance concerning the need and the importance of concentrate feeding. Discriminatory feeding of concentrates only to lactat‐ ing cows but denying it to the young, non-pregnant, as well as dry cows further sup‐

The farmers' perception of the need for concentrate feeding was only associated with the benefits of increased milk production. All these inconsistencies and irregularities of con‐ centrate feeding deny the cow access to a balanced feed type that promotes health, growth and energy [99,101]. Such varied irregularity in concentrate feeding of dairy cat‐ tle from one zero-grazing unit to the next has not been reported elsewhere, and is in sharp contrast to the more standardized dairy cattle feeding regimes in intensively man‐ aged dairy production systems in the developed countries [102]. The association ob‐ served in this study between body condition status and the level of concentrate feeding demonstrates the benefit of concentrate inclusion in the diet. It further points out to the fact that lack of, and irregular concentrate feeding has a direct negative effect on the welfare of the cows. The stronger influence of occasional (irregular) concentrate feeding than its total absence on body condition score, can be attributed to the fact that when the cow's body is denied concentrates completely, it probably adjusts through compensa‐ tory mechanisms. Conversely, occasional inconsistent feeding does not allow the cow physiological adjustment to one consistent system, but rather destabilizes it, hence nega‐

The study also indicated that good body condition of the cows was enhanced when addi‐ tional protein supplements were mixed with the concentrate feeds. These observations

bruising and white line separation [17].

the paragraphs below.

ports evidences to this ignorance.

tively affecting the general welfare of the animal.


**Table 5.** Amount of concentrates fed to cows per day in 68 of the 80 smallholder zero-grazing units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

According to data in table 5, concentrate feeding in most zero-grazing units was minimal in quantity, and particularly when considering that in many of these units it was partial since the cows were fed only when lactating.

Cows in 88.75% (n=71) of the zero-grazing units were given mineral supplements. In the remaining 11.25% (n=9) of the units, no minerals were provided for the cows. In the zero-grazing units that provided minerals, 77.5% (n=55) of them provided minerals ad li‐ bitum, 19.7% (n=14) at 200g to 500g per cow per day, and 2.8% (n=2) of them only occa‐ sionally during the lactation period. The mineral supplements were commercially bought and they included: "Unga high phosphorus"-SuperPHOS® (Danthil Enterprises) and "Maclick Super®" (Coopers Limited). The latter was available either in powder form or as a mineral lick block. The constituents of the mineral supplements included higher con‐ centrations of the major elements such as calcium, phosphorus, sodium, chloride and magnesium and lower concentrates of trace elements such as iron, copper, manganese, zinc, sulphur, cobalt, iodine, selenium and molybdenum. Regular mineral supplementa‐ tion has been shown to be protective on occurrence of some claw conditions such as sole bruising and white line separation [17].

maize stover, and in few occasions banana plant stems. Forages were fed to all the cows in all the 80 zero-grazing units. Moreover, main variations in dairy cow feeding practices in these zero-grazing units were found on concentrate feeding. Concentrates were fed to cows only in 85% (n= 68) of the zero-grazing units evaluated in this study. In the remaining 15% (n=12) of the units, cows were not fed on concentrates at all. Of the zero-grazing units that provided concentrates, 98.5% (n=67) used commercially available concentrates, while 1.5% (n=1) used farm-made concentrate mixtures. The farm-made concentrate mixtures consisted of pollard, maize germ, wheat bran, yeast, cotton seed cake and minerals. The formulation ratios of the ingredients in the farm-made mixtures were not revealed to the investigator. In the farms that provided concentrates, 83.8% (n=57) fed it only to lactating cows while 16.2% (n=11) fed it to all cows. Concentrates were provided 2-3 times per day, intentionally coin‐ ciding with milking times. In the farms that provided concentrates, 32.4% (n=22) fed each cow on an average of 2-4 kilograms of concentrates per day, 29.4% (n=20) on an average of 5-7 kilograms per day, 23.5% (n=16) on an average of 8-10 kilograms per day and in 14.7%

> **Number of Zero-grazing units**

2 – 4 22 32.4 5 – 7 20 29.4 8 – 10 16 23.5 > 10 10 14.7 Total 68 100

**Table 5.** Amount of concentrates fed to cows per day in 68 of the 80 smallholder zero-grazing units evaluated for

According to data in table 5, concentrate feeding in most zero-grazing units was minimal in quantity, and particularly when considering that in many of these units it was partial since

Cows in 88.75% (n=71) of the zero-grazing units were given mineral supplements. In the remaining 11.25% (n=9) of the units, no minerals were provided for the cows. In the zero-grazing units that provided minerals, 77.5% (n=55) of them provided minerals ad li‐ bitum, 19.7% (n=14) at 200g to 500g per cow per day, and 2.8% (n=2) of them only occa‐ sionally during the lactation period. The mineral supplements were commercially bought and they included: "Unga high phosphorus"-SuperPHOS® (Danthil Enterprises) and "Maclick Super®" (Coopers Limited). The latter was available either in powder form or as a mineral lick block. The constituents of the mineral supplements included higher con‐ centrations of the major elements such as calcium, phosphorus, sodium, chloride and magnesium and lower concentrates of trace elements such as iron, copper, manganese, zinc, sulphur, cobalt, iodine, selenium and molybdenum. Regular mineral supplementa‐

**Percentage of Zero-grazing units**

(n=10) on more than 10 kilograms per day (Table 5).

welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

the cows were fed only when lactating.

**Daily concentrate amount (Kilograms / cow)**

74 Insights from Veterinary Medicine

Additional protein supplements such as cotton seed cake, sorghum, fish-meal and high protein forage (Alfafa/Lucerne-*Medicago sativa*) were provided in 36% (n=29) of the zerograzing units. These protein supplements were added to concentrates, but the high pro‐ tein forages such as Lucerne were mixed with fodder feeds. Protein supplements were added and fed to cattle only during early lactation. Protein supplementation had no in‐ fluence on occurrence of claw lesions, but on body condition score, which is discussed in the paragraphs below.

Concentrates are rich in proteins and carbohydrates and have some levels of minerals and vitamins, hence their usefulness in supplementing forages that generally have less of these nutrients. Apart from being essential for growth and for improved milk production [6,99], concentrates also make the diet of dairy cows more complete, thus contributing to their good welfare [99]. However, if fed in large quantities, carbohydrate feeds could lead to ruminal tympany, sub-acute ruminal acidosis and subsequent laminitis [100], which consequently results in lameness that negatively impacts on the welfare of the cow [31,44]. The inconsistencies of concentrate feeding observed in this study including total failure to feed the cows on any concentrate, irregular feeding frequencies and feed‐ ing irregular amounts, demonstrated the farmers' ignorance concerning the need and the importance of concentrate feeding. Discriminatory feeding of concentrates only to lactat‐ ing cows but denying it to the young, non-pregnant, as well as dry cows further sup‐ ports evidences to this ignorance.

The farmers' perception of the need for concentrate feeding was only associated with the benefits of increased milk production. All these inconsistencies and irregularities of con‐ centrate feeding deny the cow access to a balanced feed type that promotes health, growth and energy [99,101]. Such varied irregularity in concentrate feeding of dairy cat‐ tle from one zero-grazing unit to the next has not been reported elsewhere, and is in sharp contrast to the more standardized dairy cattle feeding regimes in intensively man‐ aged dairy production systems in the developed countries [102]. The association ob‐ served in this study between body condition status and the level of concentrate feeding demonstrates the benefit of concentrate inclusion in the diet. It further points out to the fact that lack of, and irregular concentrate feeding has a direct negative effect on the welfare of the cows. The stronger influence of occasional (irregular) concentrate feeding than its total absence on body condition score, can be attributed to the fact that when the cow's body is denied concentrates completely, it probably adjusts through compensa‐ tory mechanisms. Conversely, occasional inconsistent feeding does not allow the cow physiological adjustment to one consistent system, but rather destabilizes it, hence nega‐ tively affecting the general welfare of the animal.

The study also indicated that good body condition of the cows was enhanced when addi‐ tional protein supplements were mixed with the concentrate feeds. These observations could be attributed to the fact that concentrates supply the primary nutrient requirements to the cow as well as sufficient reserves needed for secondary processes such as normal lacta‐ tion [99,101], and increased milk production [18]. Therefore, concentrates are pertinent con‐ stituents of the dairy cow diet if the stress of both body maintenance and milk production has to be avoided.

Regular mineral supplementation supplied in a majority of the zero-grazing units in this study is a reflection of good animal welfare practice, since minerals enhance animal growth, reproduction and health [99,101]. In this study, the importance of mineral supplementation was evidenced by the association between regular supplementation and fair body condition, while occasional or absence of mineral supplementation was associated with poor body con‐ dition. Irregular mineral supplementation like was found with irregular concentrate feeding destabilizes the body more than complete absence of minerals, hence affecting body condi‐ tion score that impacts negatively on the welfare of the cows. Findings from previous stud‐ ies indicate that absence or insufficient mineral supplementation impacts negatively on growth rate and reproduction, leading to anoestrus [101,103], and hence inevitably affecting animal welfare.

**Figure 15.** Slurry accumulated in animal facilities. Picture A shows slurry in an earthen floor. Picture B shows slurry in concrete floor. Picture C shows slurry and narrow walk alley. Picture D-Despite slurry accumulation on parks, the cows in this unit were clean because the cubicles had good clean bedding of sawdust, which meant the cows never lay on

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Kenya

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**Soiled body region Associated factor Chi-square value ( χ2) P value**

**Table 6.** Factors associated with soiling in various body parts of cows examined among the 80 smallholder zero-

Although more than half of the smallholder units in the current study had slurry re‐ moved frequently, the excessive slurry found in the rest of the units is likely to affect health of the claws and the udders of the cows. The holding of slurry in cow housing units for long without cleaning it out, exposes the claws to continuous wet environ‐ ment which softens the horny parts of the claws, predisposing them to development of lesions as previously reported [17,44]. Accumulated slurry also exposes udders to unhy‐ gienic conditions that predispose them to mastitis, particularly when the cows lie on it most of the time as observed previously [17,44]. The subsequent development of claw lameness and mastitis will cause pain and inevitably lead to poor welfare. Furthermore, this study was able to show that accumulation of slurry caused excessive soiling of the skin in some parts of the body mainly because most of the time the cows were found lying on it. This is supported by the strong statistical association between the presence of slurry and soiling of the hind quarters as well as the udder region. This may proba‐ bly be exacerbated by poor housing designs, which were reported previously as the main predisposing factor for soiling of these body regions [104] due to likelihood of these designs forcing the cows to lie on the walk alleys where slurry accumulates. Moreover, prolonged soiling of the skin is not only likely to cause loss of hair especial‐ ly when the matting is being removed after slurry dries on the skin, but it also inter‐ feres with normal health of the epidermis of the involved areas [54]. It is therefore important that slurry is removed from cattle houses at least once per day in order to

grazing units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

promote good animal welfare [28].

Flanks Excess slurry in cow house 80 <0.0001 Limbs Excess slurry in cow house 13.58 0.0035 Udder Excess slurry in cow house 16.06 <0.0011

the slurry.

#### **3.4. Other parameters indicating poor welfare**

Gross soiling with slurry on various areas of the bodies of the cows was observed in all the zero-grazing units evaluated in this study. In all the cows examined, all their limbs were soiled. The flanks and udder were soiled in 97% (n=297) and 90% (n=28) of the cows, respec‐ tively (Figure14). Soiling was an indicator of the management of the slurry in the zero-graz‐ ing unit, which means if the body is grossly dirty with raw or dried slurry, then possibly slurry is left to accumulate for long on the floor before being scrapped or washed off. Re‐ moval of slurry and cleaning of the cow housing floors was done at least once per day in 55% (n=44) of the units. For the remaining 45% (n=36) of the units, it was done only occa‐ sionally, either once a week or once every two weeks (Figure 15). The frequency of cleaning the slurry from the floor was significantly associated with soiling of flanks (χ<sup>2</sup> =80; p<0.0001), limbs (χ<sup>2</sup> =16.06; p<0.0011) and udder (χ<sup>2</sup> =13.58; p=0.0035) (Table 6).

**Figure 14.** Gross soiling of the whole limb area in Picture A, and the udder plus whole hind quarters in Picture B, with slurry accumulated in the unit facilities.

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi, Kenya http://dx.doi.org/10.5772/53678 77

could be attributed to the fact that concentrates supply the primary nutrient requirements to the cow as well as sufficient reserves needed for secondary processes such as normal lacta‐ tion [99,101], and increased milk production [18]. Therefore, concentrates are pertinent con‐ stituents of the dairy cow diet if the stress of both body maintenance and milk production

Regular mineral supplementation supplied in a majority of the zero-grazing units in this study is a reflection of good animal welfare practice, since minerals enhance animal growth, reproduction and health [99,101]. In this study, the importance of mineral supplementation was evidenced by the association between regular supplementation and fair body condition, while occasional or absence of mineral supplementation was associated with poor body con‐ dition. Irregular mineral supplementation like was found with irregular concentrate feeding destabilizes the body more than complete absence of minerals, hence affecting body condi‐ tion score that impacts negatively on the welfare of the cows. Findings from previous stud‐ ies indicate that absence or insufficient mineral supplementation impacts negatively on growth rate and reproduction, leading to anoestrus [101,103], and hence inevitably affecting

Gross soiling with slurry on various areas of the bodies of the cows was observed in all the zero-grazing units evaluated in this study. In all the cows examined, all their limbs were soiled. The flanks and udder were soiled in 97% (n=297) and 90% (n=28) of the cows, respec‐ tively (Figure14). Soiling was an indicator of the management of the slurry in the zero-graz‐ ing unit, which means if the body is grossly dirty with raw or dried slurry, then possibly slurry is left to accumulate for long on the floor before being scrapped or washed off. Re‐ moval of slurry and cleaning of the cow housing floors was done at least once per day in 55% (n=44) of the units. For the remaining 45% (n=36) of the units, it was done only occa‐ sionally, either once a week or once every two weeks (Figure 15). The frequency of cleaning

**Figure 14.** Gross soiling of the whole limb area in Picture A, and the udder plus whole hind quarters in Picture B, with

=13.58; p=0.0035) (Table 6).

=80; p<0.0001),

the slurry from the floor was significantly associated with soiling of flanks (χ<sup>2</sup>

has to be avoided.

76 Insights from Veterinary Medicine

animal welfare.

limbs (χ<sup>2</sup>

**3.4. Other parameters indicating poor welfare**

=16.06; p<0.0011) and udder (χ<sup>2</sup>

slurry accumulated in the unit facilities.

**Figure 15.** Slurry accumulated in animal facilities. Picture A shows slurry in an earthen floor. Picture B shows slurry in concrete floor. Picture C shows slurry and narrow walk alley. Picture D-Despite slurry accumulation on parks, the cows in this unit were clean because the cubicles had good clean bedding of sawdust, which meant the cows never lay on the slurry.


**Table 6.** Factors associated with soiling in various body parts of cows examined among the 80 smallholder zerograzing units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

Although more than half of the smallholder units in the current study had slurry re‐ moved frequently, the excessive slurry found in the rest of the units is likely to affect health of the claws and the udders of the cows. The holding of slurry in cow housing units for long without cleaning it out, exposes the claws to continuous wet environ‐ ment which softens the horny parts of the claws, predisposing them to development of lesions as previously reported [17,44]. Accumulated slurry also exposes udders to unhy‐ gienic conditions that predispose them to mastitis, particularly when the cows lie on it most of the time as observed previously [17,44]. The subsequent development of claw lameness and mastitis will cause pain and inevitably lead to poor welfare. Furthermore, this study was able to show that accumulation of slurry caused excessive soiling of the skin in some parts of the body mainly because most of the time the cows were found lying on it. This is supported by the strong statistical association between the presence of slurry and soiling of the hind quarters as well as the udder region. This may proba‐ bly be exacerbated by poor housing designs, which were reported previously as the main predisposing factor for soiling of these body regions [104] due to likelihood of these designs forcing the cows to lie on the walk alleys where slurry accumulates. Moreover, prolonged soiling of the skin is not only likely to cause loss of hair especial‐ ly when the matting is being removed after slurry dries on the skin, but it also inter‐ feres with normal health of the epidermis of the involved areas [54]. It is therefore important that slurry is removed from cattle houses at least once per day in order to promote good animal welfare [28].

None of the 80 zero-grazing units evaluated had proper milking parlour; instead the cows were milked in unsuitable improvised cubicles with protruding traumatic pieces of wood or nails in 76% (n=61) of the units, or in their sleeping cubicles in the remaining 24% (n=19) units (Figure 16). Only 12% (n=10) of the zero-grazing units had maternity stalls into which pregnant cows were transferred in the last few days prior to parturition. In the remaining 88% (n=70) of the units, cows calved in their resting cubicles (Figure 17) and in the walk al‐ leys. Lack of maternity areas constituted a poor welfare risk factor, which exposed the cow and her newborn calf to trauma by the rest of the cows in the unit. Moreover, the level of hygiene within the cattle unit is reduced due to spread of bloody fetal fluids and placental remnants [28].

**3.5. Farmers and stockmen perspective on animal welfare**

Farmers and stockmen acknowledged the need for cattle to have ready access to feed and to water in 98.75% (n=79) and 88.75% (n=71) of the zero-grazing units, respectively. In 47.5% (n=38) of the units evaluated, farmers and stockmen supported the need for alleviating un‐ necessary pain and suffering of the cattle as well as providing prompt medical attention when needed. In 31.25% (n=25) of the units, they also shared the opinion that animals suffer when mistreated and that there was the need to protect them from conditions that expose them to distress. The need for provision of a shelter and good housing systems to avoid ani‐ mal discomfort and physical stress was acknowledged by farmers in 28.75% (n=23) of the units evaluated. The need to provide sufficient housing space with adequate facilities so as to allow for expression of normal behavioral patterns of animals was acknowledged by the farmers and stockmen in 5% (n=4) of the zero-grazing units evaluated in this study (Table 7).

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

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http://dx.doi.org/10.5772/53678

**Welfare input Positive response (%) Negative response (%)**

Feed at all times 98.75 1.25

Water at all times 88.75 11.25

Medical attention when required 47.50 52.50

Appropriate treatment / handling 31.25 68.75

Comfortable housing 28.75 71.25

Adequate space for movement 5.00 95.00

and comfort sustaining needs [6,105].

**Table 7.** Percentages of responses from farmers and stockmen on their perspective of animal welfare issues in the 80 smallholder zero-grazing dairy units evaluated for the welfare of dairy cattle in peri-urban areas of Nairobi, Kenya.

The farmers and stockmen were found to have poor human-animal interaction, as exempli‐ fied by shouting and whipping of the cows particularly during milking times. Such interac‐ tions caused fear that made the cattle aggressive, leading to agonistic behavior towards making them difficult to handle contrary to good animal welfare recommendations [28]. Nevertheless, the few farmers who supported the need for alleviation of animal pain and suffering as well as provision for animal comfort were found to be better informed on other factors that also contribute to the improvement of production. Generally, the farmers and stockmen interviewed in the current study seemed to have the attitude that animal suffering and its alleviation were not important and that animal comfort was absolutely unnecessary. In spite of these attitudes, the farmers' and stockmen's perspective of animal welfare mat‐ ters tend to agree with the understanding that animal welfare is affected by a hierarchy of needs whose importance is classified in priority order as life sustaining, health sustaining

**Figure 16.** A cow being milked inside an improvised enclosure with a stack of firewood on the left, which poses a risk of injury to the cow when the animal struggles. The firewood can also slide off toward the cow from the top.

**Figure 17.** A cow calving in a lying position inside the sleeping cubicle. The cow is stuck in this position with her hind limbs having slipped under the lower side-bar (arrow), which further predisposed them to risk of injury. The head was also at risk of injury from being squeezed toward the inside wall.

#### **3.5. Farmers and stockmen perspective on animal welfare**

None of the 80 zero-grazing units evaluated had proper milking parlour; instead the cows were milked in unsuitable improvised cubicles with protruding traumatic pieces of wood or nails in 76% (n=61) of the units, or in their sleeping cubicles in the remaining 24% (n=19) units (Figure 16). Only 12% (n=10) of the zero-grazing units had maternity stalls into which pregnant cows were transferred in the last few days prior to parturition. In the remaining 88% (n=70) of the units, cows calved in their resting cubicles (Figure 17) and in the walk al‐ leys. Lack of maternity areas constituted a poor welfare risk factor, which exposed the cow and her newborn calf to trauma by the rest of the cows in the unit. Moreover, the level of hygiene within the cattle unit is reduced due to spread of bloody fetal fluids and placental

**Figure 16.** A cow being milked inside an improvised enclosure with a stack of firewood on the left, which poses a risk of injury to the cow when the animal struggles. The firewood can also slide off toward the cow from the top.

**Figure 17.** A cow calving in a lying position inside the sleeping cubicle. The cow is stuck in this position with her hind limbs having slipped under the lower side-bar (arrow), which further predisposed them to risk of injury. The head was

also at risk of injury from being squeezed toward the inside wall.

remnants [28].

78 Insights from Veterinary Medicine

Farmers and stockmen acknowledged the need for cattle to have ready access to feed and to water in 98.75% (n=79) and 88.75% (n=71) of the zero-grazing units, respectively. In 47.5% (n=38) of the units evaluated, farmers and stockmen supported the need for alleviating un‐ necessary pain and suffering of the cattle as well as providing prompt medical attention when needed. In 31.25% (n=25) of the units, they also shared the opinion that animals suffer when mistreated and that there was the need to protect them from conditions that expose them to distress. The need for provision of a shelter and good housing systems to avoid ani‐ mal discomfort and physical stress was acknowledged by farmers in 28.75% (n=23) of the units evaluated. The need to provide sufficient housing space with adequate facilities so as to allow for expression of normal behavioral patterns of animals was acknowledged by the farmers and stockmen in 5% (n=4) of the zero-grazing units evaluated in this study (Table 7).


**Table 7.** Percentages of responses from farmers and stockmen on their perspective of animal welfare issues in the 80 smallholder zero-grazing dairy units evaluated for the welfare of dairy cattle in peri-urban areas of Nairobi, Kenya.

The farmers and stockmen were found to have poor human-animal interaction, as exempli‐ fied by shouting and whipping of the cows particularly during milking times. Such interac‐ tions caused fear that made the cattle aggressive, leading to agonistic behavior towards making them difficult to handle contrary to good animal welfare recommendations [28]. Nevertheless, the few farmers who supported the need for alleviation of animal pain and suffering as well as provision for animal comfort were found to be better informed on other factors that also contribute to the improvement of production. Generally, the farmers and stockmen interviewed in the current study seemed to have the attitude that animal suffering and its alleviation were not important and that animal comfort was absolutely unnecessary. In spite of these attitudes, the farmers' and stockmen's perspective of animal welfare mat‐ ters tend to agree with the understanding that animal welfare is affected by a hierarchy of needs whose importance is classified in priority order as life sustaining, health sustaining and comfort sustaining needs [6,105].

#### **3.6. Mixing cattle of different age groups and with other species**

In 74% (n=59) of the zero-grazing units evaluated in this study, cattle were housed separate‐ ly according to their age groups, while for 26% (n=21) of the units the different groups were non-existent, with animals indiscriminately mixed irrespective of their age and lactation stage. Only male calves were separated from the rest of the cattle in all farms. In only 18% (n=14) of the studied units other species of animals such as poultry, pigs, sheep and goats were reared separately, although their houses were attached to the cattle housing facilities. However, in some of the units, chicken house was on top of the cattle house (Table 8).

**Author details**

Nairobi, Kenya

**References**

526.

Ethics 1993; 6 15-25.

ingford, UK 1998; p 152.

ternational, Oxon 1997; p437.

James Nguhiu-Mwangi\*

\*Address all correspondence to: nguhiuja@yahoo.com

\*Address all correspondence to: jamesnguhiumwangi@gmail.com

PhD Thesis. Wye College, University of London; 1999.

quences. Canadian Veterinary Journal 2003; 44 496-499.

ment. Acta Agriculturae Scandinavica 1996; 27 22-28.

Italian.Journal of.Animal Science 2009; 8 (Suppl. 1) 45-66.

Italian Journal of Animal Science 2009; 8 21-30.

and Environmental Ethics 1993; 6 37-43.

, Joshua W. Aleri, Eddy G. M. Mogoa and Peter M. F. Mbithi

Kenya

81

http://dx.doi.org/10.5772/53678

Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi,

Indicators of Poor Welfare in Dairy Cows Within Smallholder Zero-Grazing Units in the Peri-Urban Areas of Nairobi,

[1] Broom D M. Definition of animal welfare. Journal of Agricultural and Environmental

[2] Hemsworth P H., Coleman, G.J. Human Livestock Interactions. The stockperson and productivity and welfare of intensively farmed animals. In: CAB International, Wall‐

[3] Fregonesi J A.. Production and behaviour of dairy cattle in different housing systems.

[4] Broom D M. Indicators of poor welfare. British Veterinary Journal 1986; 142 524 –

[5] Fraser D., Broom D M. Farm animal behaviour and welfare. 3rd edition. In: CAB In‐

[6] Duncan I J H. Science-based assessment of animal welfare. Farm animals. Scientific

[7] Hewson C J. What is animal welfare? Common definitions and their practical conse‐

[8] Carenzi C., Verga M. Animal welfare review of the scientific concept and definition.

[9] Broom D M. Animal welfare defined in terms of attempt to cope with the environ‐

[10] Gonyou H W. Animal welfare: Definitions and assessment. Journal of Agricultural

[11] Abeni F., Bertoni G. Main causes of poor welfare in intensively reared dairy cows.

and Technical Review of Office International Epizooties 2005; 24 483-492.


**Table 8.** Animal mixing practices in the 80 smallholder zero-grazing units evaluated for welfare of dairy cattle in the peri-urban areas of Nairobi, Kenya.

Although only a small percentage (26%; n= 21) of the smallholder units in this study housed cattle of different age groups together, it still creates conditions that would en‐ hance development of negative social interactions. Such interactions create fear and dis‐ rupt feeding for the subordinate cattle. Eventually, this will inevitably affect health and productivity of the animals negatively [3,101], and subsequently lead to increased stress and poor animal welfare. In the units that had poultry production in rooms on an up‐ per floor above cow houses, particularly separated by timber from the cows below, the cow houses are likely to have accumulations of ammonia from the chicken waste (fae‐ ces). This will exacerbate effects of poor ventilation. In the rest of the cattle units that were the majority (74%; n=59), cattle were housed according to their appropriate age groups according to the universal recommendations [28,31].

## **4. Conclusions and recommendations**

The studies presented herein conclude that poor cattle welfare exist in the Kenyan smallholder zero-grazing units, and were able to identify some of the factors responsible for it, through di‐ rect indicators in the animals such as lameness lesions, body surface injuries, body condition scores and soiling of the body with slurry. The main factors resulting in poor welfare of dairy cows in the zero-grazing units within the peripheral areas of Nairobi include substandard housing designs, cattle housing in poor state, suboptimal feeding and poor husbandry practi‐ ces. In this work, physical and environmental parameters that can be used to assess the welfare level in these zero-grazing units were discussed. The farmer perceptions and ignorance on ani‐ mal welfare issues additionally precipitates cattle poor welfare.

## **Author details**

**3.6. Mixing cattle of different age groups and with other species**

In 74% (n=59) of the zero-grazing units evaluated in this study, cattle were housed separate‐ ly according to their age groups, while for 26% (n=21) of the units the different groups were non-existent, with animals indiscriminately mixed irrespective of their age and lactation stage. Only male calves were separated from the rest of the cattle in all farms. In only 18% (n=14) of the studied units other species of animals such as poultry, pigs, sheep and goats were reared separately, although their houses were attached to the cattle housing facilities. However, in some of the units, chicken house was on top of the cattle house (Table 8).

**Mixing practice Number of zero-grazing units Percentage of zero-grazing units**

**Table 8.** Animal mixing practices in the 80 smallholder zero-grazing units evaluated for welfare of dairy cattle in the

Although only a small percentage (26%; n= 21) of the smallholder units in this study housed cattle of different age groups together, it still creates conditions that would en‐ hance development of negative social interactions. Such interactions create fear and dis‐ rupt feeding for the subordinate cattle. Eventually, this will inevitably affect health and productivity of the animals negatively [3,101], and subsequently lead to increased stress and poor animal welfare. In the units that had poultry production in rooms on an up‐ per floor above cow houses, particularly separated by timber from the cows below, the cow houses are likely to have accumulations of ammonia from the chicken waste (fae‐ ces). This will exacerbate effects of poor ventilation. In the rest of the cattle units that were the majority (74%; n=59), cattle were housed according to their appropriate age

The studies presented herein conclude that poor cattle welfare exist in the Kenyan smallholder zero-grazing units, and were able to identify some of the factors responsible for it, through di‐ rect indicators in the animals such as lameness lesions, body surface injuries, body condition scores and soiling of the body with slurry. The main factors resulting in poor welfare of dairy cows in the zero-grazing units within the peripheral areas of Nairobi include substandard housing designs, cattle housing in poor state, suboptimal feeding and poor husbandry practi‐ ces. In this work, physical and environmental parameters that can be used to assess the welfare level in these zero-grazing units were discussed. The farmer perceptions and ignorance on ani‐

Separate age groups 59 74 Mixing of age groups 21 26 Only cattle reared 66 82 Cattle and other species 14 18

groups according to the universal recommendations [28,31].

mal welfare issues additionally precipitates cattle poor welfare.

**4. Conclusions and recommendations**

peri-urban areas of Nairobi, Kenya.

80 Insights from Veterinary Medicine

James Nguhiu-Mwangi\* , Joshua W. Aleri, Eddy G. M. Mogoa and Peter M. F. Mbithi

\*Address all correspondence to: nguhiuja@yahoo.com

\*Address all correspondence to: jamesnguhiumwangi@gmail.com

Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, Nairobi, Kenya

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**Section 2**

**Current Topics in Infectious Diseases**


**Current Topics in Infectious Diseases**

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**Chapter 4**

*Mycobacterium avium* **Complex in Domestic and Wild**

Mycobacteria from the *Mycobacterium avium* complex (MAC) cause a variety of diseases in‐ cluding tuberculosis-like disease in humans and birds, disseminated infections in AIDS pa‐ tients and otherwise immunocompromised patients, lymphadenitis in humans and mammals and paratuberculosis in ruminants. *M. avium* subsp. *paratuberculosis* (*Map*) is the etiologic agent of Johne´s disease in cattle and it has been identified in human patients with Crohn's disease. The MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various do‐

The aim of this study was to discuss the classification and biology, epidemiology, clinical signs, pathology, diagnostic techniques, and public health concerns in *Mycobacterium avium*

The phylum Actinobacteria is large and very complex; it contains one class (Actinobacteria), five subclasses, six orders, 14 suborders, and 40 families. The orders, suborders, and families are defined based on 16S rRNA sequences and distinctive signature nucleotides. The subor‐ der Corynebacterineae contains seven families with several well-known genera. Three of the

> © 2013 Coelho et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Coelho et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**2. Classification and biology of** *Mycobacterium avium* **complex**

most important genera are *Corynebacterium, Mycobacterium,* and *Nocardia* [2].

Ana Cláudia Coelho, Maria de Lurdes Pinto,

Additional information is available at the end of the chapter

Ana Matos, Manuela Matos and

mestic and wild mammals and birds [1].

complex in domestic and wild animals.

Maria dos Anjos Pires

http://dx.doi.org/10.5772/54323

**1. Introduction**

**Animals**

## *Mycobacterium avium* **Complex in Domestic and Wild Animals**

Ana Cláudia Coelho, Maria de Lurdes Pinto, Ana Matos, Manuela Matos and Maria dos Anjos Pires

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54323

## **1. Introduction**

Mycobacteria from the *Mycobacterium avium* complex (MAC) cause a variety of diseases in‐ cluding tuberculosis-like disease in humans and birds, disseminated infections in AIDS pa‐ tients and otherwise immunocompromised patients, lymphadenitis in humans and mammals and paratuberculosis in ruminants. *M. avium* subsp. *paratuberculosis* (*Map*) is the etiologic agent of Johne´s disease in cattle and it has been identified in human patients with Crohn's disease. The MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various do‐ mestic and wild mammals and birds [1].

The aim of this study was to discuss the classification and biology, epidemiology, clinical signs, pathology, diagnostic techniques, and public health concerns in *Mycobacterium avium* complex in domestic and wild animals.

## **2. Classification and biology of** *Mycobacterium avium* **complex**

The phylum Actinobacteria is large and very complex; it contains one class (Actinobacteria), five subclasses, six orders, 14 suborders, and 40 families. The orders, suborders, and families are defined based on 16S rRNA sequences and distinctive signature nucleotides. The subor‐ der Corynebacterineae contains seven families with several well-known genera. Three of the most important genera are *Corynebacterium, Mycobacterium,* and *Nocardia* [2].

© 2013 Coelho et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Coelho et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The species of *Mycobacterium*, sole genus of the family Mycobacteriaceae, is composed of a group of high genomic C+G content (~61 to 71%), facultative intracellular, Gram-positive microorganisms comprising more than 130 established and validated species and subspecies [3], with surprisingly diverse phenotypes related to growth rate, metabolic activity, colony appearance, environmental distribution, and pathogenic potential for eukaryotic hosts [4]. Although most of these species are saprophytic, important human and animal pathogens have been identified. Pathogenic members are usually characterized by their slow growth in culture, with generation times of 12 to 24 h, and must be incubated for 2 to 40 days after inoculation of a solidified complex medium to form a visible colony, whereas nonpathogen‐ ic members grow considerably faster [5]. Mycobacteria are acid-fast bacilli, acidophilic, small, slightly curved or straight rods that sometimes branch or form filaments. Mycobacte‐ rial filaments differ from those of actinomycetes in readily fragmenting into rods and coc‐ coid bodies when distributed. They are aerobic, immobile, non-sporulated and catalase positive bacteria. Their cell wall is lipid-rich and contain waxes with 60 to 90 carbon mycolic acids, which are complex fatty acids with a hydroxyl group on the β-carbon and an aliphatic chain attached to the α-carbon. The presence of mycolic acids and other lipids, in high con‐ centration outside the peptidoglycan, makes mycobacteria acid-fast dye resistant (basic fuchsin cannot be removed from the cell by acid alcohol treatment), as well as resistant to immune system defense mechanisms and disinfectants [2,6].

with pulmonary disease. On the basis of genotypic, phenotypic and growth characteristics, biochemical tests and historical reasons, multiple subspecies of *Mycobacterium avium* are rec‐ ognized. These include the subsp*. avium*, subsp. *paratuberculosis*, subsp. *hominissuis* and subsp. *silvaticum* [3]. All four *Mycobacterium avium* subspecies and *Mycobacterium intracellu‐ lare* are capable of infecting a diverse range of host and possess a high degree of genetic sim‐ ilarity [17]. Contemporary methods for MAC identification, e.g., high performance liquid chromatography (HPLC) of cell wall mycolic acids and genetic probes based on rRNA tar‐ gets, e.g. AccuProbe, cannot discriminate among *Mycobacterium avium* subspecies. Given the differences in pathogenicity among *Mycobacterium avium* subspecies and the implications re‐ garding the infection source, a practical and accurate method of simply identifying *Mycobac‐*

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93

Before establishing the *Mycobacterium avium* subsp. *avium (Maa)* designation, this bacterium was simply referred to as *Mycobacterium avium* and has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds as well as in a variety of fowl, game birds and water-fowl. The most common route of infection for susceptible animals is the alimentary tract. Respiratory tract is also suggested as a potential source of infection [19].

*Mycobacterium avium* subsp. *paratuberculosis (Map)* is the etiologic agent of Johne's disease or paratuberculosis, a chronic granulomatous enteritis of ruminant livestock and wildlife, with worldwide distribution having a significant impact on the world economy [5]. For veterina‐ ry medicine, *Map* is the MAC member of greatest importance, and is capable of infecting and causing disease in a wide array of animal species, including nonhuman primates, with‐ out the need for co-existent immunosuppressive infections [18]. *Map* is one of the slowest growing mycobacterial species, hence primary isolation from specimen, requires prolonged culture incubation and can take several months. Unlike most other *Mycobacterium avium* sub‐ species, isolation of *Map* requires the addition of the siderophore mycobactin to culture me‐ dia [20]. From phenotypic analysis, the *Map* group has been subdivided into two main types, bovine and ovine, that vary in hosts, diseases caused, and growth phenotypes [21]*.* Genotypically, these findings were based primarily on comparisons of the integration *loci* of the IS*900* insertion sequence (IS) and used polymorphisms in IS*1311* to separate sheep and

MAC isolates of genotypes IS*901*- and IS*1245*+ and serotypes 4 to 6, 8 to 11 and 21 are less virulent for birds and are designated *M. avium* subsp. *hominissuis* (*Mah*)*. Mah* was proposed to distinguish organisms found in humans and pigs from those isolated from birds [3]. Those are genomically diverse, the more diverse group of strains, low-virulence, opportun‐ istic pathogens for both animals and humans [18]. Considered ubiquitous in the environ‐ ment (the most likely source of infection for humans), *Mah* can cause serious systemic

*terium avium* subspecies is needed [18].

**2.2.** *Mycobacterium avium* **subsp.** *avium*

**2.3.** *Mycobacterium avium* **subsp.** *paratuberculosis*

cattle isolates into separate populations [22].

**2.4.** *Mycobacterium avium* **subsp.** *hominissuis*

#### **2.1.** *Mycobacterium avium* **complex (MAC)**

Bacteria from the *Mycobacterium avium* complex (MAC) differ in virulence and ecology, and are the most frequently isolated non-tuberculous mycobacteria [7]. *Mycobacterium* members of MAC have the capacity to survive and multiply under a wide range of environmental conditions, including low pH, extreme temperatures, chlorine or ozone treatment and low oxygen level. Thus, plus their ability to utilize many substances as nutrients, enables them to grow successfully in many biotopes [1]. The environmental sources responsible for MAC in‐ fection in different populations, the specific routes of infection and transmission, the poten‐ tial for latent infection and reactivation of disease are not yet well defined [4,8]. Ingestion of environmental organisms followed by invasion through the gastrointestinal tract has been suggested as the main route of infection because the organisms are frequently isolated from stools of different animals. There is also an important positive correlation between the pres‐ ence of MAC in respiratory samples and the subsequent development of disseminated dis‐ ease [9]. Traditionally, MAC includes two species, *Mycobacterium avium* and *Mycobacterium intracellulare* [3]. Recently, advances in molecular taxonomy have fuelled identification of novel species within the MAC, including the *Mycobacterium chimaera* incorporating sequevar MAC-A organisms isolated from humans with pulmonary cavitations, pulmonary abscess, chronic obstructive pulmonary disease and bronchiectasis [10]; the *Mycobacterium colom‐ biense* incorporating sequevar MAC-X organisms isolated from the blood and sputum of HIV infected patients in Colombia [11], and from diseased lymph nodes in children [12,13]; the *Mycobacterium arosiense,* recently described in an immunocompromised child with disse‐ minated osteomyelitic lesions [14]; the *Mycobacterium vulneris* [15], *Mycobacterium marseil‐ lense, Mycobacterium timonense* and *Mycobacterium bouchedurhonense* isolated from patients with pulmonary disease. On the basis of genotypic, phenotypic and growth characteristics, biochemical tests and historical reasons, multiple subspecies of *Mycobacterium avium* are rec‐ ognized. These include the subsp*. avium*, subsp. *paratuberculosis*, subsp. *hominissuis* and subsp. *silvaticum* [3]. All four *Mycobacterium avium* subspecies and *Mycobacterium intracellu‐ lare* are capable of infecting a diverse range of host and possess a high degree of genetic sim‐ ilarity [17]. Contemporary methods for MAC identification, e.g., high performance liquid chromatography (HPLC) of cell wall mycolic acids and genetic probes based on rRNA tar‐ gets, e.g. AccuProbe, cannot discriminate among *Mycobacterium avium* subspecies. Given the differences in pathogenicity among *Mycobacterium avium* subspecies and the implications re‐ garding the infection source, a practical and accurate method of simply identifying *Mycobac‐ terium avium* subspecies is needed [18].

#### **2.2.** *Mycobacterium avium* **subsp.** *avium*

The species of *Mycobacterium*, sole genus of the family Mycobacteriaceae, is composed of a group of high genomic C+G content (~61 to 71%), facultative intracellular, Gram-positive microorganisms comprising more than 130 established and validated species and subspecies [3], with surprisingly diverse phenotypes related to growth rate, metabolic activity, colony appearance, environmental distribution, and pathogenic potential for eukaryotic hosts [4]. Although most of these species are saprophytic, important human and animal pathogens have been identified. Pathogenic members are usually characterized by their slow growth in culture, with generation times of 12 to 24 h, and must be incubated for 2 to 40 days after inoculation of a solidified complex medium to form a visible colony, whereas nonpathogen‐ ic members grow considerably faster [5]. Mycobacteria are acid-fast bacilli, acidophilic, small, slightly curved or straight rods that sometimes branch or form filaments. Mycobacte‐ rial filaments differ from those of actinomycetes in readily fragmenting into rods and coc‐ coid bodies when distributed. They are aerobic, immobile, non-sporulated and catalase positive bacteria. Their cell wall is lipid-rich and contain waxes with 60 to 90 carbon mycolic acids, which are complex fatty acids with a hydroxyl group on the β-carbon and an aliphatic chain attached to the α-carbon. The presence of mycolic acids and other lipids, in high con‐ centration outside the peptidoglycan, makes mycobacteria acid-fast dye resistant (basic fuchsin cannot be removed from the cell by acid alcohol treatment), as well as resistant to

Bacteria from the *Mycobacterium avium* complex (MAC) differ in virulence and ecology, and are the most frequently isolated non-tuberculous mycobacteria [7]. *Mycobacterium* members of MAC have the capacity to survive and multiply under a wide range of environmental conditions, including low pH, extreme temperatures, chlorine or ozone treatment and low oxygen level. Thus, plus their ability to utilize many substances as nutrients, enables them to grow successfully in many biotopes [1]. The environmental sources responsible for MAC in‐ fection in different populations, the specific routes of infection and transmission, the poten‐ tial for latent infection and reactivation of disease are not yet well defined [4,8]. Ingestion of environmental organisms followed by invasion through the gastrointestinal tract has been suggested as the main route of infection because the organisms are frequently isolated from stools of different animals. There is also an important positive correlation between the pres‐ ence of MAC in respiratory samples and the subsequent development of disseminated dis‐ ease [9]. Traditionally, MAC includes two species, *Mycobacterium avium* and *Mycobacterium intracellulare* [3]. Recently, advances in molecular taxonomy have fuelled identification of novel species within the MAC, including the *Mycobacterium chimaera* incorporating sequevar MAC-A organisms isolated from humans with pulmonary cavitations, pulmonary abscess, chronic obstructive pulmonary disease and bronchiectasis [10]; the *Mycobacterium colom‐ biense* incorporating sequevar MAC-X organisms isolated from the blood and sputum of HIV infected patients in Colombia [11], and from diseased lymph nodes in children [12,13]; the *Mycobacterium arosiense,* recently described in an immunocompromised child with disse‐ minated osteomyelitic lesions [14]; the *Mycobacterium vulneris* [15], *Mycobacterium marseil‐ lense, Mycobacterium timonense* and *Mycobacterium bouchedurhonense* isolated from patients

immune system defense mechanisms and disinfectants [2,6].

**2.1.** *Mycobacterium avium* **complex (MAC)**

92 Insights from Veterinary Medicine

Before establishing the *Mycobacterium avium* subsp. *avium (Maa)* designation, this bacterium was simply referred to as *Mycobacterium avium* and has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds as well as in a variety of fowl, game birds and water-fowl. The most common route of infection for susceptible animals is the alimentary tract. Respiratory tract is also suggested as a potential source of infection [19].

#### **2.3.** *Mycobacterium avium* **subsp.** *paratuberculosis*

*Mycobacterium avium* subsp. *paratuberculosis (Map)* is the etiologic agent of Johne's disease or paratuberculosis, a chronic granulomatous enteritis of ruminant livestock and wildlife, with worldwide distribution having a significant impact on the world economy [5]. For veterina‐ ry medicine, *Map* is the MAC member of greatest importance, and is capable of infecting and causing disease in a wide array of animal species, including nonhuman primates, with‐ out the need for co-existent immunosuppressive infections [18]. *Map* is one of the slowest growing mycobacterial species, hence primary isolation from specimen, requires prolonged culture incubation and can take several months. Unlike most other *Mycobacterium avium* sub‐ species, isolation of *Map* requires the addition of the siderophore mycobactin to culture me‐ dia [20]. From phenotypic analysis, the *Map* group has been subdivided into two main types, bovine and ovine, that vary in hosts, diseases caused, and growth phenotypes [21]*.* Genotypically, these findings were based primarily on comparisons of the integration *loci* of the IS*900* insertion sequence (IS) and used polymorphisms in IS*1311* to separate sheep and cattle isolates into separate populations [22].

#### **2.4.** *Mycobacterium avium* **subsp.** *hominissuis*

MAC isolates of genotypes IS*901*- and IS*1245*+ and serotypes 4 to 6, 8 to 11 and 21 are less virulent for birds and are designated *M. avium* subsp. *hominissuis* (*Mah*)*. Mah* was proposed to distinguish organisms found in humans and pigs from those isolated from birds [3]. Those are genomically diverse, the more diverse group of strains, low-virulence, opportun‐ istic pathogens for both animals and humans [18]. Considered ubiquitous in the environ‐ ment (the most likely source of infection for humans), *Mah* can cause serious systemic infection in immunocompromised patients, such as those infected with HIV. Additionally, this opportunistic pathogen can cause cervical lymphadenitis in children with cystic fibrosis, and lung infections in patients with underlying lung disease [23]. Domestic water distribu‐ tion systems have been reported as possible sources of *Mah* infections in hospitals, family houses, and commercial places [24]. In animals, *Mah* is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine documented at slaughter [18], and can also lead to systemic infection of parenchymatous organs [23]. *Mah* were recovered from affected lymph nodes of red deer from Austria [25].

In cattle this disease is scored in four stages according to its evolution and symptoms, two of them evolving sub-clinically. Stage I, or silent infection, is the most observed in young ani‐ mals, without significant clinical signs and only in *post mortem* evaluation it is possible to

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Stage II remains a subclinical disease, being observed in adult animals. It may be detected by alterations in immunological serological and/or cellular parameters. Intermittently, fecal

In stage III the clinical signs can be observed, occurring after several years of incubation. The initial clinical signs are subtle with gradual weight loss despite normal appetite, intermittent diarrhea along several weeks, drop in milk production and roughness of hair coat. These symptoms are included in the differential diagnosis of multiple diseases, so it is often mis‐ diagnosed [34]. Usually, animals in this stage are positive upon ELISA and other serological tests, as for histopathological analysis of lesions, which are common in the terminal ileum

The last stage of the disease (stage IV) comprises animals that rapidly progress from the stage III with rapid condition deteriorated. They became increasingly lethargic, weak and emaciated and present intermandibular edema due to hypoproteinemia. In this stage, the culture of the agent, molecular biology techniques of PCR, ELISA, serology and histopathol‐ ogy (Figure 1), all are positive for the majority of animals tested. The gastrointestinal tract is the preferential local to sample in order to isolate the agent, but in some conditions it can even be present in extraintestinal lesions, with the liver and lymph nodes being the most

**Figure 1.** Morphological aspects of paratuberculosis lesions in the ileum of sheep. **A**- Thickening of the mucosa by an inflammatory infiltrate composed by epithelioid cells, macrophages and some multinucleated giant cells. (H&E stain, Bar=50μm) **B**- Acid-acohol resistant bacilli within the macrophages and epithelioid cells that infiltrate the mucosa. No‐ tice the abundant number of mycobacteria that are visible at moderate amplification (Ziehl-Neelsen, Bar=30μm)

culture and histopathology analysis of these animals could be positive to *Map* [33].

identify the agent by culture or histopathology analysis [33].

[35] (Figure 2A).

common sites [33].

#### **2.5.** *Mycobacterium avium* **subsp.** *silvaticum*

*Mycobacterium avium* subsp. *silvaticum* applies to the previously named wood pigeon bacil‐ lus, an acid-fast organism causing tuberculosis-like lesions in these wood pigeons. The in‐ ability to grow on egg media, the stimulation of growth by pyruvate and at pH 5.5 and their mycobactin dependency upon primary isolation, gradually losing this phenotype upon sub‐ culture, have been described as characteristics of *Mycobacterium avium* subsp. *silvaticum* [17].

#### **2.6.** *Mycobacterium intracellulare*

*Mycobacterium intracellulare*, initially named *Nocardia intracellularis,* is an environmental organ‐ ism and opportunistic pathogen, isolated from a variety of animal hosts and environmental sources. *Mycobacterium intracellulare* is a closely related pathogen of birds with a lower preva‐ lence [26]. In general, it has been subject to less study than *Mycobacterium avium*, as the latter is more prevalent in clinical and environmental samples, has a wider apparent host range, and contributes almost exclusively to disseminated MAC disease in human immunodeficiency vi‐ rus patients [3]. The type strain of *Mycobacterium intracellulare* (ATCC 13950) was isolated from a human, specifically responsible for enlarged lymph nodes in children, who died from disse‐ minated disease [3], and progressive pulmonary disease in elderly women [27]. *Mycobacterium intracellulare* appears to have a distinct ecological niche, more prevalent in biofilms and at sig‐ nificantly higher CFU numbers than *Mycobacterium avium* [28].

## **3. Clinical signs and morphology in domestic and wild species**

All ruminant species, captive or free-ranging, are susceptible to disease and death due to MAC infection [29], and a wide diversity of non-ruminant species can become infected with mycobateria belonging to MAC, especially with *Map* and *Maa*. Paratuberculosis has been de‐ scribed in cattle, small ruminants, deer, and in South American camelids (llamas and alpa‐ cas) [30,31]. This chronic disease is one of the most serious affecting dairy cattle worldwide showing symptoms of an insidious intestinal pathology responsible for significant economic losses [5]. The close relationship between wild, captive and domestic ruminants and other species like birds is, nowadays, clinically relevant as the wild population could act as reser‐ voir for this agent [32].

In cattle this disease is scored in four stages according to its evolution and symptoms, two of them evolving sub-clinically. Stage I, or silent infection, is the most observed in young ani‐ mals, without significant clinical signs and only in *post mortem* evaluation it is possible to identify the agent by culture or histopathology analysis [33].

infection in immunocompromised patients, such as those infected with HIV. Additionally, this opportunistic pathogen can cause cervical lymphadenitis in children with cystic fibrosis, and lung infections in patients with underlying lung disease [23]. Domestic water distribu‐ tion systems have been reported as possible sources of *Mah* infections in hospitals, family houses, and commercial places [24]. In animals, *Mah* is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine documented at slaughter [18], and can also lead to systemic infection of parenchymatous organs [23]. *Mah* were recovered from affected

*Mycobacterium avium* subsp. *silvaticum* applies to the previously named wood pigeon bacil‐ lus, an acid-fast organism causing tuberculosis-like lesions in these wood pigeons. The in‐ ability to grow on egg media, the stimulation of growth by pyruvate and at pH 5.5 and their mycobactin dependency upon primary isolation, gradually losing this phenotype upon sub‐ culture, have been described as characteristics of *Mycobacterium avium* subsp. *silvaticum* [17].

*Mycobacterium intracellulare*, initially named *Nocardia intracellularis,* is an environmental organ‐ ism and opportunistic pathogen, isolated from a variety of animal hosts and environmental sources. *Mycobacterium intracellulare* is a closely related pathogen of birds with a lower preva‐ lence [26]. In general, it has been subject to less study than *Mycobacterium avium*, as the latter is more prevalent in clinical and environmental samples, has a wider apparent host range, and contributes almost exclusively to disseminated MAC disease in human immunodeficiency vi‐ rus patients [3]. The type strain of *Mycobacterium intracellulare* (ATCC 13950) was isolated from a human, specifically responsible for enlarged lymph nodes in children, who died from disse‐ minated disease [3], and progressive pulmonary disease in elderly women [27]. *Mycobacterium intracellulare* appears to have a distinct ecological niche, more prevalent in biofilms and at sig‐

lymph nodes of red deer from Austria [25].

94 Insights from Veterinary Medicine

**2.5.** *Mycobacterium avium* **subsp.** *silvaticum*

nificantly higher CFU numbers than *Mycobacterium avium* [28].

**3. Clinical signs and morphology in domestic and wild species**

All ruminant species, captive or free-ranging, are susceptible to disease and death due to MAC infection [29], and a wide diversity of non-ruminant species can become infected with mycobateria belonging to MAC, especially with *Map* and *Maa*. Paratuberculosis has been de‐ scribed in cattle, small ruminants, deer, and in South American camelids (llamas and alpa‐ cas) [30,31]. This chronic disease is one of the most serious affecting dairy cattle worldwide showing symptoms of an insidious intestinal pathology responsible for significant economic losses [5]. The close relationship between wild, captive and domestic ruminants and other species like birds is, nowadays, clinically relevant as the wild population could act as reser‐

**2.6.** *Mycobacterium intracellulare*

voir for this agent [32].

Stage II remains a subclinical disease, being observed in adult animals. It may be detected by alterations in immunological serological and/or cellular parameters. Intermittently, fecal culture and histopathology analysis of these animals could be positive to *Map* [33].

In stage III the clinical signs can be observed, occurring after several years of incubation. The initial clinical signs are subtle with gradual weight loss despite normal appetite, intermittent diarrhea along several weeks, drop in milk production and roughness of hair coat. These symptoms are included in the differential diagnosis of multiple diseases, so it is often mis‐ diagnosed [34]. Usually, animals in this stage are positive upon ELISA and other serological tests, as for histopathological analysis of lesions, which are common in the terminal ileum [35] (Figure 2A).

The last stage of the disease (stage IV) comprises animals that rapidly progress from the stage III with rapid condition deteriorated. They became increasingly lethargic, weak and emaciated and present intermandibular edema due to hypoproteinemia. In this stage, the culture of the agent, molecular biology techniques of PCR, ELISA, serology and histopathol‐ ogy (Figure 1), all are positive for the majority of animals tested. The gastrointestinal tract is the preferential local to sample in order to isolate the agent, but in some conditions it can even be present in extraintestinal lesions, with the liver and lymph nodes being the most common sites [33].

**Figure 1.** Morphological aspects of paratuberculosis lesions in the ileum of sheep. **A**- Thickening of the mucosa by an inflammatory infiltrate composed by epithelioid cells, macrophages and some multinucleated giant cells. (H&E stain, Bar=50μm) **B**- Acid-acohol resistant bacilli within the macrophages and epithelioid cells that infiltrate the mucosa. No‐ tice the abundant number of mycobacteria that are visible at moderate amplification (Ziehl-Neelsen, Bar=30μm)

#### **3.1. Clinical signs and lesions in wild species**

Mycobacteria belonging to the MAC can affect a wide-range of wild animals, but little has been published on the clinical signs, which are rarely perceived or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing but often similar to those of their domesticated counterparts. The vast majority of reports on MAC species affecting wildlife mention the *Map* and the *Maa* as the mycobacteria most commonly isolated in these animals.

tous lesions in the lung and liver can also be observed [38]. A recent report in free-ranging red deer (*Cervus elaphus*) supports the possibility of multiorganic dissemination of *Map* in

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**Figure 2.** A – Paratuberculosis gross lesions in the ileum of sheep. Notice the increased thickness of the mucosa, with the characteristic folds and gyros B – Paratuberculosis lesions in the submandibularis lymph nodes of red deer (*Cervus*

**Figure 3. A** - Morphological aspects of paratuberculosis lesions in the kidneys of deer (*Cervus elaphus*). A single granu‐ loma with central caseous necrosis, surrounded by a thick fibrous capsule (H&E 100μm). **B** – Higher magnification of

In deer, the infection by *Maa* is self-limiting as in other mammalian species [36,42]. The lesions may be purulent, caseous, or granulomatous [26], and are mainly present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract (mesenteric and ileocaecal), consistent with the feco-oral route of infection. The granulomatous le‐

the previous image. Notice the presence of Langhan's MGC (Arrow. H&E 50μm).

*elaphus*) with caseous necrosis.

deer, since the agent was isolated from kidneys with granulomatous lesions [41].

#### *3.1.2. Wild ruminants*

The lesions observed in wild species of sheep and goats are identical to those of their domes‐ tic counterparts, while in the South American camelids the lesional pattern is similar to that of cattle. However in llamas and alpacas, in contrast to what is generally described in cattle, lymph node necrosis and mineralization, along with multiorganic dissemination, have also been reported [30,31]. As in the previously mentioned species, the most significant MAC species capable of causing clinical disease in free-living, captive and farmed deer are *Map* and *Maa*. Although *Mah* has been also isolated from lesions in deer [25,26] and *Mycobacteri‐ um intracellulare* was also found in deer species but they are not so common and it's infection is usually subclinical*.* Despite the occurrence of paratuberculosis in adults, outbreaks of the disease frequently occur in young deer of 8-15 months of age, contrary to the clinical disease in sheep and cattle which usually affects adults of 3-5 years of age [36]. Clinical signs of par‐ atuberculosis in deer are similar to those described in sheep and cattle, with diarrhea and loss of weight and body condition [37]. Accordingly, the intestinal lesions of paratuberculo‐ sis in deer primarily affect the jejunum and ileum, and are identical to the typical lesions ob‐ served in sheep and goats [30]; yet, necrosis and mineralization in lymph nodes draining the gastrointestinal tract, especially those draining the ileum and ileocecal valve, are a common feature (Figure 2B). The lymph nodes are often enlarged, and a range of changes from yel‐ low watery areas to caseous necrosis is observed on cut surfaces. The histologic changes in these lesions are very similar to those caused by *Mycobacterium bovis* and other members of the MAC genus [38,39]. Balseiro et al., have proposed an histopathological classification of lesions observed in natural occurring cases of paratuberculosis in free-ranging fallow deer (*Dama dama*), according to which the lesions would be graded into four categories: focal, multifocal, diffuse multibacillary, and diffuse intermediate (multibacillary-lymphocytic) le‐ sions. Focal lesions are composed of small granulomas, mainly in the jejunal and ileal lymph nodes, whereas multifocal lesions consist in well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propria. Diffuse multibacillary lesions are characterized by a severe granulomatous enteritis and lymphadenitis. Macrophages and nu‐ merous Langhan's multinucleatd giant cells (L-MGC) containing many mycobacteria are present, resulting in macroscopic changes in the normal gut morphology. These changes are found from the proximal jejunum to the ileocaecal valve, but lesions are always particularly severe in the distal jejunum. In the diffuse intermediate (multibacillary-lymphocytic) lesions, there is a prominence of lymphocytes, macrophages and L-MGC, with small numbers of mycobacteria [40]. In deer with clinical signs of paratuberculosis, disseminated granuloma‐ tous lesions in the lung and liver can also be observed [38]. A recent report in free-ranging red deer (*Cervus elaphus*) supports the possibility of multiorganic dissemination of *Map* in deer, since the agent was isolated from kidneys with granulomatous lesions [41].

**3.1. Clinical signs and lesions in wild species**

isolated in these animals.

96 Insights from Veterinary Medicine

*3.1.2. Wild ruminants*

Mycobacteria belonging to the MAC can affect a wide-range of wild animals, but little has been published on the clinical signs, which are rarely perceived or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing but often similar to those of their domesticated counterparts. The vast majority of reports on MAC species affecting wildlife mention the *Map* and the *Maa* as the mycobacteria most commonly

The lesions observed in wild species of sheep and goats are identical to those of their domes‐ tic counterparts, while in the South American camelids the lesional pattern is similar to that of cattle. However in llamas and alpacas, in contrast to what is generally described in cattle, lymph node necrosis and mineralization, along with multiorganic dissemination, have also been reported [30,31]. As in the previously mentioned species, the most significant MAC species capable of causing clinical disease in free-living, captive and farmed deer are *Map* and *Maa*. Although *Mah* has been also isolated from lesions in deer [25,26] and *Mycobacteri‐ um intracellulare* was also found in deer species but they are not so common and it's infection is usually subclinical*.* Despite the occurrence of paratuberculosis in adults, outbreaks of the disease frequently occur in young deer of 8-15 months of age, contrary to the clinical disease in sheep and cattle which usually affects adults of 3-5 years of age [36]. Clinical signs of par‐ atuberculosis in deer are similar to those described in sheep and cattle, with diarrhea and loss of weight and body condition [37]. Accordingly, the intestinal lesions of paratuberculo‐ sis in deer primarily affect the jejunum and ileum, and are identical to the typical lesions ob‐ served in sheep and goats [30]; yet, necrosis and mineralization in lymph nodes draining the gastrointestinal tract, especially those draining the ileum and ileocecal valve, are a common feature (Figure 2B). The lymph nodes are often enlarged, and a range of changes from yel‐ low watery areas to caseous necrosis is observed on cut surfaces. The histologic changes in these lesions are very similar to those caused by *Mycobacterium bovis* and other members of the MAC genus [38,39]. Balseiro et al., have proposed an histopathological classification of lesions observed in natural occurring cases of paratuberculosis in free-ranging fallow deer (*Dama dama*), according to which the lesions would be graded into four categories: focal, multifocal, diffuse multibacillary, and diffuse intermediate (multibacillary-lymphocytic) le‐ sions. Focal lesions are composed of small granulomas, mainly in the jejunal and ileal lymph nodes, whereas multifocal lesions consist in well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propria. Diffuse multibacillary lesions are characterized by a severe granulomatous enteritis and lymphadenitis. Macrophages and nu‐ merous Langhan's multinucleatd giant cells (L-MGC) containing many mycobacteria are present, resulting in macroscopic changes in the normal gut morphology. These changes are found from the proximal jejunum to the ileocaecal valve, but lesions are always particularly severe in the distal jejunum. In the diffuse intermediate (multibacillary-lymphocytic) lesions, there is a prominence of lymphocytes, macrophages and L-MGC, with small numbers of mycobacteria [40]. In deer with clinical signs of paratuberculosis, disseminated granuloma‐

**Figure 2.** A – Paratuberculosis gross lesions in the ileum of sheep. Notice the increased thickness of the mucosa, with the characteristic folds and gyros B – Paratuberculosis lesions in the submandibularis lymph nodes of red deer (*Cervus elaphus*) with caseous necrosis.

**Figure 3. A** - Morphological aspects of paratuberculosis lesions in the kidneys of deer (*Cervus elaphus*). A single granu‐ loma with central caseous necrosis, surrounded by a thick fibrous capsule (H&E 100μm). **B** – Higher magnification of the previous image. Notice the presence of Langhan's MGC (Arrow. H&E 50μm).

In deer, the infection by *Maa* is self-limiting as in other mammalian species [36,42]. The lesions may be purulent, caseous, or granulomatous [26], and are mainly present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract (mesenteric and ileocaecal), consistent with the feco-oral route of infection. The granulomatous le‐ sions are grossly and histologically identical to the lesions caused by *Mycobacterium bovis* [42]. *Mah* lesions in deer are similar to those observed in animals with *Maa* infection, and although rare, both mycobacteria can cause systemic disease [26] with hematoge‐ nous spread to the liver and lungs to produce miliary lesions and a terminal septicemia [43]. Despite of these findings, *Map* and *Maa* infections can be present in apparently asymptomatic deer herds [44,45]. Furthermore, a study of wild Tule elks (*Cervus elaphus nannodes*) from California revealed no significant associations between MAC infection and microscopic lesions, such as presence of macrophages and/or multinucleate giant cells (MGC) in tissue sections [45] (Figure 3).

of more than 1 cm in diameter, of either necrotic or necrotic calcified granulomas are ob‐ served (Figure 4). The presence of lymphocytes and caseous necrosis are the most com‐

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**Figure 4.** Morphological aspects of paratuberculosis lesions in wild boar (*Sus scrofa*). Presence of of multiple granulo‐ mas in the mesenteric lymph nodes (H&E 50μm). **A** – Notice the caseous necrosis surrounded by inflammatory cells predominantly macrophages and some fibroblasts. **B** – Higher magnification of the former image. Note the Langhan's

Despite *Maa* being widely reported in wild boar, data on clinical infection or mortality are scarce [49]. Apparently, natural infection with this mycobateria causes barely detectable clinical signs or lesions [49]. However, there are reports of *Maa* isolated from free-ranging Eurasian wild boars with tuberculous lesions in intestinal lymph nodes [60], and the experi‐ mental infection with high doses of *Maa* results in gross and histopathological lesions of tu‐ berculosis in tracheobronchial and mandibular lymph nodes. All visible lesions are less than 10 mm in diameter and consist of typical tuberculous granulomas with a central caseous ne‐ crosis, variably mineralized, surrounded by macrophages, lymphocytes, neutrophils, eosi‐ nophils and occasional MGC surrounded by fibrous tissue. Acid-fast bacilli are rarely detected in the necrotic debris of these lesions [61]. Another study also showed that in wild boars with mesenteric and submaxillar lymphadenitis, *Mycobacterium avium* subspecies type 1 and *M. avium* subspecies type 2 were the most frequently isolated mycobacteria [62].

Regarding *Mah* in wild boar, recent reports suggest that this animal species may act as a res‐ ervoir for these mycobacteria, since it was detected in lymph nodes without gross lesions or

Bird species, either domestic or free-living can be infected with MAC mycobacteria, but they are more susceptible to *Maa*, the causative agent of avian tuberculosis [19]. Infection by *M. avium* subsp. *paratuberculosis* has been also documented in birds, with and without clinical

mon findings in these lesions [59].

MGC (Arrows. H&E 50μm).

microscopic lesions [60,63].

and pathological findings [46,57]*.*

**3.2. Bird species**

#### *3.1.3. Non-ruminant species*

*Map* has been isolated in a wide range of wild mammals, from rodents, badgers, ra‐ coons, nine-banded armadillos, opossums, northern short-tailed shrew, striped skunks [46-48], wild boars [32,49] and rhinoceros [50] to bears [51], but not all of them present the same susceptibility and develop clinical signs or lesions when infected. The lesions produced by *Map*, representative or not of paratuberculosis, as well as the clinical signs seen in non-ruminant wild animals are more subtle than those observed in wild-rumi‐ nant species. Monkeys (*Macaca arctoides* and *Papio sphinx*) have demonstrated susceptibili‐ ty to *Map* infection and develop lesions that are confined to the intestine and abdominal lymph nodes, resembling the lesions of paratuberculosis in cattle and in humans Crohn's disease [52,53]. When infected by *Map* lagomorphs produce an intestinal disease similar to paratuberculosis in ruminants, and severe infection can occur naturally, in which ex‐ tensive granulomas with numerous giants' cells carrying multiple acid-fast bacilli can be observed in the small intestine [54]. In foxes and stoats, *Map* affects the intestines and mesenteric lymph nodes with microscopic changes similar to those described in rumi‐ nants with subclinical paratuberculosis. The lesions are composed of single macrophagelike cells or discrete granulomas consisting of small numbers of macrophages, in the cortex and paracortex of the mesenteric lymph nodes. In the small intestine, only few numbers of intracellular acid-fast bacteria are present within the macrophages, and Lan‐ ghan's-type MGC, irregularly scattered in the granulomas, in all layers of affected intes‐ tine [46,54,55]. The typical pathology of paratuberculosis has also been noted in wood mice, weasels, badgers [46], and rats infected with the predominant ruminant strains [56]. When present, in brushtail possum and hedgehogs infected with *Map*, the lesions observed are in the grastrointestinal tract [57]. A recent report revealed that wild Eura‐ sian otters (*Lutra lutra* L.) could be infected with *Map*. In that study, no gross lesions were found, but the retropharyngeal and mesenteric lymph nodes presented disrupted architecture, lymphoid depletion and diffuse inflammatory infiltrate composed mainly of macrophages and, to a lesser extent, neutrophils. *Map* was identified by direct PCR in several organs, including the intestine and lymph nodes [58]. Reports in which *Map* was isolated from Eurasian wild boars (*Sus scrofa*) revealed that the infection can occur with or without lesions, the later being the most frequent. If present, lesions generally consist of granulomatous enteritis and mesenteric lymphadenitis [32,49]. In mesenteric lymph nodes, lymphadenitis with multifocal lesions ranging from less than 1 cm to large areas of more than 1 cm in diameter, of either necrotic or necrotic calcified granulomas are ob‐ served (Figure 4). The presence of lymphocytes and caseous necrosis are the most com‐ mon findings in these lesions [59].

**Figure 4.** Morphological aspects of paratuberculosis lesions in wild boar (*Sus scrofa*). Presence of of multiple granulo‐ mas in the mesenteric lymph nodes (H&E 50μm). **A** – Notice the caseous necrosis surrounded by inflammatory cells predominantly macrophages and some fibroblasts. **B** – Higher magnification of the former image. Note the Langhan's MGC (Arrows. H&E 50μm).

Despite *Maa* being widely reported in wild boar, data on clinical infection or mortality are scarce [49]. Apparently, natural infection with this mycobateria causes barely detectable clinical signs or lesions [49]. However, there are reports of *Maa* isolated from free-ranging Eurasian wild boars with tuberculous lesions in intestinal lymph nodes [60], and the experi‐ mental infection with high doses of *Maa* results in gross and histopathological lesions of tu‐ berculosis in tracheobronchial and mandibular lymph nodes. All visible lesions are less than 10 mm in diameter and consist of typical tuberculous granulomas with a central caseous ne‐ crosis, variably mineralized, surrounded by macrophages, lymphocytes, neutrophils, eosi‐ nophils and occasional MGC surrounded by fibrous tissue. Acid-fast bacilli are rarely detected in the necrotic debris of these lesions [61]. Another study also showed that in wild boars with mesenteric and submaxillar lymphadenitis, *Mycobacterium avium* subspecies type 1 and *M. avium* subspecies type 2 were the most frequently isolated mycobacteria [62].

Regarding *Mah* in wild boar, recent reports suggest that this animal species may act as a res‐ ervoir for these mycobacteria, since it was detected in lymph nodes without gross lesions or microscopic lesions [60,63].

#### **3.2. Bird species**

sions are grossly and histologically identical to the lesions caused by *Mycobacterium bovis* [42]. *Mah* lesions in deer are similar to those observed in animals with *Maa* infection, and although rare, both mycobacteria can cause systemic disease [26] with hematoge‐ nous spread to the liver and lungs to produce miliary lesions and a terminal septicemia [43]. Despite of these findings, *Map* and *Maa* infections can be present in apparently asymptomatic deer herds [44,45]. Furthermore, a study of wild Tule elks (*Cervus elaphus nannodes*) from California revealed no significant associations between MAC infection and microscopic lesions, such as presence of macrophages and/or multinucleate giant

*Map* has been isolated in a wide range of wild mammals, from rodents, badgers, ra‐ coons, nine-banded armadillos, opossums, northern short-tailed shrew, striped skunks [46-48], wild boars [32,49] and rhinoceros [50] to bears [51], but not all of them present the same susceptibility and develop clinical signs or lesions when infected. The lesions produced by *Map*, representative or not of paratuberculosis, as well as the clinical signs seen in non-ruminant wild animals are more subtle than those observed in wild-rumi‐ nant species. Monkeys (*Macaca arctoides* and *Papio sphinx*) have demonstrated susceptibili‐ ty to *Map* infection and develop lesions that are confined to the intestine and abdominal lymph nodes, resembling the lesions of paratuberculosis in cattle and in humans Crohn's disease [52,53]. When infected by *Map* lagomorphs produce an intestinal disease similar to paratuberculosis in ruminants, and severe infection can occur naturally, in which ex‐ tensive granulomas with numerous giants' cells carrying multiple acid-fast bacilli can be observed in the small intestine [54]. In foxes and stoats, *Map* affects the intestines and mesenteric lymph nodes with microscopic changes similar to those described in rumi‐ nants with subclinical paratuberculosis. The lesions are composed of single macrophagelike cells or discrete granulomas consisting of small numbers of macrophages, in the cortex and paracortex of the mesenteric lymph nodes. In the small intestine, only few numbers of intracellular acid-fast bacteria are present within the macrophages, and Lan‐ ghan's-type MGC, irregularly scattered in the granulomas, in all layers of affected intes‐ tine [46,54,55]. The typical pathology of paratuberculosis has also been noted in wood mice, weasels, badgers [46], and rats infected with the predominant ruminant strains [56]. When present, in brushtail possum and hedgehogs infected with *Map*, the lesions observed are in the grastrointestinal tract [57]. A recent report revealed that wild Eura‐ sian otters (*Lutra lutra* L.) could be infected with *Map*. In that study, no gross lesions were found, but the retropharyngeal and mesenteric lymph nodes presented disrupted architecture, lymphoid depletion and diffuse inflammatory infiltrate composed mainly of macrophages and, to a lesser extent, neutrophils. *Map* was identified by direct PCR in several organs, including the intestine and lymph nodes [58]. Reports in which *Map* was isolated from Eurasian wild boars (*Sus scrofa*) revealed that the infection can occur with or without lesions, the later being the most frequent. If present, lesions generally consist of granulomatous enteritis and mesenteric lymphadenitis [32,49]. In mesenteric lymph nodes, lymphadenitis with multifocal lesions ranging from less than 1 cm to large areas

cells (MGC) in tissue sections [45] (Figure 3).

*3.1.3. Non-ruminant species*

98 Insights from Veterinary Medicine

Bird species, either domestic or free-living can be infected with MAC mycobacteria, but they are more susceptible to *Maa*, the causative agent of avian tuberculosis [19]. Infection by *M. avium* subsp. *paratuberculosis* has been also documented in birds, with and without clinical and pathological findings [46,57]*.*

*Maa* belonging to serotypes 1, 2, 3, and 6 is the most common agent of avian tuberculosis, but other species belonging to MAC, such as *M. intracellulare*, are also sporadic causes of dis‐ ease [19]. Tuberculosis affects a wide-range of bird species, and it has been documented in waterfowl, galliformes, columbiformes, passerines, psittacines, raptors, and ratites [64-67]. According to their susceptibility to the disease, it has been proposed the classification of bird species into four groups, from highly susceptible to highly resistant: (1) domestic fowl, spar‐ rows, pheasants, and partridges; (2) guinea fowl and domestic turkeys; (3) domestic goose and duck; (4) domestic pigeon [19]. The clinical signs of avian tuberculosis are well estab‐ lished but are not pathognomonic, and are different according to the phase of infection and the predominant form of the disease in the bird species, which in turn is related to the spe‐ cies susceptibility to the agent. Avian tuberculosis usually presents three stages or phases: latency, lesion development, and period of cachexia [68,69]. In most reports on the initial phase of avian tuberculosis the absence of clinical signs is a common feature. On the contra‐ ry, in advanced cases, progressive weight loss, depression, white diarrhea with soiled feath‐ ers, increased thirst, respiratory distress, fatigue, and decreased egg production may be observed [68]. Generally, avian tuberculosis is an intestinal and hepatic disease that can dis‐ seminate to other organs including the lungs, air sacs, spleen, bone marrow, and skin [69]. This is considered as the classical form of infection, and it's marked by the presence of tuber‐ cles or granulomas in multiple organs. Lesions in the intestinal tract characterize a second form of infection, and a third type of infection, especially reported finches, canaries, and psittacines occurs without the development of tuberculous lesions [19,68,69]. The clinical signs of the intestinal form of tuberculosis are characterized by chronic wasting disease. In severe cases and as disease progresses, feathers are often dull or ruffled and comb, wattle, and earlobes are paler, thinner and dry. Birds become lethargic and emaciated with marked atrophy of breast muscles, displaying the typical "knife edged" keel. In extreme cases, the body fat disappears, and the bird's face looks smaller than normal. The body temperature of the affected birds remains normal, even in severe cases [68,69]. Sudden death may also oc‐ cur, as well as dyspnea, granulomatous ocular lesions [70] and skin lesions, which are less frequent [68,69]. Sudden death may be the result of massive hemorrhage secondary to liver or spleen rupture. In such cases, the birds may exhibit good body condition but frequently show advanced lesions of tuberculosis. Lameness can be the result of bone dissemination, in particular to the bone marrow of long bones. Joints can also be affected, and as consequence, some birds may adapt a sitting position or even show paralysis. There are also reports of neurological signs, due to the involvement of vertebral or central nervous system [68]. The lesions of avian tuberculosis are mainly composed by epithelioid cells containing large num‐ bers of bacterium, that may either diffusely infiltrate the organ or form discrete tubercles or granulomas [68]. In the classical and intestinal forms of the disease, studded greyish-white to greyish-yellow nodules are frequently observed. The nodules, which appear as tumourlike masses, bulge from the serosal surface of the intestine and can be palpable. These nod‐ ules may suffer ulceration, and the caseous material within may be discharge into the intestinal lumen leading to the excretion of mycobacteria in droppings. During dissemina‐ tion, typical caseous lesions, without calcification, are always found in the liver and spleen, with considerable enlargement of the organs. Nodules are firm but can be incised easily

since mineralization is rare in avian tuberculosis. Due to this, spleen takes irregular and "knobbly" appearance. With the disease progression, tubercular nodules in the bone mar‐ row, ovaries and testes, are often seen. Pulmonary lesions, which are a striking feature of tuberculosis in other species, are rarely observed in birds [68,69]. Pulmonary avian tubercu‐ losis is not common, but it has been reported occasionally in pigeons and water-fowl [19,65,69]. In the cachexia stage of avian tuberculosis, massive tubercles with large numbers

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In accordance to the clinical signs and lesional patterns of tuberculosis in domestic spe‐ cies, captive, exotic and wild birds, including raptors, generally develop the disseminat‐ ed form of avian tuberculosis, involving the digestive tract, liver and spleen [66,71,72]. However, some studies reveal that exotic bird species may have lesions in the liver and spleen without intestinal involvement. These lesions are typical granulomas with a case‐ ous or coagulative necrotic centre and MGC. Acid-fast bacilli are numerous in the central

Infection of birds by *Map* has been reported, but it often occurs without clinical signs or le‐ sions, despite the typical lesions of paratuberculosis being described in jackdaws (*Corvus monedula*), rooks (*Corvus frugilegus*), and crows (*Corvus corone*) [46,57]. A study also reported the occurrence of diarrhea, respiratory signs, hepatomegaly and splenomegaly in a diamond sparrow (*Emblema guttata*) with liver granuloma lesions, predominantly composed of lym‐

The diagnosis of Mac is based on the clinical signs, post mortem gross lesions, and by dem‐ onstrating the presence of acid-fast bacilli using Ziehl-Neelsen staining. This is normally suf‐ ficient to establish the diagnosis [68,69,74,75]. *Ante mortem* diagnosis is based on clinical signs, leukograme, serology, culture and also acid-fast stain or biopsy samples of fluids or organs [76-80]. Radiography and ultrasonography are also useful in the medical evaluation

The golden standard test for mycobacterium diagnosis is the microbiological culture. Tissue culture seems to be slightly more sensitive than faecal culture and it allows the infection to be detected in some animals that had no specific lesions. The culture of bacteria requires weeks or months of incubation before colony growth occurs. This means that a significant amount of time is needed before a diagnosis can be made. It is also difficult to isolate bacte‐ ria in culture due to intermittent shedding and a low number of bacilli in faeces and tissues [81]. *M. genavense* is extremely difficult to isolate by culture [82,83]. *M. avium* complex grows best in media containing eggs or egg yolk and the incubation temperature should be set at 37ºC-40ºC. Culture can be performed in Dorset´s, Herrold´s egg yolk medium, Middlebrook 7H10 and 7H11 or Coletsos medium supplemented with 1% sodium piruvate [68,74,75]. For

of bacilli are observed [68,69].

zone of the tubercle [69].

phocytes and mononuclear cells [73].

**4. Diagnostic techniques**

**4.1. Isolation and identification**

of birds [79].

since mineralization is rare in avian tuberculosis. Due to this, spleen takes irregular and "knobbly" appearance. With the disease progression, tubercular nodules in the bone mar‐ row, ovaries and testes, are often seen. Pulmonary lesions, which are a striking feature of tuberculosis in other species, are rarely observed in birds [68,69]. Pulmonary avian tubercu‐ losis is not common, but it has been reported occasionally in pigeons and water-fowl [19,65,69]. In the cachexia stage of avian tuberculosis, massive tubercles with large numbers of bacilli are observed [68,69].

In accordance to the clinical signs and lesional patterns of tuberculosis in domestic spe‐ cies, captive, exotic and wild birds, including raptors, generally develop the disseminat‐ ed form of avian tuberculosis, involving the digestive tract, liver and spleen [66,71,72]. However, some studies reveal that exotic bird species may have lesions in the liver and spleen without intestinal involvement. These lesions are typical granulomas with a case‐ ous or coagulative necrotic centre and MGC. Acid-fast bacilli are numerous in the central zone of the tubercle [69].

Infection of birds by *Map* has been reported, but it often occurs without clinical signs or le‐ sions, despite the typical lesions of paratuberculosis being described in jackdaws (*Corvus monedula*), rooks (*Corvus frugilegus*), and crows (*Corvus corone*) [46,57]. A study also reported the occurrence of diarrhea, respiratory signs, hepatomegaly and splenomegaly in a diamond sparrow (*Emblema guttata*) with liver granuloma lesions, predominantly composed of lym‐ phocytes and mononuclear cells [73].

## **4. Diagnostic techniques**

*Maa* belonging to serotypes 1, 2, 3, and 6 is the most common agent of avian tuberculosis, but other species belonging to MAC, such as *M. intracellulare*, are also sporadic causes of dis‐ ease [19]. Tuberculosis affects a wide-range of bird species, and it has been documented in waterfowl, galliformes, columbiformes, passerines, psittacines, raptors, and ratites [64-67]. According to their susceptibility to the disease, it has been proposed the classification of bird species into four groups, from highly susceptible to highly resistant: (1) domestic fowl, spar‐ rows, pheasants, and partridges; (2) guinea fowl and domestic turkeys; (3) domestic goose and duck; (4) domestic pigeon [19]. The clinical signs of avian tuberculosis are well estab‐ lished but are not pathognomonic, and are different according to the phase of infection and the predominant form of the disease in the bird species, which in turn is related to the spe‐ cies susceptibility to the agent. Avian tuberculosis usually presents three stages or phases: latency, lesion development, and period of cachexia [68,69]. In most reports on the initial phase of avian tuberculosis the absence of clinical signs is a common feature. On the contra‐ ry, in advanced cases, progressive weight loss, depression, white diarrhea with soiled feath‐ ers, increased thirst, respiratory distress, fatigue, and decreased egg production may be observed [68]. Generally, avian tuberculosis is an intestinal and hepatic disease that can dis‐ seminate to other organs including the lungs, air sacs, spleen, bone marrow, and skin [69]. This is considered as the classical form of infection, and it's marked by the presence of tuber‐ cles or granulomas in multiple organs. Lesions in the intestinal tract characterize a second form of infection, and a third type of infection, especially reported finches, canaries, and psittacines occurs without the development of tuberculous lesions [19,68,69]. The clinical signs of the intestinal form of tuberculosis are characterized by chronic wasting disease. In severe cases and as disease progresses, feathers are often dull or ruffled and comb, wattle, and earlobes are paler, thinner and dry. Birds become lethargic and emaciated with marked atrophy of breast muscles, displaying the typical "knife edged" keel. In extreme cases, the body fat disappears, and the bird's face looks smaller than normal. The body temperature of the affected birds remains normal, even in severe cases [68,69]. Sudden death may also oc‐ cur, as well as dyspnea, granulomatous ocular lesions [70] and skin lesions, which are less frequent [68,69]. Sudden death may be the result of massive hemorrhage secondary to liver or spleen rupture. In such cases, the birds may exhibit good body condition but frequently show advanced lesions of tuberculosis. Lameness can be the result of bone dissemination, in particular to the bone marrow of long bones. Joints can also be affected, and as consequence, some birds may adapt a sitting position or even show paralysis. There are also reports of neurological signs, due to the involvement of vertebral or central nervous system [68]. The lesions of avian tuberculosis are mainly composed by epithelioid cells containing large num‐ bers of bacterium, that may either diffusely infiltrate the organ or form discrete tubercles or granulomas [68]. In the classical and intestinal forms of the disease, studded greyish-white to greyish-yellow nodules are frequently observed. The nodules, which appear as tumourlike masses, bulge from the serosal surface of the intestine and can be palpable. These nod‐ ules may suffer ulceration, and the caseous material within may be discharge into the intestinal lumen leading to the excretion of mycobacteria in droppings. During dissemina‐ tion, typical caseous lesions, without calcification, are always found in the liver and spleen, with considerable enlargement of the organs. Nodules are firm but can be incised easily

100 Insights from Veterinary Medicine

The diagnosis of Mac is based on the clinical signs, post mortem gross lesions, and by dem‐ onstrating the presence of acid-fast bacilli using Ziehl-Neelsen staining. This is normally suf‐ ficient to establish the diagnosis [68,69,74,75]. *Ante mortem* diagnosis is based on clinical signs, leukograme, serology, culture and also acid-fast stain or biopsy samples of fluids or organs [76-80]. Radiography and ultrasonography are also useful in the medical evaluation of birds [79].

#### **4.1. Isolation and identification**

The golden standard test for mycobacterium diagnosis is the microbiological culture. Tissue culture seems to be slightly more sensitive than faecal culture and it allows the infection to be detected in some animals that had no specific lesions. The culture of bacteria requires weeks or months of incubation before colony growth occurs. This means that a significant amount of time is needed before a diagnosis can be made. It is also difficult to isolate bacte‐ ria in culture due to intermittent shedding and a low number of bacilli in faeces and tissues [81]. *M. genavense* is extremely difficult to isolate by culture [82,83]. *M. avium* complex grows best in media containing eggs or egg yolk and the incubation temperature should be set at 37ºC-40ºC. Culture can be performed in Dorset´s, Herrold´s egg yolk medium, Middlebrook 7H10 and 7H11 or Coletsos medium supplemented with 1% sodium piruvate [68,74,75]. For

the isolation of *Map* or *M. silvaticum* addition of mycobactin is required in all media. *Map* is the slowest growing of the culturable mycobacteria [84]. Cultures should be incubated for at least 8 weeks. Typically, *M. avium* produces smooth colonies within 2-4 weeks and rough variants can occur [74]. Culture of *Map* from faeces or tissues of other animals such as sheep and goats is less successful due to the "S" strains that usually infect these animals [84]. The best organs to use for culture are usually liver and spleen but bone marrow can be used if carcass is decomposed, as it could be less contaminated [74]. Non-sterile specimens need to be processed with detergent alkali or acid to eliminate rapidly growing microorganisms be‐ fore culture decontamination in order to remove faster growing microbial species. Incuba‐ tion with various decontamination agents such as 0.6-0.75% hexadecylpryridinium chloride (HPC) or NaOH for 3 hours to overnight, have been used. It is important that decontamina‐ tion does not remove too many viable mycobacterium cells [85,86]. Other methods like sedi‐ mentation and centrifugation can be employed if small numbers of mycobacteria are expected in the sample [85,87]. Shorter incubation times can be achieved using automated broth based systems, like liquid culture BACTEC system MGIT 960 [84]. These systems have been reported to be highly sensitive for culture [88]. For *M. genavense* the use of BACTEC system with no additives and pH 6.0 is recommended [89,90]. Middlebrook 7H11 with pH 6.0 supplemented with blood and charcoal is also recommended to promote growth of *M. genavense* [91]. In human AIDS patients, laboratory diagnosis of MAC infection is usually made by the BACTEC blood culture [92]. Conventional biochemical tests for species identifi‐ cation are lengthy and fail to distinguish between *M. avium* and *M. intracelullare* [74]. Identi‐ fication of isolates by phenotypic characteristics of majority of clinically relevant mycobacteria can be based on growth rates, colony pigmentation and biochemical tests such as niacin production, nitrate reduction, tween 80 hydrolysis arylsulphatase, urease, tellurite reduction, thiophen-2-carboxylic acid hydrazide sensitivity catalase (qualitative and quanti‐ tative) growth on MacConkey and sodium chloride tolerance [88,93].

tween different mycobacterial infections probably due to close antigenic relationship be‐

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Tuberculin test is the most widely used method in domestic fowl and the only for which an international standard for the reagent exists. Birds are tested by intradermal inocula‐ tion in the wattle with 0.05 ml or 0.1 ml of tuberculin (avian purified protein derivate – PPD) [74]. A positive reaction is identified as a hot and oedematous swelling at the site or by the presence of a small firm nodule of approximately 5 mm in diameter after 48 hours [19]. The tuberculin test and the haemagglutination test (stained antigen) are the immunological methods most frequently used for export testing [74]. In the stained anti‐ gen test an antigen stained with 1% malachite green is used for the rapid blood plate agglutination test [96].The diagnosis of *Mycobacterial* infections in live wild animals re‐ mains a challenge [95]. The comparative cervical tuberculin (CCT) skin test has been ap‐ plied in wild animals such as in cervids [97], but the test presents two major limitations. It has been proved that CCT cannot detect some stages of infection [98], and wild rumi‐ nants must be captured twice increasing the stress and the risk of accidents for the ani‐ mals and for handlers [95]. Alternative probes like the detection of interferon-γ (IFN- γ) assay and ELISA, which employ blood and serum respectively, could be an alternative in wild animals [95]. Advantages of ELISA and IFN-γ assay is that they enable testing without handling the animals twice and allow repeated testing, which are important ad‐ vantages in case of wild ruminants [99]. ELISA detects humoral immune response whereas the CCT and IFN-γ assay detect cellular immune response [95]. The dominant response to mycobacterial infections in ruminants is cell-mediated. However, a recent study demonstrated that the IFN-γ assay may be of limited usefulness in some species

During the past several years, many molecular methods have been developed for direct de‐ tection, species identification, and drug susceptibility testing of mycobacteria. These meth‐ ods can potentially reduce the diagnostic time from weeks to days with a higher sensibility. Molecular biology methods offer new opportunities to differentiate, identify and type bacte‐ rial species and strains. These methods use the variability of nucleic sequences of genes such as 16S rDNA, beta subunit RNA-ase (rpoB), gyrase (gyrB), rDNA internal transcribed spacer among other genes. Some of the methods available to differentiate and identify species of mycobacteria at the DNA sequence level are PCR, PCR-REA, sequencing analysis, spoligo‐ typing and DNA fingerprinting. These methods have been applied to both the "universal"

Isolation of mycobacterial DNA can be done from living mycobacteria, not only from myco‐ bacterial isolates but also directly from body fluids (sputum, bronchoalveolar lavages, and bronchial and tracheal aspirates, semen, milk, blood, cerebrospinal fluid), from tissues and from faeces and can be done using dead mycobacterial cells, namely from formalin-fixed

tween *Maa* and *Map* [95].

of cervids [100].

**4.3. Genetic methods**

*4.3.1. The contribution of molecular biology to MAC research*

part of the genome and to specific mycobacterial genes.

Classification of MAC organisms has been made by seroagglutination [19,74]. Seroagglu‐ tination is based on sugar residue specificity of surface glycopeptidolipids, and allows classification of MAC organisms into 28 serovars: 1 to 6, 8 to 11 and 21 are currently as‐ cribed as *M. avium*, while serovars 7, 12 to 20 and 25 to *M. intracellulare*. However, no consensus was achieved on the other serovars [74]. MAC colonies can also be identified using high performance liquid chromatography (HPLC) for detecting mycolic acid [19]. HPLC and the use of monoclonal antibodies to major serovars in ELISA also facilitate typing of mycobacteria [19,75].

#### **4.2. Immunological methods**

The enzyme linked immunosorbent assay (ELISA) has been used for detecting antimycobac‐ terial antibodies in the serum of ruminants [94]. However, serological assays for detecting *Mycobacterium* are problematic. The sensitivity of ELISA is dependent on the stage of the dis‐ ease with a higher sensitivity of the test in case of higher bacterial load. The test can detect the most severe infections in multibacillar lesions but shows lower sensitivity in animals with paucibacillar lesions [84]. One important disadvantage is the inability to distinguish be‐ tween different mycobacterial infections probably due to close antigenic relationship be‐ tween *Maa* and *Map* [95].

Tuberculin test is the most widely used method in domestic fowl and the only for which an international standard for the reagent exists. Birds are tested by intradermal inocula‐ tion in the wattle with 0.05 ml or 0.1 ml of tuberculin (avian purified protein derivate – PPD) [74]. A positive reaction is identified as a hot and oedematous swelling at the site or by the presence of a small firm nodule of approximately 5 mm in diameter after 48 hours [19]. The tuberculin test and the haemagglutination test (stained antigen) are the immunological methods most frequently used for export testing [74]. In the stained anti‐ gen test an antigen stained with 1% malachite green is used for the rapid blood plate agglutination test [96].The diagnosis of *Mycobacterial* infections in live wild animals re‐ mains a challenge [95]. The comparative cervical tuberculin (CCT) skin test has been ap‐ plied in wild animals such as in cervids [97], but the test presents two major limitations. It has been proved that CCT cannot detect some stages of infection [98], and wild rumi‐ nants must be captured twice increasing the stress and the risk of accidents for the ani‐ mals and for handlers [95]. Alternative probes like the detection of interferon-γ (IFN- γ) assay and ELISA, which employ blood and serum respectively, could be an alternative in wild animals [95]. Advantages of ELISA and IFN-γ assay is that they enable testing without handling the animals twice and allow repeated testing, which are important ad‐ vantages in case of wild ruminants [99]. ELISA detects humoral immune response whereas the CCT and IFN-γ assay detect cellular immune response [95]. The dominant response to mycobacterial infections in ruminants is cell-mediated. However, a recent study demonstrated that the IFN-γ assay may be of limited usefulness in some species of cervids [100].

#### **4.3. Genetic methods**

the isolation of *Map* or *M. silvaticum* addition of mycobactin is required in all media. *Map* is the slowest growing of the culturable mycobacteria [84]. Cultures should be incubated for at least 8 weeks. Typically, *M. avium* produces smooth colonies within 2-4 weeks and rough variants can occur [74]. Culture of *Map* from faeces or tissues of other animals such as sheep and goats is less successful due to the "S" strains that usually infect these animals [84]. The best organs to use for culture are usually liver and spleen but bone marrow can be used if carcass is decomposed, as it could be less contaminated [74]. Non-sterile specimens need to be processed with detergent alkali or acid to eliminate rapidly growing microorganisms be‐ fore culture decontamination in order to remove faster growing microbial species. Incuba‐ tion with various decontamination agents such as 0.6-0.75% hexadecylpryridinium chloride (HPC) or NaOH for 3 hours to overnight, have been used. It is important that decontamina‐ tion does not remove too many viable mycobacterium cells [85,86]. Other methods like sedi‐ mentation and centrifugation can be employed if small numbers of mycobacteria are expected in the sample [85,87]. Shorter incubation times can be achieved using automated broth based systems, like liquid culture BACTEC system MGIT 960 [84]. These systems have been reported to be highly sensitive for culture [88]. For *M. genavense* the use of BACTEC system with no additives and pH 6.0 is recommended [89,90]. Middlebrook 7H11 with pH 6.0 supplemented with blood and charcoal is also recommended to promote growth of *M. genavense* [91]. In human AIDS patients, laboratory diagnosis of MAC infection is usually made by the BACTEC blood culture [92]. Conventional biochemical tests for species identifi‐ cation are lengthy and fail to distinguish between *M. avium* and *M. intracelullare* [74]. Identi‐ fication of isolates by phenotypic characteristics of majority of clinically relevant mycobacteria can be based on growth rates, colony pigmentation and biochemical tests such as niacin production, nitrate reduction, tween 80 hydrolysis arylsulphatase, urease, tellurite reduction, thiophen-2-carboxylic acid hydrazide sensitivity catalase (qualitative and quanti‐

tative) growth on MacConkey and sodium chloride tolerance [88,93].

typing of mycobacteria [19,75].

102 Insights from Veterinary Medicine

**4.2. Immunological methods**

Classification of MAC organisms has been made by seroagglutination [19,74]. Seroagglu‐ tination is based on sugar residue specificity of surface glycopeptidolipids, and allows classification of MAC organisms into 28 serovars: 1 to 6, 8 to 11 and 21 are currently as‐ cribed as *M. avium*, while serovars 7, 12 to 20 and 25 to *M. intracellulare*. However, no consensus was achieved on the other serovars [74]. MAC colonies can also be identified using high performance liquid chromatography (HPLC) for detecting mycolic acid [19]. HPLC and the use of monoclonal antibodies to major serovars in ELISA also facilitate

The enzyme linked immunosorbent assay (ELISA) has been used for detecting antimycobac‐ terial antibodies in the serum of ruminants [94]. However, serological assays for detecting *Mycobacterium* are problematic. The sensitivity of ELISA is dependent on the stage of the dis‐ ease with a higher sensitivity of the test in case of higher bacterial load. The test can detect the most severe infections in multibacillar lesions but shows lower sensitivity in animals with paucibacillar lesions [84]. One important disadvantage is the inability to distinguish be‐

#### *4.3.1. The contribution of molecular biology to MAC research*

During the past several years, many molecular methods have been developed for direct de‐ tection, species identification, and drug susceptibility testing of mycobacteria. These meth‐ ods can potentially reduce the diagnostic time from weeks to days with a higher sensibility. Molecular biology methods offer new opportunities to differentiate, identify and type bacte‐ rial species and strains. These methods use the variability of nucleic sequences of genes such as 16S rDNA, beta subunit RNA-ase (rpoB), gyrase (gyrB), rDNA internal transcribed spacer among other genes. Some of the methods available to differentiate and identify species of mycobacteria at the DNA sequence level are PCR, PCR-REA, sequencing analysis, spoligo‐ typing and DNA fingerprinting. These methods have been applied to both the "universal" part of the genome and to specific mycobacterial genes.

Isolation of mycobacterial DNA can be done from living mycobacteria, not only from myco‐ bacterial isolates but also directly from body fluids (sputum, bronchoalveolar lavages, and bronchial and tracheal aspirates, semen, milk, blood, cerebrospinal fluid), from tissues and from faeces and can be done using dead mycobacterial cells, namely from formalin-fixed and paraffin-embedded tissues and from forensic and archaeological samples [101]. One of the challenges with molecular detection of *Map* is to get the genomic DNA out of the bacte‐ ria, which is protected by its thick and waxy cell wall. One method commonly used is the mechanical disruption by the use of bead beating, a general term for using small beads mixed with the sample, usually in the presence of a proteolytic enzyme and lysis buffer, to break tissues or tough cell walls and spores by forceful shaking in a cell disrupter, or "bead beater". It is one of several suggested methods to lyse *Map* [102-105]. Others include homo‐ genized (grinded) sample under liquid nitrogen in a mortar and pestle, combinations of en‐ zymatic treatment, freeze-thaw/boiling and kits for plant DNA purification or for animal tissues DNA purification [103,105-109]. Commercially available kits, developed for DNA isolation from different matrices, are commonly used for the rapid isolation and detection of *Map* in milk.

to *Maa*. Serovars 7, 12–20, and 25 have been ascribed to *Mycobacterium intracellulare*. Se‐ rovar-1 is the most common organism isolated from birds and from human. Serotypes 1 and 2 are most commonly isolated from domestic birds, and serovar 3 is recovered spor‐ adically from wild birds. Serotypes 1, 2, and 3 are considered virulent for chickens. Se‐ rotypes 1, 4, and 8 have been reported to predominate among isolates from AIDS

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105

Other approach to the differentiation of MAC strains was obtained with the description of repetitive insertion sequence IS*900* in *Map* strains and IS*901* or IS*902* in *M. avium* subsp. *sil‐ vaticum* strains [115]. Examination of serotyped strains revealed IS*901* only in strains of sero‐

The discovery of insertion sequences in mycobacterial genomes, e.g. IS*900* in *Map* [116], IS*901* [117], IS*1245* [118], IS*1311* [119] in the MAC strains was a major breakthrough in the study of mycobacterial infections. When characterized and used in the proper context, the species-specific IS (insertion sequences) elements can be useful classification tools to distin‐ guish subsets of the MAC [18,120]. However, there are two problems described that can question their utility for this purpose. First, a number of IS elements have been uncovered in strains considered to be MAC organisms, but without adequate strain characterization, it is difficult to judge which organisms harbour such elements. Second, IS elements are by nature mobile elements, so there is a risk that similar elements are found in unrelated bacteria be‐

IS*900* was the first IS characterized within the *Mycobacterium* genus [116,121] and is the most widely used target sequence for detection of *Map* and presently considered specific for this agent. The *Map* genome is reported to have 15 to 20 copies of the insertion element, and the sequenced strain K-10 has 17 copies [122]. Cousins et al. recommended that restriction diges‐ tion should be used to confirm the profile of the IS of the amplified product [123]. However, Englund et al. identified a *Mycobacterium* sp. with an IS*900* like sequence in which the re‐ striction sites after amplification with the original primers were identical to the restriction sites in amplified DNA of *Map* [124]. Therefore, restriction endonucleases analysis did not solve the problem of false positives. Englund et al. recommended that a positive IS*900* PCR should be confirmed by subsequent sequencing or by a PCR assay targeting another gene in *Map* [124]. In a study performed by Vansnick et al. two sets of newly developed PCR pri‐ mers based on the insertion sequence IS*900* and the unique sequence f57 were developed and the combination of the two PCR assays has proven to be useful for the identification of *Map* [125]. *Map* genome has revealed the presence of 17 IS*900*, 7 IS*1311*, and 3 IS*1245* inser‐ tion elements. The IS*900* element seems unique to *Map* and has been widely used as a diag‐

nostic tool to detect *Map* in clinical samples from both animals and humans [5].

The specific DNA sequence IS*900* was also used as the target for *Map* detection in Nested PCR. In 2002, IS*900*-nested PCR was used to determine the specificity and sensitivity of a commercial ELISA test [126]. However, the nested PCR method is now being replaced by

patients [19].

types 1, 2, and 3 [19].

Real-Time PCR [127].

cause of mobility to or from MAC organisms [3].

#### *4.3.2. Polymerase Chain Reaction (PCR)*

The polymerase chain reaction (PCR) is an in vitro method for the amplification of DNA that was introduced in 1985 [110]. With the performance of a previous reverse transcrip‐ tion step, PCR can also be applied to RNA [111]. Reverse transcription PCR is a modifi‐ cation of this method used when the initial template is RNA rather than DNA, the reverse transcriptase enzyme first converts the RNA target into a complementary DNA copy (cDNA), that can be used to amplify the much higher numbers of copies of mes‐ senger or ribosomal RNA than the number of DNA copies present in bacteria, and it may detect specific expression of certain genes. Some modifications to single PCR were done to improve results and were used for MAC species detection, the multiplex PCR, the assay that include several primer pairs specific to different DNA targets to allow amplification and detection of several pathogens at the same time, and nested PCR, the product from one PCR reaction serves as template in a second reaction with fresh re‐ agents, thus diluting any PCR inhibiting substances and increasing the sensitivity. As ex‐ ample differentiation of *M. tuberculosis* complex, *M. avium* and other non-tuberculous mycobacteria (NTM) has been done by using hybridization probes [112]. Targeting the 16S rRNA gene, 3 different probes, specific for mycobacteria, *M. tuberculosis* complex and *M. avium,* were constructed and the thermal melting temperature (Tm) was differ‐ ent for *M. tuberculosis*, *M. kansasii*, *M. avium, M. intracellulare* and *M. marinum* allowing the differentiation. TaqMan PCR assay targeting 65 kD heat shock protein gene has been used for the detection of *M. genavense* and *M. avium* complex species in avian tissue samples [83].

Specific probes are available for the identification of *Mycobacterium avium* and *Mycobacte‐ rium intracellulare*. Amplification of the DT1 and DT6 fragments was considered equally sensitive for species identification [113]. Recently, numerous isolates suspected of be *My‐ cobacterium intracellulare* were reclassified as *Mycobacterium chimaera* sp. nov., as part of the MAC [114]. Strains of MAC can be identified by serological procedures, on the basis of differences in the C-mycoside glycopeptidolipids. To date, using 16S rRNA probes, 28 MAC serotypes have been identified from which the serotypes 1–6, 8–11, and 21 belong to *Maa*. Serovars 7, 12–20, and 25 have been ascribed to *Mycobacterium intracellulare*. Se‐ rovar-1 is the most common organism isolated from birds and from human. Serotypes 1 and 2 are most commonly isolated from domestic birds, and serovar 3 is recovered spor‐ adically from wild birds. Serotypes 1, 2, and 3 are considered virulent for chickens. Se‐ rotypes 1, 4, and 8 have been reported to predominate among isolates from AIDS patients [19].

and paraffin-embedded tissues and from forensic and archaeological samples [101]. One of the challenges with molecular detection of *Map* is to get the genomic DNA out of the bacte‐ ria, which is protected by its thick and waxy cell wall. One method commonly used is the mechanical disruption by the use of bead beating, a general term for using small beads mixed with the sample, usually in the presence of a proteolytic enzyme and lysis buffer, to break tissues or tough cell walls and spores by forceful shaking in a cell disrupter, or "bead beater". It is one of several suggested methods to lyse *Map* [102-105]. Others include homo‐ genized (grinded) sample under liquid nitrogen in a mortar and pestle, combinations of en‐ zymatic treatment, freeze-thaw/boiling and kits for plant DNA purification or for animal tissues DNA purification [103,105-109]. Commercially available kits, developed for DNA isolation from different matrices, are commonly used for the rapid isolation and detection of

The polymerase chain reaction (PCR) is an in vitro method for the amplification of DNA that was introduced in 1985 [110]. With the performance of a previous reverse transcrip‐ tion step, PCR can also be applied to RNA [111]. Reverse transcription PCR is a modifi‐ cation of this method used when the initial template is RNA rather than DNA, the reverse transcriptase enzyme first converts the RNA target into a complementary DNA copy (cDNA), that can be used to amplify the much higher numbers of copies of mes‐ senger or ribosomal RNA than the number of DNA copies present in bacteria, and it may detect specific expression of certain genes. Some modifications to single PCR were done to improve results and were used for MAC species detection, the multiplex PCR, the assay that include several primer pairs specific to different DNA targets to allow amplification and detection of several pathogens at the same time, and nested PCR, the product from one PCR reaction serves as template in a second reaction with fresh re‐ agents, thus diluting any PCR inhibiting substances and increasing the sensitivity. As ex‐ ample differentiation of *M. tuberculosis* complex, *M. avium* and other non-tuberculous mycobacteria (NTM) has been done by using hybridization probes [112]. Targeting the 16S rRNA gene, 3 different probes, specific for mycobacteria, *M. tuberculosis* complex and *M. avium,* were constructed and the thermal melting temperature (Tm) was differ‐ ent for *M. tuberculosis*, *M. kansasii*, *M. avium, M. intracellulare* and *M. marinum* allowing the differentiation. TaqMan PCR assay targeting 65 kD heat shock protein gene has been used for the detection of *M. genavense* and *M. avium* complex species in avian tissue

Specific probes are available for the identification of *Mycobacterium avium* and *Mycobacte‐ rium intracellulare*. Amplification of the DT1 and DT6 fragments was considered equally sensitive for species identification [113]. Recently, numerous isolates suspected of be *My‐ cobacterium intracellulare* were reclassified as *Mycobacterium chimaera* sp. nov., as part of the MAC [114]. Strains of MAC can be identified by serological procedures, on the basis of differences in the C-mycoside glycopeptidolipids. To date, using 16S rRNA probes, 28 MAC serotypes have been identified from which the serotypes 1–6, 8–11, and 21 belong

*Map* in milk.

104 Insights from Veterinary Medicine

samples [83].

*4.3.2. Polymerase Chain Reaction (PCR)*

Other approach to the differentiation of MAC strains was obtained with the description of repetitive insertion sequence IS*900* in *Map* strains and IS*901* or IS*902* in *M. avium* subsp. *sil‐ vaticum* strains [115]. Examination of serotyped strains revealed IS*901* only in strains of sero‐ types 1, 2, and 3 [19].

The discovery of insertion sequences in mycobacterial genomes, e.g. IS*900* in *Map* [116], IS*901* [117], IS*1245* [118], IS*1311* [119] in the MAC strains was a major breakthrough in the study of mycobacterial infections. When characterized and used in the proper context, the species-specific IS (insertion sequences) elements can be useful classification tools to distin‐ guish subsets of the MAC [18,120]. However, there are two problems described that can question their utility for this purpose. First, a number of IS elements have been uncovered in strains considered to be MAC organisms, but without adequate strain characterization, it is difficult to judge which organisms harbour such elements. Second, IS elements are by nature mobile elements, so there is a risk that similar elements are found in unrelated bacteria be‐ cause of mobility to or from MAC organisms [3].

IS*900* was the first IS characterized within the *Mycobacterium* genus [116,121] and is the most widely used target sequence for detection of *Map* and presently considered specific for this agent. The *Map* genome is reported to have 15 to 20 copies of the insertion element, and the sequenced strain K-10 has 17 copies [122]. Cousins et al. recommended that restriction diges‐ tion should be used to confirm the profile of the IS of the amplified product [123]. However, Englund et al. identified a *Mycobacterium* sp. with an IS*900* like sequence in which the re‐ striction sites after amplification with the original primers were identical to the restriction sites in amplified DNA of *Map* [124]. Therefore, restriction endonucleases analysis did not solve the problem of false positives. Englund et al. recommended that a positive IS*900* PCR should be confirmed by subsequent sequencing or by a PCR assay targeting another gene in *Map* [124]. In a study performed by Vansnick et al. two sets of newly developed PCR pri‐ mers based on the insertion sequence IS*900* and the unique sequence f57 were developed and the combination of the two PCR assays has proven to be useful for the identification of *Map* [125]. *Map* genome has revealed the presence of 17 IS*900*, 7 IS*1311*, and 3 IS*1245* inser‐ tion elements. The IS*900* element seems unique to *Map* and has been widely used as a diag‐ nostic tool to detect *Map* in clinical samples from both animals and humans [5].

The specific DNA sequence IS*900* was also used as the target for *Map* detection in Nested PCR. In 2002, IS*900*-nested PCR was used to determine the specificity and sensitivity of a commercial ELISA test [126]. However, the nested PCR method is now being replaced by Real-Time PCR [127].

RFLP analysis of the IS*900* element has been used a molecular tool to type *Map* isolates and allowed the division of *Map* into different groups, associated to different host species [3]. The IS*900* element is by far the most widely used target for the molecular detection of *Map* and has been used in the form of direct PCR [108,125], *in situ* PCR [128] nested PCR [126,129,130], and real-time PCR [131-133]. Sequencing of the amplified product for IS*900* is therefore necessary to confirm that the amplicon is truly IS*900*.

for MAC molecular epidemiology [143]. Some *M. avium* isolates have been documented as being IS*1245* negative, but only a few such reports have presented further documentation of strain identity by a sequence-based method [144]. Beggs et al. found IS*1245* in strains of *M. intracellulare*, demonstrating that the element is present in other species of the *M. avium* com‐ plex [144]. In some reports, IS*1245*-negative isolates have been described that contain an hsp65 sequence identical to that of *M. avium* but that differs from *M. avium* in other taxo‐

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Other identification methods of *M. avium* species or its subspecies are based on 16S rDNA [145], PCR-REA (Restriction Endonuclease Analysis) [146], sequence analysis of hsp65 [8] or a strategy based on large sequence polymorphisms [147]. Semret et al. evaluating the distri‐ bution of genomic polymorphisms across a panel of strains, verified that it was possible to assign unique genomic signatures to host-associated variants and based on these polymor‐ phisms proposed a simple PCR-based strategy that can rapidly type *M. avium* isolates into

The sequence of the 16S rDNA gene is specific at the species level and is also a stable prop‐ erty of microorganisms. Wilton and Cousins described a method for the simultaneous iden‐ tification of genus, species and strains of *Mycobacterium* sp. using conserved and variable sequences of the 16S rDNA gene [145]. Comparing the 16S rDNA sequences of mycobacteri‐ al pathogens, they found variable regions specific for individual species and used the infor‐ mation to develop a duplex amplification system, which makes it possible to identify the genus *Mycobacterium*, and the species *M. a. avium* and *M. intracellulare*. If combining the pri‐ mers for 16S rDNA with primers specific to the gene that encodes the secretion protein MPB70 (specific for *Mycobacterium tuberculosis* complex) this system permits the detection and identification of clinically important mycobacteria in one single PCR. The disadvantage of this method is that it does not distinguish between *M. a. avium* and *M. a. paratuberculosis*. Combining PCR amplification of the 16S rDNA gene and subsequent restriction analysis we have the PCR-REA (or PRA) method. Using the 16S rDNA gene primers according to Thier‐ ry et al. and the resulting PCR products, 1 300 bp in size digested with *Rsa I* it is possible to distinguish species of MAC (*M. a. avium* serotypes 1–3, 8–11 and 21, *M. a. paratuberculosis*)

Standard (housekeeping) genes offer a higher level of sequence variation than do ribosomal genes but are nonetheless useful for taxonomic purposes due to the relative sequence con‐ servation imposed to maintain function. In this category, the stress protein gene hsp65 is a preferred target for mycobacterial identification to the species level, having been routinely used in diagnostics since the development of rapid PCR-restriction enzyme analysis (PRA) methods. The *dnaJ* gene encodes a stress chaperone protein and is highly conserved among the bacterial genera [149]. Morakova et al. designed primers specific for the *dnaJ* gene in the *M. avium* species that allow amplification of the *dnaJ* gene in all isolates of all *M. avium* sub‐ species and the authors suggest using them as an internal standard in the multiplex PCR to control inhibition of the amplification, and consequently false negatives, because are highly specific for at least *M. avium* [108]. The same team designed a fast and specific PCR strategy for the detection and differentiation of *M. avium* subspecies for use in routine veterinary di‐

nomic targets, such as the 16S rRNA gene and the ITS sequence [3].

and *M. intracellulare* (serotypes 7, 12–20, 22–28) [148].

these subgroups [147].

Additional gene loci specific for *Map* have been identified and suggested for use in diagnos‐ tics: ISMav2 [134], f57 [135], IS*Map*02 [136], and other *Map* specific coding sequences [137].

To identify the methods which are best suited for diagnostics, eight single-round and five nested PCR systems including twelve different primer pairs based on IS*900* (9x), 7 ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared by Möbius et al., which conclud‐ ed that stringent selection of IS*900*-specific primers ensures that IS*900* remains a favourite target sequence for amplification of *Map* specific loci [138]. A PCR system targeting two dif‐ ferent *Map* specific regions would have a still higher specificity. The following six single round PCR-systems were recommended by Möbius et al. [138]: IS*900* based PCRs of En‐ glund et al. [139], and Doran et al. [140], the f57 based PCR assay [125], and the locus 255 based PCR assay [137]. However, despite their advantages, such as a hundredfold enhance‐ ment of sensitivity, nested PCR assays bear a high risk of contamination and crossing over and, therefore, cannot be recommended as a reliable method for routine diagnosis of *Map*.

The insertion sequence IS901 was discovered by Kunze et al. and shows around 60% se‐ quence homology to IS*900* [117]. The stability of IS*901* in strains isolated primarily from clin‐ ical material from birds, domestic animals and from the environment is used for the rapid identification of IS*901*+ strains using the PCR method. Screening across a larger panel of iso‐ lates revealed that most isolates from birds and some animals contained the element, where‐ as isolates obtained from AIDS patients or the environment did not. Furthermore, it was found that most bird isolates had similar IS*901* patterns [3].

IS*1311* was first reported as a GenBank entry in 1994 (U16276) and was subsequently used for RFLP analyses [119,141]. This element is present in all members of the *M. avium* subspe‐ cies, including *Maa*, *Mah*, and *Map* [142], and is not present in *M. intracellulare* [22,141]. The element itself has 85% sequence identity to IS*1245*. With the wide range of *M. avium* hosting for this element, it is possible that IS*1311* represents an "older" IS element which may have been present prior to subspecies divergence [3]. RFLP analysis of IS*1311* and the use of IS*1311* PCR-REA also revealed distinct pattern types, corresponding to different genotype species strains of *M. avium* subsp. *paratuberculosis* [22,142].

The IS*1245* was first described in 1995, is present in up to 27 copies in *Mycobacterium avium* [118], was presented as having a more restricted range than IS*1311*, and was found to be sta‐ ble during in vivo and in vitro passage, making it a popular target for restriction fragment length polymorphism strain typing. It is described as limited to the subspecies of *M. avium*, i.e., *Maa* (that would include *Mah*), *Map* subsp. *paratuberculosis*, and *M. avium* subsp. *silvati‐ cum*. By PCR analysis, this element was not found in *M. intracellulare* or in 17 other mycobac‐ terium species [3]. Standardization of IS*1245* RFLP analysis was proposed in 1998 as a tool for MAC molecular epidemiology [143]. Some *M. avium* isolates have been documented as being IS*1245* negative, but only a few such reports have presented further documentation of strain identity by a sequence-based method [144]. Beggs et al. found IS*1245* in strains of *M. intracellulare*, demonstrating that the element is present in other species of the *M. avium* com‐ plex [144]. In some reports, IS*1245*-negative isolates have been described that contain an hsp65 sequence identical to that of *M. avium* but that differs from *M. avium* in other taxo‐ nomic targets, such as the 16S rRNA gene and the ITS sequence [3].

RFLP analysis of the IS*900* element has been used a molecular tool to type *Map* isolates and allowed the division of *Map* into different groups, associated to different host species [3]. The IS*900* element is by far the most widely used target for the molecular detection of *Map* and has been used in the form of direct PCR [108,125], *in situ* PCR [128] nested PCR [126,129,130], and real-time PCR [131-133]. Sequencing of the amplified product for IS*900* is

Additional gene loci specific for *Map* have been identified and suggested for use in diagnos‐ tics: ISMav2 [134], f57 [135], IS*Map*02 [136], and other *Map* specific coding sequences [137].

To identify the methods which are best suited for diagnostics, eight single-round and five nested PCR systems including twelve different primer pairs based on IS*900* (9x), 7 ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared by Möbius et al., which conclud‐ ed that stringent selection of IS*900*-specific primers ensures that IS*900* remains a favourite target sequence for amplification of *Map* specific loci [138]. A PCR system targeting two dif‐ ferent *Map* specific regions would have a still higher specificity. The following six single round PCR-systems were recommended by Möbius et al. [138]: IS*900* based PCRs of En‐ glund et al. [139], and Doran et al. [140], the f57 based PCR assay [125], and the locus 255 based PCR assay [137]. However, despite their advantages, such as a hundredfold enhance‐ ment of sensitivity, nested PCR assays bear a high risk of contamination and crossing over and, therefore, cannot be recommended as a reliable method for routine diagnosis of *Map*.

The insertion sequence IS901 was discovered by Kunze et al. and shows around 60% se‐ quence homology to IS*900* [117]. The stability of IS*901* in strains isolated primarily from clin‐ ical material from birds, domestic animals and from the environment is used for the rapid identification of IS*901*+ strains using the PCR method. Screening across a larger panel of iso‐ lates revealed that most isolates from birds and some animals contained the element, where‐ as isolates obtained from AIDS patients or the environment did not. Furthermore, it was

IS*1311* was first reported as a GenBank entry in 1994 (U16276) and was subsequently used for RFLP analyses [119,141]. This element is present in all members of the *M. avium* subspe‐ cies, including *Maa*, *Mah*, and *Map* [142], and is not present in *M. intracellulare* [22,141]. The element itself has 85% sequence identity to IS*1245*. With the wide range of *M. avium* hosting for this element, it is possible that IS*1311* represents an "older" IS element which may have been present prior to subspecies divergence [3]. RFLP analysis of IS*1311* and the use of IS*1311* PCR-REA also revealed distinct pattern types, corresponding to different genotype

The IS*1245* was first described in 1995, is present in up to 27 copies in *Mycobacterium avium* [118], was presented as having a more restricted range than IS*1311*, and was found to be sta‐ ble during in vivo and in vitro passage, making it a popular target for restriction fragment length polymorphism strain typing. It is described as limited to the subspecies of *M. avium*, i.e., *Maa* (that would include *Mah*), *Map* subsp. *paratuberculosis*, and *M. avium* subsp. *silvati‐ cum*. By PCR analysis, this element was not found in *M. intracellulare* or in 17 other mycobac‐ terium species [3]. Standardization of IS*1245* RFLP analysis was proposed in 1998 as a tool

therefore necessary to confirm that the amplicon is truly IS*900*.

106 Insights from Veterinary Medicine

found that most bird isolates had similar IS*901* patterns [3].

species strains of *M. avium* subsp. *paratuberculosis* [22,142].

Other identification methods of *M. avium* species or its subspecies are based on 16S rDNA [145], PCR-REA (Restriction Endonuclease Analysis) [146], sequence analysis of hsp65 [8] or a strategy based on large sequence polymorphisms [147]. Semret et al. evaluating the distri‐ bution of genomic polymorphisms across a panel of strains, verified that it was possible to assign unique genomic signatures to host-associated variants and based on these polymor‐ phisms proposed a simple PCR-based strategy that can rapidly type *M. avium* isolates into these subgroups [147].

The sequence of the 16S rDNA gene is specific at the species level and is also a stable prop‐ erty of microorganisms. Wilton and Cousins described a method for the simultaneous iden‐ tification of genus, species and strains of *Mycobacterium* sp. using conserved and variable sequences of the 16S rDNA gene [145]. Comparing the 16S rDNA sequences of mycobacteri‐ al pathogens, they found variable regions specific for individual species and used the infor‐ mation to develop a duplex amplification system, which makes it possible to identify the genus *Mycobacterium*, and the species *M. a. avium* and *M. intracellulare*. If combining the pri‐ mers for 16S rDNA with primers specific to the gene that encodes the secretion protein MPB70 (specific for *Mycobacterium tuberculosis* complex) this system permits the detection and identification of clinically important mycobacteria in one single PCR. The disadvantage of this method is that it does not distinguish between *M. a. avium* and *M. a. paratuberculosis*.

Combining PCR amplification of the 16S rDNA gene and subsequent restriction analysis we have the PCR-REA (or PRA) method. Using the 16S rDNA gene primers according to Thier‐ ry et al. and the resulting PCR products, 1 300 bp in size digested with *Rsa I* it is possible to distinguish species of MAC (*M. a. avium* serotypes 1–3, 8–11 and 21, *M. a. paratuberculosis*) and *M. intracellulare* (serotypes 7, 12–20, 22–28) [148].

Standard (housekeeping) genes offer a higher level of sequence variation than do ribosomal genes but are nonetheless useful for taxonomic purposes due to the relative sequence con‐ servation imposed to maintain function. In this category, the stress protein gene hsp65 is a preferred target for mycobacterial identification to the species level, having been routinely used in diagnostics since the development of rapid PCR-restriction enzyme analysis (PRA) methods. The *dnaJ* gene encodes a stress chaperone protein and is highly conserved among the bacterial genera [149]. Morakova et al. designed primers specific for the *dnaJ* gene in the *M. avium* species that allow amplification of the *dnaJ* gene in all isolates of all *M. avium* sub‐ species and the authors suggest using them as an internal standard in the multiplex PCR to control inhibition of the amplification, and consequently false negatives, because are highly specific for at least *M. avium* [108]. The same team designed a fast and specific PCR strategy for the detection and differentiation of *M. avium* subspecies for use in routine veterinary di‐ agnosis [108]. They have developed a multiplex PCR based on IS*900*, IS*901*, IS*1245* and the *dnaJ* gene. This method allows the detection of *M. a. paratuberculosis*, *M. a. hominissuis* and *M. a. avium*/*M. a. silvaticum* in one PCR reaction (PCR multiplex) and theoretically enables the detection of mixed infections of *M. a. paratuberculosis* and *M. a. avium* or M*. a. paratuberculosis* and *M. a. hominissuis*. The sensitivity of this multiplex PCR is 103 CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.

high grade fever, high sweats, anaemia and weight loss in addition to nonspecific symptoms of malaise, anorexia, diarrhea, myalgia and occasional painful adenophaty [88]. Epidemiolo‐ gy of MAC complex in patients without HIV infection remains somewhat difficult to deter‐ mine since the disease is relatively uncommon, it is not a reportable health event and environmental exposure varies greatly according to the geographic region [156]. In an epide‐ miological survey in USA from 2000 to 2003 performed in [156] in patients without HIV in‐ fection the rate of positive non-tuberculous cultures was 17.7 per 100,000. Surveys conducted in Europe estimated the rate of disease anywhere in the human body to be 0.8-3.1 per 100,000 [157,158]. In a recent study, Bodle et al. estimated the incidence of non-tubercu‐ lous mycobacteria in the respiratory tract disease in 2.0 per 100,000 and the disease in any‐ where in the human body in 2.7 per 100,000 [156]. Another recently published study showed that the patient´s lung disease was likely acquired by inhalation of aerosols while showering [159]. *M. avium* may reach the lungs by aspiration because a considerable percentage of pa‐ tients with non-tuberculous mycobacteria (NMT) disease have been found to experience

*Mycobacterium avium* Complex in Domestic and Wild Animals

http://dx.doi.org/10.5772/54323

109

Disease patterns of MAC are different in immunocompromised patients. In adults, infection is mainly pulmonary [1,151]. MAC is the most common of the nontuberculous mycobacteria found in apparently healthy children [162] and it's infection is characterized by a chronic granulomatous lymphadenopathy in the neck region that preferably is treated by excision of the affected lymph node [162,163]. The main hypothesis of infection is that oral contact with *M. avium*-infected water courses causes lymphadenitis in the head and neck region in chil‐ dren [162,164]. Among the members of MAC, *Maa* is predominant (87-98%) in AIDS patients and induces disseminated mycobacteriemia rather than restrict the bacteria to the lungs [1]. Since the advent of AIDS, HIV has become the major risk factor for MAC infection. In AIDS patients the main route of infection is the gastrointestinal tract and *M. avium* is naturally tol‐ erant to the low pH that exists in the stomach [19,165]. Regarding therapeutics, *M. avium* is of special concern because drugs commonly used from treating tuberculosis in humans are not effective [166] and MAC strains are resistant to isoniazid, the most popular anti-tubercu‐ losis drug [19,167]. Preexisting pulmonary conditions, patients with current illness or immu‐ nosuppressive medication are the most important risk factors for MAC infection amongst patients without HIV infection. MAC was also reported as the most common pathogen caus‐ ing post transplant non-tuberculous mycobacteria disease [156]. Other factors are local trau‐ mas and surgical procedures injuries [168]. Chronic obstructive pulmonary disease, emphysema, pneumoconiosis, aspiration due to oesophageal disease, previous gastrectomy and chronic alcoholism are some of the conditions which have been linked to disease [88,154]. Disseminated MAC infection is more frequent in caucasians compared with His‐ panic or African-americans individuals. However, there are no differences related with age [169,170]. Infection is more frequent in men than in women, particularly in homosexual and

The zoonotic potential of *Map* has been debated for almost a century because of similarities between Johne´s disease in cattle and Crohn´s disease in humans. A quarter century later since *Map* was first proposed as an etiologic agent in Crohn´s disease based on the isolation

gastroesophageal reflux disease [160,161].

bisexual men, when compared to other HIV risk categories.

Shin et al. designed a five-target multiplex PCR to discriminate MAC organisms isolated. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all *Myco‐ bacterium species*, a chromosomal target called DT1 that is unique to *Maa* serotypes 2 and 3, to *M. avium* subsp. *silvaticum*, and to *M. intracellulare*, and three insertion sequences, IS*900*, IS*901*, and IS*1311*. The results for the pattern of amplification allowed to determine whether isolates were mycobacteria, or members of MAC, and to classify them into one of the three major MAC subspecies, *Map*, *Maa*, *Mah* [18].

#### **5. Public health concerns**

Zoonotic aspects of mycobacteria transmitted by the environment and wildlife highlights a major health problem. MAC causes a variety of disorders including tuberculosis-like diseas‐ es in animals and in human immunocompetent or immunocompromised patients. Suscepti‐ bility to mycobacterial infections depends of risk factors since they are ubiquous in the soil and water [150]. Human exposure to mycobacterium present in wildlife and in nature can occur by a variety of routes. Humans are continuously exposed at a low level (50 to 5000 bacilli per day). Contact with water, municipal or natural are also important routes for my‐ cobacteria infection. Birds are major excretors of the agent in their faeces and the bacteria can persist in the soil and in water for long [1].

Healthy humans have a low susceptibility to MAC infection and only a very small percent‐ age of mycobacteria progress trough to infection, but in immunocompromised individuals infected with HIV or leukaemia patients, treated with steroid therapy, chemotherapy or oth‐ er immunosuppressive medication, should be carefully considered regarding their possibili‐ ty to come in contact with birds with mycobacterial infection [1,151]. Prior to the introduction of highly active antiretroviral treatment more than 40% of patients developed *M. avium* complex bacteriemia two years after the AIDS diagnosis [152] and a disseminated MAC infection was found in as much as 50% of autopsied AIDS patients [153,154]. This has predominantly been attributed to the impairment of the adaptative part of the immune sys‐ tems in HIV-1 infected individuals due to the loss of CD4+ T cells, as the susceptibility to opportunistic infections including *M. avium* infection is correlated with a decline in this cell type [155]. MAC usually produces clinical disease only when CD4+ are very low (< 50 cells/ ml), which is seen in 4 to 5 per cent of HIV infected patients [88]. A recent study showed that exposure of dendritic cells to HIV-1 promotes or facilitates the intracellular growth of *M. avium* [153]. Signs and symptoms associated with MAC disease in AIDS cases are persistent high grade fever, high sweats, anaemia and weight loss in addition to nonspecific symptoms of malaise, anorexia, diarrhea, myalgia and occasional painful adenophaty [88]. Epidemiolo‐ gy of MAC complex in patients without HIV infection remains somewhat difficult to deter‐ mine since the disease is relatively uncommon, it is not a reportable health event and environmental exposure varies greatly according to the geographic region [156]. In an epide‐ miological survey in USA from 2000 to 2003 performed in [156] in patients without HIV in‐ fection the rate of positive non-tuberculous cultures was 17.7 per 100,000. Surveys conducted in Europe estimated the rate of disease anywhere in the human body to be 0.8-3.1 per 100,000 [157,158]. In a recent study, Bodle et al. estimated the incidence of non-tubercu‐ lous mycobacteria in the respiratory tract disease in 2.0 per 100,000 and the disease in any‐ where in the human body in 2.7 per 100,000 [156]. Another recently published study showed that the patient´s lung disease was likely acquired by inhalation of aerosols while showering [159]. *M. avium* may reach the lungs by aspiration because a considerable percentage of pa‐ tients with non-tuberculous mycobacteria (NMT) disease have been found to experience gastroesophageal reflux disease [160,161].

agnosis [108]. They have developed a multiplex PCR based on IS*900*, IS*901*, IS*1245* and the *dnaJ* gene. This method allows the detection of *M. a. paratuberculosis*, *M. a. hominissuis* and *M. a. avium*/*M. a. silvaticum* in one PCR reaction (PCR multiplex) and theoretically enables the detection of mixed infections of *M. a. paratuberculosis* and *M. a. avium* or M*. a. paratuberculosis* and *M. a. hominissuis*. The sensitivity of this multiplex PCR is 103 CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated

Shin et al. designed a five-target multiplex PCR to discriminate MAC organisms isolated. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all *Myco‐ bacterium species*, a chromosomal target called DT1 that is unique to *Maa* serotypes 2 and 3, to *M. avium* subsp. *silvaticum*, and to *M. intracellulare*, and three insertion sequences, IS*900*, IS*901*, and IS*1311*. The results for the pattern of amplification allowed to determine whether isolates were mycobacteria, or members of MAC, and to classify them into one of the three

Zoonotic aspects of mycobacteria transmitted by the environment and wildlife highlights a major health problem. MAC causes a variety of disorders including tuberculosis-like diseas‐ es in animals and in human immunocompetent or immunocompromised patients. Suscepti‐ bility to mycobacterial infections depends of risk factors since they are ubiquous in the soil and water [150]. Human exposure to mycobacterium present in wildlife and in nature can occur by a variety of routes. Humans are continuously exposed at a low level (50 to 5000 bacilli per day). Contact with water, municipal or natural are also important routes for my‐ cobacteria infection. Birds are major excretors of the agent in their faeces and the bacteria

Healthy humans have a low susceptibility to MAC infection and only a very small percent‐ age of mycobacteria progress trough to infection, but in immunocompromised individuals infected with HIV or leukaemia patients, treated with steroid therapy, chemotherapy or oth‐ er immunosuppressive medication, should be carefully considered regarding their possibili‐ ty to come in contact with birds with mycobacterial infection [1,151]. Prior to the introduction of highly active antiretroviral treatment more than 40% of patients developed *M. avium* complex bacteriemia two years after the AIDS diagnosis [152] and a disseminated MAC infection was found in as much as 50% of autopsied AIDS patients [153,154]. This has predominantly been attributed to the impairment of the adaptative part of the immune sys‐ tems in HIV-1 infected individuals due to the loss of CD4+ T cells, as the susceptibility to opportunistic infections including *M. avium* infection is correlated with a decline in this cell type [155]. MAC usually produces clinical disease only when CD4+ are very low (< 50 cells/ ml), which is seen in 4 to 5 per cent of HIV infected patients [88]. A recent study showed that exposure of dendritic cells to HIV-1 promotes or facilitates the intracellular growth of *M. avium* [153]. Signs and symptoms associated with MAC disease in AIDS cases are persistent

from the tissue of the heavily infected sheep.

108 Insights from Veterinary Medicine

major MAC subspecies, *Map*, *Maa*, *Mah* [18].

can persist in the soil and in water for long [1].

**5. Public health concerns**

Disease patterns of MAC are different in immunocompromised patients. In adults, infection is mainly pulmonary [1,151]. MAC is the most common of the nontuberculous mycobacteria found in apparently healthy children [162] and it's infection is characterized by a chronic granulomatous lymphadenopathy in the neck region that preferably is treated by excision of the affected lymph node [162,163]. The main hypothesis of infection is that oral contact with *M. avium*-infected water courses causes lymphadenitis in the head and neck region in chil‐ dren [162,164]. Among the members of MAC, *Maa* is predominant (87-98%) in AIDS patients and induces disseminated mycobacteriemia rather than restrict the bacteria to the lungs [1]. Since the advent of AIDS, HIV has become the major risk factor for MAC infection. In AIDS patients the main route of infection is the gastrointestinal tract and *M. avium* is naturally tol‐ erant to the low pH that exists in the stomach [19,165]. Regarding therapeutics, *M. avium* is of special concern because drugs commonly used from treating tuberculosis in humans are not effective [166] and MAC strains are resistant to isoniazid, the most popular anti-tubercu‐ losis drug [19,167]. Preexisting pulmonary conditions, patients with current illness or immu‐ nosuppressive medication are the most important risk factors for MAC infection amongst patients without HIV infection. MAC was also reported as the most common pathogen caus‐ ing post transplant non-tuberculous mycobacteria disease [156]. Other factors are local trau‐ mas and surgical procedures injuries [168]. Chronic obstructive pulmonary disease, emphysema, pneumoconiosis, aspiration due to oesophageal disease, previous gastrectomy and chronic alcoholism are some of the conditions which have been linked to disease [88,154]. Disseminated MAC infection is more frequent in caucasians compared with His‐ panic or African-americans individuals. However, there are no differences related with age [169,170]. Infection is more frequent in men than in women, particularly in homosexual and bisexual men, when compared to other HIV risk categories.

The zoonotic potential of *Map* has been debated for almost a century because of similarities between Johne´s disease in cattle and Crohn´s disease in humans. A quarter century later since *Map* was first proposed as an etiologic agent in Crohn´s disease based on the isolation of the organism from several patients the association as a causative agent or in an incidental away remains unresolved. Milk and water are potential sources for acquiring *Map* infection [171]. However, only a few samples of milk, positive by PCR for the presence of *Map* have yielded positive results in culture, suggesting that *Map* remains undetectable probably be‐ cause the low number of viable *Map* in samples [172]. Published reports indicate that *Map* may not be completely inactivated by pasteurization of milk [173]. Serological response to *Map* in humans is also not conclusive [172]. Traditional methods of detecting bacteria, cul‐ ture and stain are largely ineffective in detecting *Map* in humans. Bacteria are very difficult to culture and *Map* is able to exist in a spheroplast (cell wall deficient form) in humans so it cannot be identified by Ziehl-Neelsen staining [174-176]. Polymerase chain reaction has identified *Map* in greater than 90% of biopsy specimens from Crohn´s patients [177] and via‐ ble *Map* was detected in peripheral blood and serum in a higher proportion of individuals with Crohn's disease [178,179]. However, culture of *Map* from human specimens is a contro‐ versial question since some authors pointed out the difficulty of culturing *Map* [180,181]. At this moment is not possible to know if *Map* is a primary etiological agent or secondary in‐ vader and further research is need to understand the possible links between this agent and Crohn disease [1].

The evidence for the zoonotic potential should not be neglected particularly in immunocom‐

Recent reports, suggesting an association between MAC and autoimmune and other chronic human diseases, alert to the importance of developing new studies on MAC biology, molec‐

Research to understand the impact of MAC in public health is needed as well as the deter‐ mination of transmission routes between humans and wildlife, which requires interdiscipli‐

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐

1 CECAV- Animal and Veterinary Research Center, University of Trás-os-Montes and Alto-

3 Department of Genetics and Biotechnology, IBB-Institute for Biotechnology and Bioengin‐ eering, Centre of Genomic and Biotechnology, University of Trás-os-Montes and Alto-

[1] Biet F, Boschiroli ML, Thorel MF, Guilloteau LA. Zoonotic Aspects of Mycobacteri‐ um bovis and Mycobacterium avium-intracellulare Complex (MAC). Veterinary re‐

[3] Turenne CY, Wallace R, Behr MA. Mycobacterium avium in the Postgenomic Era.

[4] Smole SC, McAleese F, Ngampasutadol J, von Reyn CF, Arbeit RD. Clinical and Epi‐ demiological Correlates of Genotypes within the Mycobacterium avium Complex

[2] Prescott LM, Harley JP, Klein DA. Microbiology. New York: McGraw-Hill; 2002.

2 School of Agriculture, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal

, Ana Matos2

, Manuela Matos3

*Mycobacterium avium* Complex in Domestic and Wild Animals

http://dx.doi.org/10.5772/54323

111

and

nary collaboration among medical, veterinary and other public health officials.

nanced by the Foundation for Science and Technology (FCT).

, Maria de Lurdes Pinto1

Douro, Department of Veterinary Sciences, Vila Real, Portugal

Clinical Microbiology Reviews. 2007;20(2) 205-229.

promised patients, both humans and animals.

ular diagnosis and epidemiology.

**Acknowledgements**

**Author details**

Ana Cláudia Coelho1

Maria dos Anjos Pires1

Douro, Vila Real, Portugal

search. 2005;36(3) 411-436.

**References**

There is a recent interest in *Map* as an immune trigger of several autoimmune diseases [182,183]. Environmental agents are postulated to trigger autism. Recently, a theory propos‐ ing a mechanism by which *Map* triggers autism through molecular mimicry to the heat shock protein HSP65, which stimulates antibodies that cross react with myelin basic protein, a common feature of autism [184]. Another recent study has associated the presence of *Map* with Blau syndrome, an autossomal dominant, and systemic inflammatory disease. The mu‐ tations of Blau syndrome are on the same gene on chromosome 16 (CARD 15) that confers susceptibility to Crohns´disease [185]. *Map* was also implicated in sarcoidosis [186,187], which is a multisystemic granulomatous disease with many features in common with myco‐ bacterial infection, and that, like Crohn´s disease, can be mimicked by slow bacterial infec‐ tions [188,189].

*Map* was also linked to ulcerative colitis, irritable bowel syndrome, autoimmune (type 1) diabetes, Hashimoto thyroiditis and multiple sclerosis [179,184]. Increasing evidence sug‐ gests a role for *Map* in autoimmune (type I) diabetes. It is postulated that this bacterium acts via molecular mimicry between its antigens (HSP65) and the pancreatic enzyme glutamic acid decarboxylase (GAD) [190-195].

## **6. Conclusion**

MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various domestic and wild mammals and birds. MAC can affect a wide-range of wild animals, but little has been pub‐ lished up to the moment on the clinical signs, which are rarely exhibited or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing, though often similar to those of their domesticated counterparts.

The evidence for the zoonotic potential should not be neglected particularly in immunocom‐ promised patients, both humans and animals.

Recent reports, suggesting an association between MAC and autoimmune and other chronic human diseases, alert to the importance of developing new studies on MAC biology, molec‐ ular diagnosis and epidemiology.

Research to understand the impact of MAC in public health is needed as well as the deter‐ mination of transmission routes between humans and wildlife, which requires interdiscipli‐ nary collaboration among medical, veterinary and other public health officials.

## **Acknowledgements**

of the organism from several patients the association as a causative agent or in an incidental away remains unresolved. Milk and water are potential sources for acquiring *Map* infection [171]. However, only a few samples of milk, positive by PCR for the presence of *Map* have yielded positive results in culture, suggesting that *Map* remains undetectable probably be‐ cause the low number of viable *Map* in samples [172]. Published reports indicate that *Map* may not be completely inactivated by pasteurization of milk [173]. Serological response to *Map* in humans is also not conclusive [172]. Traditional methods of detecting bacteria, cul‐ ture and stain are largely ineffective in detecting *Map* in humans. Bacteria are very difficult to culture and *Map* is able to exist in a spheroplast (cell wall deficient form) in humans so it cannot be identified by Ziehl-Neelsen staining [174-176]. Polymerase chain reaction has identified *Map* in greater than 90% of biopsy specimens from Crohn´s patients [177] and via‐ ble *Map* was detected in peripheral blood and serum in a higher proportion of individuals with Crohn's disease [178,179]. However, culture of *Map* from human specimens is a contro‐ versial question since some authors pointed out the difficulty of culturing *Map* [180,181]. At this moment is not possible to know if *Map* is a primary etiological agent or secondary in‐ vader and further research is need to understand the possible links between this agent and

There is a recent interest in *Map* as an immune trigger of several autoimmune diseases [182,183]. Environmental agents are postulated to trigger autism. Recently, a theory propos‐ ing a mechanism by which *Map* triggers autism through molecular mimicry to the heat shock protein HSP65, which stimulates antibodies that cross react with myelin basic protein, a common feature of autism [184]. Another recent study has associated the presence of *Map* with Blau syndrome, an autossomal dominant, and systemic inflammatory disease. The mu‐ tations of Blau syndrome are on the same gene on chromosome 16 (CARD 15) that confers susceptibility to Crohns´disease [185]. *Map* was also implicated in sarcoidosis [186,187], which is a multisystemic granulomatous disease with many features in common with myco‐ bacterial infection, and that, like Crohn´s disease, can be mimicked by slow bacterial infec‐

*Map* was also linked to ulcerative colitis, irritable bowel syndrome, autoimmune (type 1) diabetes, Hashimoto thyroiditis and multiple sclerosis [179,184]. Increasing evidence sug‐ gests a role for *Map* in autoimmune (type I) diabetes. It is postulated that this bacterium acts via molecular mimicry between its antigens (HSP65) and the pancreatic enzyme glutamic

MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various domestic and wild mammals and birds. MAC can affect a wide-range of wild animals, but little has been pub‐ lished up to the moment on the clinical signs, which are rarely exhibited or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing,

though often similar to those of their domesticated counterparts.

Crohn disease [1].

110 Insights from Veterinary Medicine

tions [188,189].

**6. Conclusion**

acid decarboxylase (GAD) [190-195].

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐ nanced by the Foundation for Science and Technology (FCT).

## **Author details**

Ana Cláudia Coelho1 , Maria de Lurdes Pinto1 , Ana Matos2 , Manuela Matos3 and Maria dos Anjos Pires1

1 CECAV- Animal and Veterinary Research Center, University of Trás-os-Montes and Alto-Douro, Department of Veterinary Sciences, Vila Real, Portugal

2 School of Agriculture, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal

3 Department of Genetics and Biotechnology, IBB-Institute for Biotechnology and Bioengin‐ eering, Centre of Genomic and Biotechnology, University of Trás-os-Montes and Alto-Douro, Vila Real, Portugal

## **References**


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*Mycobacterium avium* Complex in Domestic and Wild Animals

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[17] Thorel M-F, Krichevsky M, Vincent Lévy-Frébault V. Numerical Taxonomy of Myco‐ bactin-Dependent Mycobacteria, Emended Description of Mycobacterium avium, and Description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvati‐ cum subsp. nov. International Journal of Systematic Bacteriology. 1990;40(3) 254-260.

[18] Shin SJ, Lee BS, Koh W-J, Manning EJB, Anklam K, Sreevatsan S, Lambrecht RS, Col‐ lins MT. Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR. Journal of Clinical Microbiology.

[19] Dhama K, Mahendran M, Tiwari R, Dayal Singh S, Kumar D, Singh S, Sawant PM. Tuberculosis in Birds: Insights into the Mycobacterium avium Infections. Veterinary

Medicine International. 2011; 2011:1-14. http://dx.doi.org/10.4061/2011/712369. [20] Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, Collins JE, Cernicchiaro N, Whitlock RH. Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle. Clinical and Vaccine

[21] Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall DJ, Fraser CA. Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of My‐ cobacterium avium subsp. paratuberculosis from Sheep. Journal of Clinical Microbi‐

[22] Whittington R, I., Marsh EC, Cousins D. Polymorphisms In IS1311, an Insertion Se‐ quence Common to Mycobacterium avium and M. avium subsp. paratuberculosis, Can Be Used to Distinguish Between and within these Species. Molecular and Cellu‐

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and Type-1 Diabetes Mellitus. PLoS ONE. 2009; 4(9):e7109. http://dx.doi.org/ 10.1371%2Fjournal.pone.0007109.

**Chapter 5**

**Psittacosis**

Maria dos Anjos Pires

http://dx.doi.org/10.5772/53720

**1. Introduction**

ed [8].

disease [18,19].

João Morais, Ana Cláudia Coelho and

Additional information is available at the end of the chapter

emphasizes the scientific reports worldwide [12-15].

animals are showing clinical signs of disease [16,17].

There are three morphologically distinct forms of *Chlamydophila*:

*Chlamydophila psittaci* (Bacteria kingdom, Chlamydiae phylum, Chlamydiae class, Chlamy‐ diales order, Chlamydiaceae family) is part of the genus *Chlamydophila*, where *Cp. abortus*, *Cp.*

Avian chlamydiosis induced by this Gram-negative obligate intracellular bacteria [2] is tradi‐ tionally known as ornithosis or psittacosis [3-5]. It is an infectious disease capable of infecting domestic and wild birds [6,7], being the Psittacidae family where most occurrences are report‐

*Chlamydophila psittaci* infects primarily birds [9], but mammals, including humans, are also sus‐ ceptible of infection [10-11]. In fact, zoonotic status of this organism is largely described and

*Chlamydophila psittaci* can be found in bird feathers, excrements and blood, whether or not these

Psittacosis can be transmitted by vertical and horizontal via [2]. The agent is excreted on faeces and ingested from the food or inhaled via aerosols [13]. At the lungs of newly infected animals, the organism gets an infecting status becoming capable to replicate and causing clinical signs of

**•** The elementary body, which is small, spherical, of about 0.2-0.3 mm in diameter;

**•** The reticular body, wider, of about 0.5-2.0 mm, which is able to replicate by binary division;

**•** And the intermediate body, with 0.3-2.0 mm in diameter, seen in infected host cells [18,20].

© 2013 Morais et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Morais et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

*caviae*, *Cp. felis*, *Cp. pecorum* and *Cp. pneumoniae* are the other five species of bacteria [1].


**Chapter 5**

## **Psittacosis**

and Type-1 Diabetes Mellitus. PLoS ONE. 2009; 4(9):e7109. http://dx.doi.org/

LA. Specific Immunoassays Confirm Association of Mycobacterium avium Subsp. paratuberculosis with Type-1 but Not Type-2 Diabetes Mellitus. PLoS ONE. 2009;

are specific tags of Mycobacterium avium subsp. paratuberculosis infection in type I

[194] Rosu V, Ahmed N, Paccagnini D, Gerlach G, Fadda G, Hasnain SE, Zanetti S, Sechi

[195] Cossu A, Rosu V, Paccagnini D, Cossu D, Pacifico A, Sechi LA. MAP3738c and MptD

4(2):e4386. http://dx.doi.org/10.1371%2Fjournal.pone.0004386.

diabetes mellitus. Clinical Immunology. 2011;141(1) 49-57.

10.1371%2Fjournal.pone.0007109.

128 Insights from Veterinary Medicine

João Morais, Ana Cláudia Coelho and Maria dos Anjos Pires

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53720

## **1. Introduction**

*Chlamydophila psittaci* (Bacteria kingdom, Chlamydiae phylum, Chlamydiae class, Chlamy‐ diales order, Chlamydiaceae family) is part of the genus *Chlamydophila*, where *Cp. abortus*, *Cp. caviae*, *Cp. felis*, *Cp. pecorum* and *Cp. pneumoniae* are the other five species of bacteria [1].

Avian chlamydiosis induced by this Gram-negative obligate intracellular bacteria [2] is tradi‐ tionally known as ornithosis or psittacosis [3-5]. It is an infectious disease capable of infecting domestic and wild birds [6,7], being the Psittacidae family where most occurrences are report‐ ed [8].

*Chlamydophila psittaci* infects primarily birds [9], but mammals, including humans, are also sus‐ ceptible of infection [10-11]. In fact, zoonotic status of this organism is largely described and emphasizes the scientific reports worldwide [12-15].

*Chlamydophila psittaci* can be found in bird feathers, excrements and blood, whether or not these animals are showing clinical signs of disease [16,17].

Psittacosis can be transmitted by vertical and horizontal via [2]. The agent is excreted on faeces and ingested from the food or inhaled via aerosols [13]. At the lungs of newly infected animals, the organism gets an infecting status becoming capable to replicate and causing clinical signs of disease [18,19].

There are three morphologically distinct forms of *Chlamydophila*:


© 2013 Morais et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Morais et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Parrots are often infected animals but they show no signs of disease unless they are stressed out [21], and thus can be a source of contamination to other birds and mam‐ mals, so as the man [3,4,22]. Infections in turkeys have been described long ago, with a mortality range of 5-40% without any treatment [23], and more recently in ducks, with a mortality of 30%, which was highlighted as an economic problem and health impairment [24]. The mortality rate in humans is low if treatment is appropriate. Therefore, setting up a quick prognosis is essential [20,23].

Later, Everett and collaborators (1999), proposed a new classification, where the *Chlamy‐ dophila* genus would contain six species: *Chlamydophila pneumoniae, Chlamydophila peco‐ rum, Chlamydophila abortus, Chlamydophila felis, Chlamydophila caviae* and *Chlamydophila psittaci* [1]. This taxonomic classification was accepted and is still used worldwide [17].

Psittacosis

131

http://dx.doi.org/10.5772/53720

The aim of this study was to discuss phylogeny, epidemiology, clinical signs, pathology, di‐ agnostic techniques, treatment, prevention and public health concerns in psittacosis with

Bacteria kingdom consists of different phyla, including the Chlamydiae, holding the class with the same name. The order Chlamydiales belongs to the class Chlamydiae and the fami‐ ly Chlamydiaceae to the order described. The genus *Chlamydophila* belongs to the family Chlamydiaceae and accordingly with the National Center For Biotechnology Information Taxonomy Browser (www.ncbi.nlm.nih.gov), it has six species of bacteria [1]. The Chlamy‐

In the genus *Chlamydophila, Chlamydophila abortus* is a bacterium whose disease is se‐ vere, especially in small ruminants [37], but it is also described as an important zoonot‐

*Chlamydophila pneumoniae* has been primarily described as an infection in humans but later has been reported in other animals as well, such as mammals, marsupials, reptiles and am‐ phibians [41-44]. The transmission of *Cp. pneumoniae* among animals and humans was not yet described; however, Myers and collaborators proved that human could be infected from

*Chlamydophila caviae* was found in animals such as rabbits, guinea pigs, horses, cats and dogs [45-48]. In 2006, it was reported the identification of this bacteria in humans, raising the pos‐

*Chlamydophila felis* affects cats mainly under one year old and is very often associated with conjunctivitis [49-52]. Although there is evidence that this bacterium can cause keratocon‐ junctivitis in humans, there is still little evidence that can create systemic disease or severe

*Chlamydophila pecorum* affects several small and large mammals such as ruminants, swine and koalas [54-59], but little or no studies have been published to date that can prove human

*Chlamydophila psittaci* is the causative agent of Psittacosis and is capable of infecting domes‐ tic and wild birds [6, 60], but also reptiles [61] and mammals such as man [10,11]. This bacte‐ rium is definitely an important zoonosis and such potential has been widely reported across

diae genus contains three species: *C. trachomatis, C. suis* and *C. muridarum* [1].

animal isolated bacteria following adaptation to the new host [44].

sibility that it has a zoonotic potential, which has not been yet clarified [45].

special attention due to *Chlamydophila psittaci* infection.

ic disease [38-40].

pneumonia in the man [53].

infection from this agent.

the years [12-15].

**2. Phylogeny and biology of Chlamydiacea family**

Clinical symptoms are very extensive in number and vary depending on the serotype of the bacteria and the animal affected. General infections may cause fever, anorexia, lethargy, diar‐ rhoea and sometimes shock or even death. Chlamydiosis in psittacines is mostly chronic and cause conjunctivitis, enteritis, sacculitis, pneumonitis, hepatomegaly and droppings can range from green to yellow-green in colour [20]. Clinical signs in humans range from unapparent to severe systemic disease with interstitial pneumonia and encephalitis [12,20]. Signs of disease are headache, chills, malaise and myalgia with possible respiratory involvement. Infection of pigeons by *Chlamydophila* is vast and well reported [15]. These animals are a major spreader of the disease, and present clinical signs such as conjunctivitis, blepharitis and rhinitis [20]. *Chla‐ mydophila psittaci* antibodies can be detected by several laboratory methodologies. The comple‐ ment fixation test (CFT) is the most frequently used test [25,26]. Other serological tests such as PCR (polymerase chain reaction) and ELISA are used as well for its diagnosis [8,22,27].

Psittacosis or "parrot fever" was documented for first time in 1879, when Jakob Ritter descri‐ bed an epidemic and unusual pneumonia, associated with exposure to tropical birds in sev‐ en Switch individuals causing flu-like symptoms and pneumonia [28]. The term "psittacosis" (from the Latin word for parrot - *psittacus*) was firstly used by Morange, in 1895, when trans‐ mission of an infectious agent to humans from parrots was notorious [29-30].

Investigations of chlamydiosis began in 1907 when microorganisms within intracytoplasmic vacuoles in conjunctival scraping cells from humans with trachoma were found. Trachoma is a chronic infectious disease of the conjunctiva and cornea [18] a disease well known since ancient civilizations [31]. These organisms were named *Chlamydozoa*, from the characteristic shape of the mantle or "*chlamys*" in Greek [32].

A psittacosis outbreak occurred during the winters of 1929 and 1930 in Europe and in the United States. The causative agent of psittacosis was isolated from birds and infected hu‐ mans and the source of contamination traced to parrots of the genus *Amazona*, originated in South America [33]. At the same time, a microorganism causing lymphogranuloma venere‐ um (LGV) was isolated in humans [34]. Until then, it was believed avian psittacosis was re‐ stricted to *Psittaciformes* birds [18]. However, a number of studies demonstrated a much higher number of species concerned or potentially infected by this microorganism. In fact, Meyer and Eddie, in 1932, described a case of transmission of human psittacosis from do‐ mestic fowl [35] and Haagen and Mauer, six years later, reported an infection in fulmar (*Fulmarus glacialis)*. Pinkerton and Swank, in 1940, proved the existence of this agent in do‐ mestic pigeons, as Wolins, in 1948, described the same disease in ducks [33]. Later studies of this matter enhanced the idea that psittacosis was not restricted to parrots, reporting infec‐ tions in humans by contact with other affected birds [18,36].

Later, Everett and collaborators (1999), proposed a new classification, where the *Chlamy‐ dophila* genus would contain six species: *Chlamydophila pneumoniae, Chlamydophila peco‐ rum, Chlamydophila abortus, Chlamydophila felis, Chlamydophila caviae* and *Chlamydophila psittaci* [1]. This taxonomic classification was accepted and is still used worldwide [17].

The aim of this study was to discuss phylogeny, epidemiology, clinical signs, pathology, di‐ agnostic techniques, treatment, prevention and public health concerns in psittacosis with special attention due to *Chlamydophila psittaci* infection.

## **2. Phylogeny and biology of Chlamydiacea family**

Parrots are often infected animals but they show no signs of disease unless they are stressed out [21], and thus can be a source of contamination to other birds and mam‐ mals, so as the man [3,4,22]. Infections in turkeys have been described long ago, with a mortality range of 5-40% without any treatment [23], and more recently in ducks, with a mortality of 30%, which was highlighted as an economic problem and health impairment [24]. The mortality rate in humans is low if treatment is appropriate. Therefore, setting

Clinical symptoms are very extensive in number and vary depending on the serotype of the bacteria and the animal affected. General infections may cause fever, anorexia, lethargy, diar‐ rhoea and sometimes shock or even death. Chlamydiosis in psittacines is mostly chronic and cause conjunctivitis, enteritis, sacculitis, pneumonitis, hepatomegaly and droppings can range from green to yellow-green in colour [20]. Clinical signs in humans range from unapparent to severe systemic disease with interstitial pneumonia and encephalitis [12,20]. Signs of disease are headache, chills, malaise and myalgia with possible respiratory involvement. Infection of pigeons by *Chlamydophila* is vast and well reported [15]. These animals are a major spreader of the disease, and present clinical signs such as conjunctivitis, blepharitis and rhinitis [20]. *Chla‐ mydophila psittaci* antibodies can be detected by several laboratory methodologies. The comple‐ ment fixation test (CFT) is the most frequently used test [25,26]. Other serological tests such as

PCR (polymerase chain reaction) and ELISA are used as well for its diagnosis [8,22,27].

mission of an infectious agent to humans from parrots was notorious [29-30].

Psittacosis or "parrot fever" was documented for first time in 1879, when Jakob Ritter descri‐ bed an epidemic and unusual pneumonia, associated with exposure to tropical birds in sev‐ en Switch individuals causing flu-like symptoms and pneumonia [28]. The term "psittacosis" (from the Latin word for parrot - *psittacus*) was firstly used by Morange, in 1895, when trans‐

Investigations of chlamydiosis began in 1907 when microorganisms within intracytoplasmic vacuoles in conjunctival scraping cells from humans with trachoma were found. Trachoma is a chronic infectious disease of the conjunctiva and cornea [18] a disease well known since ancient civilizations [31]. These organisms were named *Chlamydozoa*, from the characteristic

A psittacosis outbreak occurred during the winters of 1929 and 1930 in Europe and in the United States. The causative agent of psittacosis was isolated from birds and infected hu‐ mans and the source of contamination traced to parrots of the genus *Amazona*, originated in South America [33]. At the same time, a microorganism causing lymphogranuloma venere‐ um (LGV) was isolated in humans [34]. Until then, it was believed avian psittacosis was re‐ stricted to *Psittaciformes* birds [18]. However, a number of studies demonstrated a much higher number of species concerned or potentially infected by this microorganism. In fact, Meyer and Eddie, in 1932, described a case of transmission of human psittacosis from do‐ mestic fowl [35] and Haagen and Mauer, six years later, reported an infection in fulmar (*Fulmarus glacialis)*. Pinkerton and Swank, in 1940, proved the existence of this agent in do‐ mestic pigeons, as Wolins, in 1948, described the same disease in ducks [33]. Later studies of this matter enhanced the idea that psittacosis was not restricted to parrots, reporting infec‐

up a quick prognosis is essential [20,23].

130 Insights from Veterinary Medicine

shape of the mantle or "*chlamys*" in Greek [32].

tions in humans by contact with other affected birds [18,36].

Bacteria kingdom consists of different phyla, including the Chlamydiae, holding the class with the same name. The order Chlamydiales belongs to the class Chlamydiae and the fami‐ ly Chlamydiaceae to the order described. The genus *Chlamydophila* belongs to the family Chlamydiaceae and accordingly with the National Center For Biotechnology Information Taxonomy Browser (www.ncbi.nlm.nih.gov), it has six species of bacteria [1]. The Chlamy‐ diae genus contains three species: *C. trachomatis, C. suis* and *C. muridarum* [1].

In the genus *Chlamydophila, Chlamydophila abortus* is a bacterium whose disease is se‐ vere, especially in small ruminants [37], but it is also described as an important zoonot‐ ic disease [38-40].

*Chlamydophila pneumoniae* has been primarily described as an infection in humans but later has been reported in other animals as well, such as mammals, marsupials, reptiles and am‐ phibians [41-44]. The transmission of *Cp. pneumoniae* among animals and humans was not yet described; however, Myers and collaborators proved that human could be infected from animal isolated bacteria following adaptation to the new host [44].

*Chlamydophila caviae* was found in animals such as rabbits, guinea pigs, horses, cats and dogs [45-48]. In 2006, it was reported the identification of this bacteria in humans, raising the pos‐ sibility that it has a zoonotic potential, which has not been yet clarified [45].

*Chlamydophila felis* affects cats mainly under one year old and is very often associated with conjunctivitis [49-52]. Although there is evidence that this bacterium can cause keratocon‐ junctivitis in humans, there is still little evidence that can create systemic disease or severe pneumonia in the man [53].

*Chlamydophila pecorum* affects several small and large mammals such as ruminants, swine and koalas [54-59], but little or no studies have been published to date that can prove human infection from this agent.

*Chlamydophila psittaci* is the causative agent of Psittacosis and is capable of infecting domes‐ tic and wild birds [6, 60], but also reptiles [61] and mammals such as man [10,11]. This bacte‐ rium is definitely an important zoonosis and such potential has been widely reported across the years [12-15].

All species of the genera *Chlamydophila* and Chlamydiae have one or more serotypes, whose sequence are known [62-63] or are still under intense study [64].

Some studies have shown the existence of hemispherical-shaped projections of the cyto‐ plasm on the surface of the elementary bodies and ridge-shaped projections of the reticular forms for this bacterium [76-78]. These projections range from the bacteria to the surface of the membrane inclusion and it is speculated that can possess pores of type III secretion [32].

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133

http://dx.doi.org/10.5772/53720

Infection of *Chlamydophila psittaci* begins with the attachment of the elementary body to the host cell and subsequent parasite-mediated endocytosis [79]. The bacterium binds to the cell membrane receptor of the host cell, often associated with a cytoplasm protein known as cla‐ thrin [20]. This connection stimulates the cell to emit pseudopods; these increase in size until all bacteria have been surrounded, forming a vacuole delimited by clathrin, referred to as inclusion [18, 80]. In the vacuole, the bacterium has a biphasic cycle (Figure 1), alternating

Once inside the vacuole, the EB are redistributed from the periphery of the cell and join in the region of the Golgi apparatus, which corresponds to the MTOC (Microtubule Organiz‐ ing Centre) [19, 80]. The non-acid inclusions are firstly too small, allowing to avoid its fusion with lysosomes, and are formed by endocytic constituents of the plasma membrane of the host cell such as proteins and lipids [19, 80]. All of these conditions lead to a more efficient

On the first hours, the elementary bodies initiate to differentiate into reticular bodies. These forms of *Chlamydophila* are not infectious, have a non-condensed nucleoid and a greater size than EB [19, 65]. From eight to ten hours of cycle, the reticular bodies start to replicate by binary fission, remaining in contact with the inclusion membrane [20, 65]. In this moment, it's possible to see numerous mitochondria surrounding the inclusion [18, 20], possibly due to protein motors function as kinesins [81]. *Cp. psittaci* may not be able to generate as much energy in the form of ATP as other species of the genus, so a deeper association with the host cell mitochondria is necessary for the bacteria to recruit the necessary ATP by an alter‐ native mechanism [20]. The migration of nutrients from the host cell into the inclusion is es‐ sential. Amino acids and nucleotides cross the inclusion membrane [20, 82]. Since the vacuole is not in the vicinity of lysosomes leads to the assumption that the releasing of nu‐ trients by fusion with endocytic vesicles is unlikely [20]. It is known that exocytic vesicles containing sphingomyelin fuse with the membrane of the inclusion, delivering nutrients and lipids to the bacteria [80]. It was demonstrated that the inclusion membrane is permeable to small molecules of a molecular weight ranging from 100-520 Da [83,84]. After releasing the nutrients into the lumen of the bacterium by passive diffusion, membrane-specific carriers/ transporters located in the membrane would facilitate the entry of new nutrients and would be indispensable for the entry of molecules larger than 520 Da, but such carriers have not

Throughout the cycle, the size of the vesicles increases, as they accumulate growing num‐ bers of bacteria inside [20, 84]. The surface of the inclusion membrane also increases, while it intercepts biosynthetic transport pathways of the host cell membrane and acquires the abili‐ ty to fuse with a subset of Golgi derived vesicles [20, 84]. Within 20 hours of cycle, reticular

between states of elementary body and reticular body [19].

survival and infection by the bacterium [20].

been yet described [20].

**c.** Pathogenesis

#### **a.** *Chlamydophila psittaci*

*Chlamydophila psittaci* is negative to Gram test bacterium [2, 49, 57], having a cytoplasmic membrane and an outer three-layer membrane [65-66], with a significant cell wall fraction insoluble to ionic detergents [67]. In bacteria of the genus *Chlamydophila*, this portion is refer‐ red to as COMC (*Chlamydia* outer membrane complex), which is composed of MOMP (major outer membrane protein) and other small proteins [68]. MOMP is composed of cysteine-rich proteins and takes approximately 60% of the total weight of the outer membrane [69]. This constituent is important to maintain the integrity and rigidity of the bacteria, and the main antigen to host immune system [70].

#### **b.** *Chlamydophila psittaci* biology

*Chlamydophila bacteria* have three distinct forms during its life cycle: elementary body (EB), reticular body (RB) and intermediate body (IB) [19,20, 71].

The EB is small, electro-dense, spherical and about 0.2 to 0.3 mm in diameter [18,20]. The outer membrane is composed of proteins, lipids, lipopolysaccharides and proteins. However, unlike the Gram-negative bacteria, this species is devoid of muramic acid [72]. This is an active form of the bacterium capable of binding with the host cell allowing to reach its inside [19]. This first form is characterized by a highly electro-dense nucleoid [71, 73], located in the periphery and clearly separated from the cytoplasm [19, 71]. The Hc1 histone maintains the chromatin highly condensed [74]. Within the cell, the elemen‐ tary body increases in size to form the reticular body, which is the metabolic active in‐ tracellular form of the organism [19, 20]. At this stage, the chromatin is dispersed, since the bacteria begin a process of transcription [20, 75].

The RB is of about 0.5 to 2.0 mm in diameter and its inner and outer membranes are relative‐ ly nearby, thereby reducing the virtual space between them [18, 20]. This form of the bacteri‐ um has the ability to split by binary fission, resulting in new RB [19, 65]. During this process, intermediate bodies can be observed within the host cell, measuring between 0.3 and 1.0 mm in diameter [18, 20].

This third form of the bacterium (IB) has a very specific presentation with an electro-dense core nucleoid surrounded by fibres dispersed radially [65, 73]. In the periphery, there exists an agglomerate of cytoplasmic granules, separated from the core by a translucent area [71]. When the replication process is over, several elementary forms are observed, which can be somewhat condensed, depending on their conformations [20]. The less condensed are more immature form, have further fibrous elements in the granular cytoplasm, bearing for the electro-dense nucleoid, which will become progressively highly condensed [71]. The nucle‐ oid is close to the inner membrane [20].

The mature elementary bodies are condensed, having a homogeneous oval shaped nucleoid, irregular or an elongated, separated from the cytoplasmic organisms by a well visible elec‐ tro-transparent area [20, 75].

Some studies have shown the existence of hemispherical-shaped projections of the cyto‐ plasm on the surface of the elementary bodies and ridge-shaped projections of the reticular forms for this bacterium [76-78]. These projections range from the bacteria to the surface of the membrane inclusion and it is speculated that can possess pores of type III secretion [32].

#### **c.** Pathogenesis

All species of the genera *Chlamydophila* and Chlamydiae have one or more serotypes, whose

*Chlamydophila psittaci* is negative to Gram test bacterium [2, 49, 57], having a cytoplasmic membrane and an outer three-layer membrane [65-66], with a significant cell wall fraction insoluble to ionic detergents [67]. In bacteria of the genus *Chlamydophila*, this portion is refer‐ red to as COMC (*Chlamydia* outer membrane complex), which is composed of MOMP (major outer membrane protein) and other small proteins [68]. MOMP is composed of cysteine-rich proteins and takes approximately 60% of the total weight of the outer membrane [69]. This constituent is important to maintain the integrity and rigidity of the bacteria, and the main

*Chlamydophila bacteria* have three distinct forms during its life cycle: elementary body (EB),

The EB is small, electro-dense, spherical and about 0.2 to 0.3 mm in diameter [18,20]. The outer membrane is composed of proteins, lipids, lipopolysaccharides and proteins. However, unlike the Gram-negative bacteria, this species is devoid of muramic acid [72]. This is an active form of the bacterium capable of binding with the host cell allowing to reach its inside [19]. This first form is characterized by a highly electro-dense nucleoid [71, 73], located in the periphery and clearly separated from the cytoplasm [19, 71]. The Hc1 histone maintains the chromatin highly condensed [74]. Within the cell, the elemen‐ tary body increases in size to form the reticular body, which is the metabolic active in‐ tracellular form of the organism [19, 20]. At this stage, the chromatin is dispersed, since

The RB is of about 0.5 to 2.0 mm in diameter and its inner and outer membranes are relative‐ ly nearby, thereby reducing the virtual space between them [18, 20]. This form of the bacteri‐ um has the ability to split by binary fission, resulting in new RB [19, 65]. During this process, intermediate bodies can be observed within the host cell, measuring between 0.3 and 1.0

This third form of the bacterium (IB) has a very specific presentation with an electro-dense core nucleoid surrounded by fibres dispersed radially [65, 73]. In the periphery, there exists an agglomerate of cytoplasmic granules, separated from the core by a translucent area [71]. When the replication process is over, several elementary forms are observed, which can be somewhat condensed, depending on their conformations [20]. The less condensed are more immature form, have further fibrous elements in the granular cytoplasm, bearing for the electro-dense nucleoid, which will become progressively highly condensed [71]. The nucle‐

The mature elementary bodies are condensed, having a homogeneous oval shaped nucleoid, irregular or an elongated, separated from the cytoplasmic organisms by a well visible elec‐

sequence are known [62-63] or are still under intense study [64].

reticular body (RB) and intermediate body (IB) [19,20, 71].

the bacteria begin a process of transcription [20, 75].

**a.** *Chlamydophila psittaci*

132 Insights from Veterinary Medicine

antigen to host immune system [70].

**b.** *Chlamydophila psittaci* biology

mm in diameter [18, 20].

oid is close to the inner membrane [20].

tro-transparent area [20, 75].

Infection of *Chlamydophila psittaci* begins with the attachment of the elementary body to the host cell and subsequent parasite-mediated endocytosis [79]. The bacterium binds to the cell membrane receptor of the host cell, often associated with a cytoplasm protein known as cla‐ thrin [20]. This connection stimulates the cell to emit pseudopods; these increase in size until all bacteria have been surrounded, forming a vacuole delimited by clathrin, referred to as inclusion [18, 80]. In the vacuole, the bacterium has a biphasic cycle (Figure 1), alternating between states of elementary body and reticular body [19].

Once inside the vacuole, the EB are redistributed from the periphery of the cell and join in the region of the Golgi apparatus, which corresponds to the MTOC (Microtubule Organiz‐ ing Centre) [19, 80]. The non-acid inclusions are firstly too small, allowing to avoid its fusion with lysosomes, and are formed by endocytic constituents of the plasma membrane of the host cell such as proteins and lipids [19, 80]. All of these conditions lead to a more efficient survival and infection by the bacterium [20].

On the first hours, the elementary bodies initiate to differentiate into reticular bodies. These forms of *Chlamydophila* are not infectious, have a non-condensed nucleoid and a greater size than EB [19, 65]. From eight to ten hours of cycle, the reticular bodies start to replicate by binary fission, remaining in contact with the inclusion membrane [20, 65]. In this moment, it's possible to see numerous mitochondria surrounding the inclusion [18, 20], possibly due to protein motors function as kinesins [81]. *Cp. psittaci* may not be able to generate as much energy in the form of ATP as other species of the genus, so a deeper association with the host cell mitochondria is necessary for the bacteria to recruit the necessary ATP by an alter‐ native mechanism [20]. The migration of nutrients from the host cell into the inclusion is es‐ sential. Amino acids and nucleotides cross the inclusion membrane [20, 82]. Since the vacuole is not in the vicinity of lysosomes leads to the assumption that the releasing of nu‐ trients by fusion with endocytic vesicles is unlikely [20]. It is known that exocytic vesicles containing sphingomyelin fuse with the membrane of the inclusion, delivering nutrients and lipids to the bacteria [80]. It was demonstrated that the inclusion membrane is permeable to small molecules of a molecular weight ranging from 100-520 Da [83,84]. After releasing the nutrients into the lumen of the bacterium by passive diffusion, membrane-specific carriers/ transporters located in the membrane would facilitate the entry of new nutrients and would be indispensable for the entry of molecules larger than 520 Da, but such carriers have not been yet described [20].

Throughout the cycle, the size of the vesicles increases, as they accumulate growing num‐ bers of bacteria inside [20, 84]. The surface of the inclusion membrane also increases, while it intercepts biosynthetic transport pathways of the host cell membrane and acquires the abili‐ ty to fuse with a subset of Golgi derived vesicles [20, 84]. Within 20 hours of cycle, reticular bodies continue their replication and each body can give rise to a thousand of new bacteria [20]. As this process occurs, the inclusion becomes increasingly overloaded with bacteria and as such they are obligated to leave the inclusion [19, 20]. This may be the reason for the reticular bodies to turn into intermediate bodies and new infectious elementary bodies, which occurs about 36 hours after infection [19]. During the final stage, at 50 hours of cycle, the host cell and the inclusion undergo a process of lysis or, very often, the elementary bod‐ ies are released by reverse endocytosis [20], which leaves the host cell intact, allowing per‐ sistence of a chronic and silent infection [19].

After treatment with cytokines, antibiotics or restriction to particular nutrients [19, 85], this cycle may undergo a modification with the emergence of "persistent" bacteria [85-86]. This kind of bacterium does not complete its continuing transformation from reticular bodies to infectious elementary form. Instead, it remain with low metabolic activity [19]. These RB are morphologically aberrant, appearing with a dilated oval shape within inclusions of small size [20, 85-86]. Excessive accumulation of chromosomes is due to the continuous DNA rep‐ lication of the bacteria that lack the ability to divide [20]. These persistent forms of *Cp. psitta‐ ci* are associated with chronic infections, and it has been shown that aberrant reticular bodies can quickly develop into normal forms and subsequently into infective elementary bodies [85-86]. The way this bacteria model passes through such transformations between aberrant forms and normal forms of reticular bodies is not yet completely understood [75, 87-88].

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135

http://dx.doi.org/10.5772/53720

Parrots were the first animals linked to *Cp. psittaci* in 1929-1930 and 1930-1938 when they were confirmed to be the source of outbreaks of psittacosis [33]. However, thereafter it became clear that this disease was not confined to these birds. In 1939, the agent was isolated from two pigeons in South Africa and later two new cases of psittacosis arose in citizens who contacted with pigeons in the U.S. [89]. Ducks and turkeys are given as possible sources of infection since the early fifties, although in the seventies there was a large decline in the prevalence of bacteria, but without ever ceasing [20]. Confirmation of new U.S. cases of chlamydiosis in turkeys in the following decade [20] and in Europe, al‐ ready in the nineties [90,91], proved the continuing problematic status of this disease. In the last thirty years it was reported several cases in humans who have contracted the disease through direct contact with ducks [92-95] and more recently the number of these

Studies on *Chlamydophila'*s prevalence increased and psittacosis was classified as an en‐ demic disease in Belgium [97-98] and other European countries, such as France and

Psittacines and pigeons are the most disturbing cases, being the range of prevalence within the first, 16% to 81% and mortality frequently above 50% [99-101]. Other studies have shown that parrots are the largest sources of *Cp. psittaci*, especially when they are

The wild pigeons have a broad seropositivity that ranges from 12.5% up to 95.6%. These da‐ ta were obtained from studies conducted from 1966 to 2005 [20,104-105]. The seropositivity in this species is alarming, once they live in urban and rural areas throughout the world, in close contact with human [60]. Carrier pigeons are reported as having a lower seropositivity

Kaleta and Taday (2003) reported that seabirds are more often infected with *Cp. psittaci* than other birds like chickens, quails and pheasants, but virtually all species of birds can

**3. Epidemiology**

cases increased dramatically [96].

than wild pigeons, between 35.9 and 60% [20].

Germany [97].

in captivity [102-103].

**Figure 1.** Chlamydia life cycle. Infection begins with the attachment of the elementary bodies (EB) to the surface of target epithelial cells. These cells promote a pseudopod formation to engulf the EB. Inside the cytoplasm this bacterium inhibits the fusion of the vesicle with the cell lysosomes. The nascent inclusion is accompanied by the transition from EBs to reticu‐ late bodies (RB). Late in the cycle, RBs replicate by binary fission to generate both RBs and intermediate bodies (IB). At this stage, antigenic proteins are exposed into the cell surface. An elongated, aberrant RB could be formed at this time with an arrest on chlamydia cycle originating a persistent infection, or continuing the cycle. The various intracytoplasmic inclusions with bacterium inside, can also be fused in this phase, and the agent develop into intermediate bodies (IB), before DNA condensation and RB transformation into a newly EB. The mature inclusion increases in size with EB formation, until be‐ coming infectious and released into the extracellular space to continue a new intracellular cycle. N – nucleus; G – Golgi ap‐ paratus: EB – elementary bodies; RB – reticulate bodies; IB – intermediate bodies.

After treatment with cytokines, antibiotics or restriction to particular nutrients [19, 85], this cycle may undergo a modification with the emergence of "persistent" bacteria [85-86]. This kind of bacterium does not complete its continuing transformation from reticular bodies to infectious elementary form. Instead, it remain with low metabolic activity [19]. These RB are morphologically aberrant, appearing with a dilated oval shape within inclusions of small size [20, 85-86]. Excessive accumulation of chromosomes is due to the continuous DNA rep‐ lication of the bacteria that lack the ability to divide [20]. These persistent forms of *Cp. psitta‐ ci* are associated with chronic infections, and it has been shown that aberrant reticular bodies can quickly develop into normal forms and subsequently into infective elementary bodies [85-86]. The way this bacteria model passes through such transformations between aberrant forms and normal forms of reticular bodies is not yet completely understood [75, 87-88].

## **3. Epidemiology**

bodies continue their replication and each body can give rise to a thousand of new bacteria [20]. As this process occurs, the inclusion becomes increasingly overloaded with bacteria and as such they are obligated to leave the inclusion [19, 20]. This may be the reason for the reticular bodies to turn into intermediate bodies and new infectious elementary bodies, which occurs about 36 hours after infection [19]. During the final stage, at 50 hours of cycle, the host cell and the inclusion undergo a process of lysis or, very often, the elementary bod‐ ies are released by reverse endocytosis [20], which leaves the host cell intact, allowing per‐

**Figure 1.** Chlamydia life cycle. Infection begins with the attachment of the elementary bodies (EB) to the surface of target epithelial cells. These cells promote a pseudopod formation to engulf the EB. Inside the cytoplasm this bacterium inhibits the fusion of the vesicle with the cell lysosomes. The nascent inclusion is accompanied by the transition from EBs to reticu‐ late bodies (RB). Late in the cycle, RBs replicate by binary fission to generate both RBs and intermediate bodies (IB). At this stage, antigenic proteins are exposed into the cell surface. An elongated, aberrant RB could be formed at this time with an arrest on chlamydia cycle originating a persistent infection, or continuing the cycle. The various intracytoplasmic inclusions with bacterium inside, can also be fused in this phase, and the agent develop into intermediate bodies (IB), before DNA condensation and RB transformation into a newly EB. The mature inclusion increases in size with EB formation, until be‐ coming infectious and released into the extracellular space to continue a new intracellular cycle. N – nucleus; G – Golgi ap‐

paratus: EB – elementary bodies; RB – reticulate bodies; IB – intermediate bodies.

sistence of a chronic and silent infection [19].

134 Insights from Veterinary Medicine

Parrots were the first animals linked to *Cp. psittaci* in 1929-1930 and 1930-1938 when they were confirmed to be the source of outbreaks of psittacosis [33]. However, thereafter it became clear that this disease was not confined to these birds. In 1939, the agent was isolated from two pigeons in South Africa and later two new cases of psittacosis arose in citizens who contacted with pigeons in the U.S. [89]. Ducks and turkeys are given as possible sources of infection since the early fifties, although in the seventies there was a large decline in the prevalence of bacteria, but without ever ceasing [20]. Confirmation of new U.S. cases of chlamydiosis in turkeys in the following decade [20] and in Europe, al‐ ready in the nineties [90,91], proved the continuing problematic status of this disease. In the last thirty years it was reported several cases in humans who have contracted the disease through direct contact with ducks [92-95] and more recently the number of these cases increased dramatically [96].

Studies on *Chlamydophila'*s prevalence increased and psittacosis was classified as an en‐ demic disease in Belgium [97-98] and other European countries, such as France and Germany [97].

Psittacines and pigeons are the most disturbing cases, being the range of prevalence within the first, 16% to 81% and mortality frequently above 50% [99-101]. Other studies have shown that parrots are the largest sources of *Cp. psittaci*, especially when they are in captivity [102-103].

The wild pigeons have a broad seropositivity that ranges from 12.5% up to 95.6%. These da‐ ta were obtained from studies conducted from 1966 to 2005 [20,104-105]. The seropositivity in this species is alarming, once they live in urban and rural areas throughout the world, in close contact with human [60]. Carrier pigeons are reported as having a lower seropositivity than wild pigeons, between 35.9 and 60% [20].

Kaleta and Taday (2003) reported that seabirds are more often infected with *Cp. psittaci* than other birds like chickens, quails and pheasants, but virtually all species of birds can contract the disease, even without apparent symptoms, such as for instance, the cranes and seagull. Psittacosis in arthropods can be detected, however without initiation of in‐ fection in these animals [106].

General clinical signs of disease and highlights of the most common within the species are listed on table 1, being generally flu-like symptoms, CNS disorders, pericarditis, sacculitis

**Psittacines** Anorexia, diarrhoea, difficulty breathing, sinusitis, conjunctivitis, yellowish droppings and, perhaps,

Chronic Infection - lameness, stiff neck, opisthotonos, tremor and convulsions.

production, conjunctivitis, sinusitis, sneezing and mortality between 10 and 30%.

*Chlamydophila psittaci* post-mortem lesions are not specific enough to be able to differentiate this disease from other systemic diseases [113]. The severity of injury depends on several factors including the virulence of serotype, the susceptibility and age of the host, the form

In most cases of psittacosis, lesions are limited to three structures: spleen, liver and air sacs [113]. Table 2 summarizes the post-mortem lesions in these specific organs and points addi‐

**Clinical signs**

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Difficulty breathing, fever, lethargy, anorexia, ruffled feathers, diarrhoea, oral and nasal discharges, decreased egg laying, polyuria, pericarditis, sacculitis, pneumonia, lateral nasal adenitis, peritonitis,

Acute Infection - anorexia, diarrhoea, conjunctivitis, rhinitis, swollen eyelids and a decrease in flight

D serotype of the bacteria - Anorexia, cachexia, diarrhoea gelatinous yellow-green, low egg

Agitation, unsteady gait, conjunctivitis, serous to purulent nasal discharge and depression.

and occasionally shock and death, signs of infection [18, 20,113].

hepatitis and splenitis, occasionally shock and death.

Signs appear only when there is another competitor disease.

B serotype of the bacteria - Anorexia and green manure

CNS disorders.

performance.

**Ducks** It affects mostly the young ones.

**Chickens** Blindness, anorexia and occasionally death.

**Table 1.** General clinical signs of disease (adapted from [18,20,113].

and time of exposure and the presence of concurrent diseases [18].

tional possible lesions of *Cp. psittaci* found in animals [18, 108,113].

**Overall**

**Species**

**Pigeons**

**Turkeys**

Although the horizontal transmission is the most common way of infection [24], the vertical transmission was also described [18].

*Chlamydophila psittaci* are most excreted in the faeces [21, 107], nasal [12] and oral dis‐ charges of infected animals [108]. Sareyyupoglu and collaborators (2007) indicates that excretion of the bacteria may occur intermittently in sub-clinical infections for a long time, being activated in situations of stress such as transportation and handling [21] or nutritional restrictions and egg laying [108]. Bacteria are found in the powder of feathers [106], in excrements, secretions and respiratory exudates from infected animals, and when dry, become spread in the air [20-21] being the aerosol transmission extremely fea‐ sible [109]. The contact of non infected animals with bacteria or its proximity to animals with *Cp. psittaci* gain greater importance, being this an essential criteria for the develop‐ ment of new infections [22].

Animals become infected by ingestion [110] or inhalation of the bacteria [13, 60], and the iso‐ lation of this agent is much more substantial from choanal and throat swabs collection rath‐ er than from faeces collection [111], especially in the early stages of the disease [20]. Thus, contamination by aerosol exudates must be considered the primary form of infection [89].

Birds who share contaminated water are also susceptible of infection [18], as well as predators eating carcasses contaminated with *Cp. psittaci* [108]. The nest environment is also very susceptible to disease transmission, since this is a place where are deposited loads debris that may contain multiple bacteria [20]. Granivorous animals, such as pi‐ geons or pheasants, that are often found in corrals or stables contaminated with faeces, and also grain storage areas can become infected by inhalation of dust from grain or from aerosols of faeces [18, 20].

Ectoparasites such as fleas, mites and lice may also serve as vectors for the transmission of disease from animal to animal [112].

## **4. Clinical signs and pathology**

After 4 hours of infection via aerosol, bacteria can be found in the respiratory system of the animal [113].

This disease may be acute, sub-acute, chronic or subclinical [113], being the last one found when the animal is showing no signs of disease, and elimination of the bacteria can occur intermittently due to stress [21], inadequate nutrition or other diseases [108]. These animals are persistently infected and named as "source of infection" to other animals [20]. The acute form, in turn, is a generalized form, affecting all the organs of the animal [113].

General clinical signs of disease and highlights of the most common within the species are listed on table 1, being generally flu-like symptoms, CNS disorders, pericarditis, sacculitis and occasionally shock and death, signs of infection [18, 20,113].

contract the disease, even without apparent symptoms, such as for instance, the cranes and seagull. Psittacosis in arthropods can be detected, however without initiation of in‐

Although the horizontal transmission is the most common way of infection [24], the vertical

*Chlamydophila psittaci* are most excreted in the faeces [21, 107], nasal [12] and oral dis‐ charges of infected animals [108]. Sareyyupoglu and collaborators (2007) indicates that excretion of the bacteria may occur intermittently in sub-clinical infections for a long time, being activated in situations of stress such as transportation and handling [21] or nutritional restrictions and egg laying [108]. Bacteria are found in the powder of feathers [106], in excrements, secretions and respiratory exudates from infected animals, and when dry, become spread in the air [20-21] being the aerosol transmission extremely fea‐ sible [109]. The contact of non infected animals with bacteria or its proximity to animals with *Cp. psittaci* gain greater importance, being this an essential criteria for the develop‐

Animals become infected by ingestion [110] or inhalation of the bacteria [13, 60], and the iso‐ lation of this agent is much more substantial from choanal and throat swabs collection rath‐ er than from faeces collection [111], especially in the early stages of the disease [20]. Thus, contamination by aerosol exudates must be considered the primary form of infection [89].

Birds who share contaminated water are also susceptible of infection [18], as well as predators eating carcasses contaminated with *Cp. psittaci* [108]. The nest environment is also very susceptible to disease transmission, since this is a place where are deposited loads debris that may contain multiple bacteria [20]. Granivorous animals, such as pi‐ geons or pheasants, that are often found in corrals or stables contaminated with faeces, and also grain storage areas can become infected by inhalation of dust from grain or

Ectoparasites such as fleas, mites and lice may also serve as vectors for the transmission of

After 4 hours of infection via aerosol, bacteria can be found in the respiratory system of the

This disease may be acute, sub-acute, chronic or subclinical [113], being the last one found when the animal is showing no signs of disease, and elimination of the bacteria can occur intermittently due to stress [21], inadequate nutrition or other diseases [108]. These animals are persistently infected and named as "source of infection" to other animals [20]. The acute

form, in turn, is a generalized form, affecting all the organs of the animal [113].

fection in these animals [106].

136 Insights from Veterinary Medicine

ment of new infections [22].

from aerosols of faeces [18, 20].

disease from animal to animal [112].

**4. Clinical signs and pathology**

animal [113].

transmission was also described [18].


**Table 1.** General clinical signs of disease (adapted from [18,20,113].

*Chlamydophila psittaci* post-mortem lesions are not specific enough to be able to differentiate this disease from other systemic diseases [113]. The severity of injury depends on several factors including the virulence of serotype, the susceptibility and age of the host, the form and time of exposure and the presence of concurrent diseases [18].

In most cases of psittacosis, lesions are limited to three structures: spleen, liver and air sacs [113]. Table 2 summarizes the post-mortem lesions in these specific organs and points addi‐ tional possible lesions of *Cp. psittaci* found in animals [18, 108,113].


The samples for subsequent isolation of *Cp. psittaci* must be handled carefully to prevent the loss of infectivity of bacteria during transport and handling, but also due to its high zoonotic potential [89]. Thus, the safety of the operator must be respected (use of gloves, gown and mask) and the samples and swabs are placed in SPG (sucrose phosphate glutamate) trans‐ port medium, which is also suitable for bacteria of the Rickettsia genus [89, 114]. SPG is

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SPG is also used for samples dilution and freezing, if they will be processed with adelay of four or more days after harvest; otherwise samples should not be frozen [89, 114]. Freezing of material must be performed within the first 24 hours, in a stabilized 7.2 pH phosphatebuffered saline medium and kept at a minimum temperature of -20 ° C [89, 114]. Spencer and Johnson (1983) report that *Cp. psittaci* can survive in SPG to just over 30 days at 4 ° C, but their infectivity decreased by 1.55% [115]. Detection of *Cp. psittaci* in the blood, liver, spleen and kidney is only possible two whole days after, whereas in faeces is only possible

*Chlamydophila psittaci* can be identified by direct observation of the organism using different staining techniques such as the Giemsa, Stamp and Gimenez stainings [116]. Giemsa stain‐ ing could be performed from faeces, exudates, or liver and spleen cytologies [20]. Although cytological staining can be useful and quick, it is less sensitive and less specific than immu‐ nochemical staining or molecular detection methods [18, 20]. With Giemsa staining the methanol fixed material can be observed under the microscope. Inside the cells a basophilic inclusion composed by EB and IB located near the nucleus is found in infected cells [117]. Hayashi visualized bacteria using this technique, once isolated from organs such as liver, spleen, kidney, heart and intestine [118]. This technique is a powerful tool in the *Chlamydia*

This staining is often used, although it is not specific to this bacterium and has reduced sen‐ sitivity. Other agents beyond *Chlamydophila* (for instance, *Coxiella burnetii, Helicobacter pylori*)

are stained red with Gimenez staining in contrast to background in light green [119].

Sucrose 74.6 g / L K2HPO4 1.237 g / L Glutamic Acid 0.721 g / L Fetal calf serum 10% Streptomycin 100 µg / mL Vancomycin 100 µg / mL Nnystatin 50 µg / mL Gentamicin 50 µg / mL

formed according to Table 3 [115].

**Table 3.** Composition of SPG [115].

beyond the 72nd hour [113].

Giemsa cytological staining

Gimenez staining

**b.** *Chlamydophila psittaci* identification

diagnosis, but it requires a vast experience from the observer.

**Table 2.** Post-mortem lesions in specific organs (Adapted from [18,108,113]).

## **5. Diagnostic techniques**

There are several available methods for the diagnosis of *Chlamydophila psittaci*, including the agent direct visualization with a specific staining technique, isolation of the agent followed by agent identification, detection of bacteria specific antigen or genes in samples and, final‐ ly, serology tests that identify antibodies against this organism [20,89].

**a.** Harvesting and storage of samples

Sampling should be carried out aseptically to avoid contaminating bacteria [89]. At nec‐ ropsy, the main structures to isolate this bacterium are the air sacs, spleen, pericardium, heart, areas of hyperemia of the intestine, liver and kidney. On live animals the choanal, oropharyngeal and cloacal swabs are the principal material for *Chlamydophila psittaci* search [52,89,111]. Other samples can be taken from blood, conjunctiva and peritoneal exudates [20].

The samples for subsequent isolation of *Cp. psittaci* must be handled carefully to prevent the loss of infectivity of bacteria during transport and handling, but also due to its high zoonotic potential [89]. Thus, the safety of the operator must be respected (use of gloves, gown and mask) and the samples and swabs are placed in SPG (sucrose phosphate glutamate) trans‐ port medium, which is also suitable for bacteria of the Rickettsia genus [89, 114]. SPG is formed according to Table 3 [115].


#### **Table 3.** Composition of SPG [115].

**Spleen**

138 Insights from Veterinary Medicine

**Liver**

**Air Sacs**

**Injuries and/or changes in other**

exudates [20].

**organs**

**Size**: Increased (splenomegaly).

**Size**: Increased (hepatomegaly).

other organs show no alterations

**Colour**: Colour ranging from yellow to green.

mononuclear cell and heterophils infiltrate.

with fibrinous or fibrinopurulent exudate.

**Kidney** Acute necrosis and nephritis with inflammatory infiltrate of mixed type.

Pneumonia although not frequent is possible.

ly, serology tests that identify antibodies against this organism [20,89].

Infectious pericarditis, myocarditis, adenitis and peritonitis.

Inflammation of adrenals and gonads sporadically found.

Medium increase in granulocytic cells series in spinal cord.

There are several available methods for the diagnosis of *Chlamydophila psittaci*, including the agent direct visualization with a specific staining technique, isolation of the agent followed by agent identification, detection of bacteria specific antigen or genes in samples and, final‐

Sampling should be carried out aseptically to avoid contaminating bacteria [89]. At nec‐ ropsy, the main structures to isolate this bacterium are the air sacs, spleen, pericardium, heart, areas of hyperemia of the intestine, liver and kidney. On live animals the choanal, oropharyngeal and cloacal swabs are the principal material for *Chlamydophila psittaci* search [52,89,111]. Other samples can be taken from blood, conjunctiva and peritoneal

**Consistency**: soft.

**Consistency**: friable.

surfaces.

Brain injuries are rare.

**Table 2.** Post-mortem lesions in specific organs (Adapted from [18,108,113]).

**Intestine** Lymphocytic enteritis.

**5. Diagnostic techniques**

**a.** Harvesting and storage of samples

**Colour:** blackish, sometimes with greyish white necrotic foci and petechial haemorrhages.

Increased macrophages numbers and decreased number of lymphocytes.

Increased activity of Kupffer cells, stimulated by hemosiderin accumulation.

Perivascular sheaths of macrophages in the arterioles transforming the architecture of the organ.

**Acute infection** – often the presence of a multifocal necrosis is the only sign of disease, and the

**Subacute or chronic infections** - hyperplasia of the biliary ducts and sinus histiocytosis with

**Acute and subacute infections** – changes in thickness of the membranes that were covered

Chronic infections - presence of pyogranulomatous infections or diffuse granulomas on serous

SPG is also used for samples dilution and freezing, if they will be processed with adelay of four or more days after harvest; otherwise samples should not be frozen [89, 114]. Freezing of material must be performed within the first 24 hours, in a stabilized 7.2 pH phosphatebuffered saline medium and kept at a minimum temperature of -20 ° C [89, 114]. Spencer and Johnson (1983) report that *Cp. psittaci* can survive in SPG to just over 30 days at 4 ° C, but their infectivity decreased by 1.55% [115]. Detection of *Cp. psittaci* in the blood, liver, spleen and kidney is only possible two whole days after, whereas in faeces is only possible beyond the 72nd hour [113].

#### **b.** *Chlamydophila psittaci* identification

#### Giemsa cytological staining

*Chlamydophila psittaci* can be identified by direct observation of the organism using different staining techniques such as the Giemsa, Stamp and Gimenez stainings [116]. Giemsa stain‐ ing could be performed from faeces, exudates, or liver and spleen cytologies [20]. Although cytological staining can be useful and quick, it is less sensitive and less specific than immu‐ nochemical staining or molecular detection methods [18, 20]. With Giemsa staining the methanol fixed material can be observed under the microscope. Inside the cells a basophilic inclusion composed by EB and IB located near the nucleus is found in infected cells [117]. Hayashi visualized bacteria using this technique, once isolated from organs such as liver, spleen, kidney, heart and intestine [118]. This technique is a powerful tool in the *Chlamydia* diagnosis, but it requires a vast experience from the observer.

#### Gimenez staining

This staining is often used, although it is not specific to this bacterium and has reduced sen‐ sitivity. Other agents beyond *Chlamydophila* (for instance, *Coxiella burnetii, Helicobacter pylori*) are stained red with Gimenez staining in contrast to background in light green [119].

This staining technique is based on carbol-fuchsin (basic fuchsin) reactions contrasting the background with malachite green [120]. The Gimenez staining technique is considered as a rapid diagnostic for the detection of *Cp. psittaci* on dead birds [121]. Some authors compared the use of DGMB (dark-ground methylene blue) staining with MZN (modified Ziehl-Neel‐ sen) and DGG (Giemsa dark-ground) using infected goat foetal membranes, concluding that DGMB is a more specific staining for *Chlamydia* elementary bodies than MZN and DGG [123]. Woodland and colleagues (1982) developed a new staining technique for inclusions of *Chlamydia psittaci* and *Chlamydia trachomatis* and concluded that it was significantly better sensitivity than Giemsa staining. This technique consists on using a methyl green staining with neutral red and washing at pH 5.0 [124].

Methods of elementary body agglutination (EBA)

reduced sensitivity [20].

Complement fixation test (CF)

Micro-indirect immunofluorescence (MIF)

Direct fluorescent antibody (FA)

traviolet microscopy [96].

ELISA technique

albeit it shows cross-reactivity with other chlamydial species [20].

EBA detects IgM [12], therefore diagnosing recent (acute) cases of infection [121]. A negative result does not mean, however, that the animal is not infected, having this technique a very

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Although CF is the most used serological method [26, 89, 114, 130], it has several disadvan‐ tages in the use in psittacines because the immunoglobulins do not fixate the complement [131-132]. In such cases, an indirect test for complement fixation using the spot-*Chlamydophi‐ la psittaci* IF (bioMérieux, Basingstoke, Hants) reagent [89] or other serological test such as MIF should be used [114].. This test has also low sensitivity [114] and the technique is very laborious, being discarded whenever multiple samples need to be tested at same time [20].

This method detects all isotopes of immunoglobulin produced against the genus *Chlamydo‐ phila.* Thus, it is widely used in the detection of antibodies against *C. trachomatis*, *Cp. psittaci* and *Cp. pneumoniae* serotypes in serum or plasma [20]. The indirect immunofluorescence test seems to be more sensitive than the complement fixation test and histochemical techniques,

The preferably staining method to detect the *Chlamydophila* is the direct FA [89,133]. In this method, an anti-chlamydia fluorescein-conjugated is applied to the smear [134] fol‐ lowing the incubation for 30 minutes at 37 ° C [20]. Thereafter, the slides are washed up with PBS and distilled water, dried on air and finally mounted with a resinous com‐ pound like Entellan® [20]. The inclusions of the bacteria show a bright green under ul‐

The primary purpose of a great number of commercial ELISA developed in the last 25 years was for *Chlamydia trachomatis* in humans, but such tests are also suitable for diagnosing *Chlamydo‐ phila psittaci*, as they are specific to the LPS antigen of all species of the family Chlamydiales [1,27,135-136]. Commercial ELISA tests were, over the years, widely inquired and several stud‐ ies about the low specificity and high sensitivity of these methods came up [20], with the first dramatically reducing in situations where the prevalence of the bacterium is low and hence where there is a low number of detected bodies [136]. In fact, it takes hundreds of bacteria so that the results are positive [20]. One of the major disadvantages of this technique is the possible occurrence of false positives, since have cross-reaction with other bacterias (such *Acinetobacter calcoaceticus*, *Escherichia coli*, *Staphylococcus aureus* and *Klebsiella pneumoniae)* have been demon‐ strated in humans [34,137]. Nevertheless, the development and improvement of these commer‐ cial tests increased with the use of monoclonal antibodies and advanced blockers methods [20, 136] that improve the specificity and reduced the false positive cases. There are already availa‐ ble specific commercial ELISA tests for *C. trachomatis, Cp. pneumonia, Cp. abortus* antibodies [132,138-139], and for *Chlamydophila psittaci's* as well [101]. These methods are as reliable as the

#### **c.** Immunohistochemistry

Immunohistochemistry is a method based on specificity of immune complex formation by the specific antibodies union with their specific antigen, being the IgG much more frequent‐ ly used in comparison to IgM [20]. Nevertheless, a variability in Immunohistochemistry methods exits, and the choice of appropriated variations depends mainly on the available equipment and characteristics of existing antibody, since virtually all techniques on paraffin sections are suitable for detection of *Cp. psittaci* [20].This technique is more sensitive than routine histochemical staining; however, the user experience is essential to the identification of the bacteria. The body morphology of the agent must be remembered to avoid false posi‐ tives in situations of cross-reactions with other bacteria and fungi [125]. In some cases, he‐ mosiderin can lead an inexperienced user into errors, directing him into an incorrect positive diagnosis. Antigen positive tissues and sections stained with hematoxylin-eosin must be used as control [20].The antibody used in this method could be polyclonal [126] or monoclonal for the genus of the family *Chlamydophila* [127]. Some antibodies detect the spe‐ cific epitope of the family Chlamydiaceae, located in the LPS of the bacteria [128]. Other au‐ thors reported the degree of sensitivity of this technique with peroxidase-antiperoxidase in the identification of *Chlamydia* and point out the positive samples to Chlamydiae when un‐ apparent to the hematoxylin-eosin technique [129].

#### **d.** Serological Tests

Serological tests are still widely used, yet these are not particularly useful for the disease di‐ agnosis on birds, since they show a high prevalence of bacteria [106] and *Chlamydophila psit‐ taci* antibodies are maintained in circulation for months or even years. Due to lack of information and studies on the direct identification of bacteria by serological tests in some birds, these tests are still viewed with some uncertainty concerning the interpretation of the results [20].A positive diagnosis does not mean active infection, but confirms that the animal had contact with the bacteria in the past [12, 114]. Further, in case of acute infections, these tests easily lead to false negative results since the serum sample may be carried out before the seroconversion [18], which is necessary to the method efficiency. Another reason for false negatives is the treatment of animals with antibiotics that reduce or delay the antibody response [12].The serological methods most usually used include the methods of elementary body agglutination (EBA), the complement fixation test (CF), the indirect immunofluores‐ cence (MIF) and commercial ELISA tests [22, 114, 130].

### Methods of elementary body agglutination (EBA)

EBA detects IgM [12], therefore diagnosing recent (acute) cases of infection [121]. A negative result does not mean, however, that the animal is not infected, having this technique a very reduced sensitivity [20].

#### Complement fixation test (CF)

This staining technique is based on carbol-fuchsin (basic fuchsin) reactions contrasting the background with malachite green [120]. The Gimenez staining technique is considered as a rapid diagnostic for the detection of *Cp. psittaci* on dead birds [121]. Some authors compared the use of DGMB (dark-ground methylene blue) staining with MZN (modified Ziehl-Neel‐ sen) and DGG (Giemsa dark-ground) using infected goat foetal membranes, concluding that DGMB is a more specific staining for *Chlamydia* elementary bodies than MZN and DGG [123]. Woodland and colleagues (1982) developed a new staining technique for inclusions of *Chlamydia psittaci* and *Chlamydia trachomatis* and concluded that it was significantly better sensitivity than Giemsa staining. This technique consists on using a methyl green staining

Immunohistochemistry is a method based on specificity of immune complex formation by the specific antibodies union with their specific antigen, being the IgG much more frequent‐ ly used in comparison to IgM [20]. Nevertheless, a variability in Immunohistochemistry methods exits, and the choice of appropriated variations depends mainly on the available equipment and characteristics of existing antibody, since virtually all techniques on paraffin sections are suitable for detection of *Cp. psittaci* [20].This technique is more sensitive than routine histochemical staining; however, the user experience is essential to the identification of the bacteria. The body morphology of the agent must be remembered to avoid false posi‐ tives in situations of cross-reactions with other bacteria and fungi [125]. In some cases, he‐ mosiderin can lead an inexperienced user into errors, directing him into an incorrect positive diagnosis. Antigen positive tissues and sections stained with hematoxylin-eosin must be used as control [20].The antibody used in this method could be polyclonal [126] or monoclonal for the genus of the family *Chlamydophila* [127]. Some antibodies detect the spe‐ cific epitope of the family Chlamydiaceae, located in the LPS of the bacteria [128]. Other au‐ thors reported the degree of sensitivity of this technique with peroxidase-antiperoxidase in the identification of *Chlamydia* and point out the positive samples to Chlamydiae when un‐

Serological tests are still widely used, yet these are not particularly useful for the disease di‐ agnosis on birds, since they show a high prevalence of bacteria [106] and *Chlamydophila psit‐ taci* antibodies are maintained in circulation for months or even years. Due to lack of information and studies on the direct identification of bacteria by serological tests in some birds, these tests are still viewed with some uncertainty concerning the interpretation of the results [20].A positive diagnosis does not mean active infection, but confirms that the animal had contact with the bacteria in the past [12, 114]. Further, in case of acute infections, these tests easily lead to false negative results since the serum sample may be carried out before the seroconversion [18], which is necessary to the method efficiency. Another reason for false negatives is the treatment of animals with antibiotics that reduce or delay the antibody response [12].The serological methods most usually used include the methods of elementary body agglutination (EBA), the complement fixation test (CF), the indirect immunofluores‐

with neutral red and washing at pH 5.0 [124].

apparent to the hematoxylin-eosin technique [129].

cence (MIF) and commercial ELISA tests [22, 114, 130].

**c.** Immunohistochemistry

140 Insights from Veterinary Medicine

**d.** Serological Tests

Although CF is the most used serological method [26, 89, 114, 130], it has several disadvan‐ tages in the use in psittacines because the immunoglobulins do not fixate the complement [131-132]. In such cases, an indirect test for complement fixation using the spot-*Chlamydophi‐ la psittaci* IF (bioMérieux, Basingstoke, Hants) reagent [89] or other serological test such as MIF should be used [114].. This test has also low sensitivity [114] and the technique is very laborious, being discarded whenever multiple samples need to be tested at same time [20].

#### Micro-indirect immunofluorescence (MIF)

This method detects all isotopes of immunoglobulin produced against the genus *Chlamydo‐ phila.* Thus, it is widely used in the detection of antibodies against *C. trachomatis*, *Cp. psittaci* and *Cp. pneumoniae* serotypes in serum or plasma [20]. The indirect immunofluorescence test seems to be more sensitive than the complement fixation test and histochemical techniques, albeit it shows cross-reactivity with other chlamydial species [20].

#### Direct fluorescent antibody (FA)

The preferably staining method to detect the *Chlamydophila* is the direct FA [89,133]. In this method, an anti-chlamydia fluorescein-conjugated is applied to the smear [134] fol‐ lowing the incubation for 30 minutes at 37 ° C [20]. Thereafter, the slides are washed up with PBS and distilled water, dried on air and finally mounted with a resinous com‐ pound like Entellan® [20]. The inclusions of the bacteria show a bright green under ul‐ traviolet microscopy [96].

#### ELISA technique

The primary purpose of a great number of commercial ELISA developed in the last 25 years was for *Chlamydia trachomatis* in humans, but such tests are also suitable for diagnosing *Chlamydo‐ phila psittaci*, as they are specific to the LPS antigen of all species of the family Chlamydiales [1,27,135-136]. Commercial ELISA tests were, over the years, widely inquired and several stud‐ ies about the low specificity and high sensitivity of these methods came up [20], with the first dramatically reducing in situations where the prevalence of the bacterium is low and hence where there is a low number of detected bodies [136]. In fact, it takes hundreds of bacteria so that the results are positive [20]. One of the major disadvantages of this technique is the possible occurrence of false positives, since have cross-reaction with other bacterias (such *Acinetobacter calcoaceticus*, *Escherichia coli*, *Staphylococcus aureus* and *Klebsiella pneumoniae)* have been demon‐ strated in humans [34,137]. Nevertheless, the development and improvement of these commer‐ cial tests increased with the use of monoclonal antibodies and advanced blockers methods [20, 136] that improve the specificity and reduced the false positive cases. There are already availa‐ ble specific commercial ELISA tests for *C. trachomatis, Cp. pneumonia, Cp. abortus* antibodies [132,138-139], and for *Chlamydophila psittaci's* as well [101]. These methods are as reliable as the indirect immunofluorescence tests, but are faster, easier to perform and less expensive [20], as well as more sensitive than the complement fixation technique, easier to standardize and, above all, more suitable for large epidemiological studies [22,101]. They also do not depend on the viability of the elementary bodies, or of soluble antigens in secretions [136]. An ELISA based on a recombinant ELISA (rMOMP ELISA) was developed and tested, giving a sensitivity and specificity of 100% in cases of psittacosis [18,140].

imal, samples must be homogenized in an antibiotic solution and remain there for 24 hours [150]. In case of deep contamination of samples, such as faecal samples, they must be homogen‐ ized in an antibiotic solution and then subjected to centrifugation at 1000-2000x *g* for 30 min [148]. Thereafter the upper and lower layers must be discarded and the supernatant inoculated into the culture fluid cells or laboratory animals. In case of persistent contamination, the sample

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The cell culture is the method of choice used to isolate *Chlamydophila psittaci* [7]. Although other cultures can be used, the most frequently used are the BGM (*Buffalo green monkey*), Mc‐ Coy, Vero, HeLa or L-929 [24]. In a 1992, a study reports that, within these, BGM has the

Bacteria can be isolated from cells that have its normal cell cycle of replication, but stable cell lines without reproduction are more satisfactory, since they provide additional nutrients for the *Cp. psittaci* replication and are much easier to observe [148]. For that, suppression of the cells is made by irradiation or by cytotoxic chemicals such as 5-iodo-2-deoxiodine [149], cytoca‐ lasine, B, cycloheximide, cycloheximide and emetine hydrochloride (0.5 - 2.0mg/ml) [148]. The type of material used will mainly depend on the available material in the laboratory [20].

To increase the binding of bacteria with cells, after the inoculation of the bacterium into the cell line a centrifugation (500-1500x *g*) for 30-90 minutes is needed [150]. This union will be stronger when incubation is performed at 35°C to 39°C [152]. Cultures are checked for bacte‐

Some laboratories use chicken embryos to isolate the organism [96, 153], usually injecting up to 0.3 ml of inoculum in the yolk sac of 6 days embryos [89]. Thereafter, bacterium repli‐ cates, which leads to the death of the embryo within a maximum period of 12 days. In case of failure, two additional inoculations should be made before considering the *Cp. psittaci*

Usually, the organism causes vascular congestion in the yolk sac membranes, which is ho‐ mogenized in a 20% suspension membrane [20]. This suspension may be frozen in order to preserve bacteria or inoculated into new eggs or cell culture monolayers [148]. The identifi‐ cation of inclusions is done through cytological or immunohistochemical methods [152], like the indirect fluorescent antibody technique, the immunoperoxidase technique [129], or by histochemical stainings [134] such as Gimenez and Macchiavello based-stains, allowing the

Fixation of monolayer cells was made with acetone for 2 to10 minutes, after transport media has been removed and washed with PBS [154-155]. If the support is made of plastic material,

Molecular methods such as PCR allow the direct detection of *Chlamydophila psittaci* from clinical specimens within one day [157]. In literature, the amplification is made from the ribosomal RNA gene region [128,158,159] and the gene encoding the antigen, known as omp1 or ompA [132, 160].

should be subjected to filtration pores of 450 to 800 micrometre [148].

**f.** Cell culture and embryonated SPF eggs

highest sensitivity [151].

ria on days 2, 3, 4 and 5 [148].

sample as negative [20].

visualization of the bacteria [20].

**g.** Polymerase chain reaction (PCR)

alcohol should be used instead of acetona for fixation [156].

In our opinion, Immunocomb® (Biogal, Kibbutz Galed, Israel) commercial kit is nowadays the most suitable ELISA test available, as it has a nearly 100% sensibility and specificity [101]. This performance is enjoyable, but the labourer must understand that the choice for the most suitable laboratorial test depends on each case (Figure 2).

**Figure 2.** Commercial kit Immunocomb® (Biogal, Kibbutz Galed, Israel) ELISA test, with the reference sample (>) and examples of strong positive animals (\*).

#### **e.** Isolation of *Cp. psittaci*

Isolation of bacteria may be performed from tissue and faecal specimens or scrapings [145]. A 20% to 40% homogenized suspension sample is prepared using diluents such as phos‐ phate buffer (PBS) at pH 7.2 and the culture media [20,146]. When samples are inoculated within 24 hours after harvested and not frozen, these solvents are used with antibiotics [20]. Whenever samples are chilled or frozen, a transport media such as SPG or Bovarnick's should be used as stabilization agent [15,147].

Before cell cultures or animal inoculation, potentially contaminated samples must be treated by three possible methods: with antibiotics [133], followed or not by low speed centrifugation [148] or filtration [133,149]. The sample is afterwards submitted to a standard procedure, where it is homogenized in diluent containing 1 mg/ml streptomycin, 1mg/ml of vancomycin and 1mg/ml kanamycin [20]. It's possible to select other antibiotics, however penicillin, tetracycline and chloramphenicol should be avoided, since they inhibit the growth of these bacteria [148]. If the sample is slightly contaminated, before the inoculation into the cell culture or on the test an‐ imal, samples must be homogenized in an antibiotic solution and remain there for 24 hours [150]. In case of deep contamination of samples, such as faecal samples, they must be homogen‐ ized in an antibiotic solution and then subjected to centrifugation at 1000-2000x *g* for 30 min [148]. Thereafter the upper and lower layers must be discarded and the supernatant inoculated into the culture fluid cells or laboratory animals. In case of persistent contamination, the sample should be subjected to filtration pores of 450 to 800 micrometre [148].

**f.** Cell culture and embryonated SPF eggs

indirect immunofluorescence tests, but are faster, easier to perform and less expensive [20], as well as more sensitive than the complement fixation technique, easier to standardize and, above all, more suitable for large epidemiological studies [22,101]. They also do not depend on the viability of the elementary bodies, or of soluble antigens in secretions [136]. An ELISA based on a recombinant ELISA (rMOMP ELISA) was developed and tested, giving a sensitivity and

In our opinion, Immunocomb® (Biogal, Kibbutz Galed, Israel) commercial kit is nowadays the most suitable ELISA test available, as it has a nearly 100% sensibility and specificity [101]. This performance is enjoyable, but the labourer must understand that the choice for

Isolation of bacteria may be performed from tissue and faecal specimens or scrapings [145]. A 20% to 40% homogenized suspension sample is prepared using diluents such as phos‐ phate buffer (PBS) at pH 7.2 and the culture media [20,146]. When samples are inoculated within 24 hours after harvested and not frozen, these solvents are used with antibiotics [20]. Whenever samples are chilled or frozen, a transport media such as SPG or Bovarnick's

Before cell cultures or animal inoculation, potentially contaminated samples must be treated by three possible methods: with antibiotics [133], followed or not by low speed centrifugation [148] or filtration [133,149]. The sample is afterwards submitted to a standard procedure, where it is homogenized in diluent containing 1 mg/ml streptomycin, 1mg/ml of vancomycin and 1mg/ml kanamycin [20]. It's possible to select other antibiotics, however penicillin, tetracycline and chloramphenicol should be avoided, since they inhibit the growth of these bacteria [148]. If the sample is slightly contaminated, before the inoculation into the cell culture or on the test an‐

(Biogal, Kibbutz Galed, Israel) ELISA test, with the reference sample (>) and

specificity of 100% in cases of psittacosis [18,140].

142 Insights from Veterinary Medicine

**Figure 2.** Commercial kit Immunocomb®

should be used as stabilization agent [15,147].

examples of strong positive animals (\*).

**e.** Isolation of *Cp. psittaci*

the most suitable laboratorial test depends on each case (Figure 2).

The cell culture is the method of choice used to isolate *Chlamydophila psittaci* [7]. Although other cultures can be used, the most frequently used are the BGM (*Buffalo green monkey*), Mc‐ Coy, Vero, HeLa or L-929 [24]. In a 1992, a study reports that, within these, BGM has the highest sensitivity [151].

Bacteria can be isolated from cells that have its normal cell cycle of replication, but stable cell lines without reproduction are more satisfactory, since they provide additional nutrients for the *Cp. psittaci* replication and are much easier to observe [148]. For that, suppression of the cells is made by irradiation or by cytotoxic chemicals such as 5-iodo-2-deoxiodine [149], cytoca‐ lasine, B, cycloheximide, cycloheximide and emetine hydrochloride (0.5 - 2.0mg/ml) [148]. The type of material used will mainly depend on the available material in the laboratory [20].

To increase the binding of bacteria with cells, after the inoculation of the bacterium into the cell line a centrifugation (500-1500x *g*) for 30-90 minutes is needed [150]. This union will be stronger when incubation is performed at 35°C to 39°C [152]. Cultures are checked for bacte‐ ria on days 2, 3, 4 and 5 [148].

Some laboratories use chicken embryos to isolate the organism [96, 153], usually injecting up to 0.3 ml of inoculum in the yolk sac of 6 days embryos [89]. Thereafter, bacterium repli‐ cates, which leads to the death of the embryo within a maximum period of 12 days. In case of failure, two additional inoculations should be made before considering the *Cp. psittaci* sample as negative [20].

Usually, the organism causes vascular congestion in the yolk sac membranes, which is ho‐ mogenized in a 20% suspension membrane [20]. This suspension may be frozen in order to preserve bacteria or inoculated into new eggs or cell culture monolayers [148]. The identifi‐ cation of inclusions is done through cytological or immunohistochemical methods [152], like the indirect fluorescent antibody technique, the immunoperoxidase technique [129], or by histochemical stainings [134] such as Gimenez and Macchiavello based-stains, allowing the visualization of the bacteria [20].

Fixation of monolayer cells was made with acetone for 2 to10 minutes, after transport media has been removed and washed with PBS [154-155]. If the support is made of plastic material, alcohol should be used instead of acetona for fixation [156].

**g.** Polymerase chain reaction (PCR)

Molecular methods such as PCR allow the direct detection of *Chlamydophila psittaci* from clinical specimens within one day [157]. In literature, the amplification is made from the ribosomal RNA gene region [128,158,159] and the gene encoding the antigen, known as omp1 or ompA [132, 160].

The quality and quantity of extracted DNA is essential for the sensitivity of the test [20]. The efficient extraction of DNA samples and removal of PCR inhibitors ensure the proper func‐ tioning of the method [20]. The efficient extraction of DNA is much more difficult in bacte‐ ria, when compared to viruses, since bacteria possessing outer membranes are very resistant to destruction [20].

**Diagnostic methods Advantages Inconveniences**




antigen in formalinfixed samples needed - more time consuming than immunocytochemistry - more expensive






Hsp60

critical


http://dx.doi.org/10.5772/53720

Psittacosis

145























samples



Immunohistochemistry - automation possible

Cytological staining

Immunocytochemistry

Antigen-ELISA

Culture

Molecular diagnosis (PCR, Micro array)

There are several methods of commercial DNA extraction capable of extracting *Chlamydophi‐ la* DNA. The test QIAamp® DNA Mini Kit (Qiagen) was reported as being the one with best results for pharyngeal scrapings, whereas the test High Pure PCR Template Preparation Kit® (Roche) was the top performer for faecal and chloacal samples [20].

These commercial tests are suitable when working with PCR inhibitors [20]. They also con‐ tain a particular reagent for the bacterial and eukaryotic cells lysis. One of these reagents is GIT (Guanidine IsoThiocyanate) [20].

The cellular RNA is digested with an RNase and then the lysate is centrifuged in a minicolumn, where the DNA is joined to a solid phase that can be modified by silica hydroxyapatite or special filters membranes. Then, the elution takes place by immersing DNA of high purity and free of PCR inhibitors [20]. Cold storage leads to a rapid loss of DNA from organisms, so samples with low levels of target DNA often become negative. To avoid this problem, DNA stabilizers are used in PCR analysis [161]. Some reagents are available com‐ mercially as the RNA/DNA Stabilization Reagent for Blood/Bone Marrow® from Roche Ap‐ plied Science [161,162].

In 2005, a PCR assay proved sensitive enough to detect *Chlamydophila psittaci* in samples from birds [97]. Sachse and colleagues (2005) detected the bacteria based on the amplifica‐ tion, resulting on a specific ompA product for the genus, followed by a second amplification using a specific primer for the genus and a specific primer for the species. At the end, it is created a specific amplicon for *Chlamydophila psittaci* [20, 163].

The PCR results are visualized by electrophoresis and the sensitivity of PCR-EIA was set at 0.1 IFU (infection forming unit) [97].

These tests are in progress and recently new types of PCR emerged. One such case is the SYBR Green-based real-time PCR that targets the rDNA spacer of *Chlamydophila psittaci* [20]. This test detects 10rDNA copies/ml of extracted DNA and all ompA genotypes of the bacteria [20].

**h.** DNA microarray-based detection

Sachse and colleagues (2005) developed a method to identify *Chlamydia* and *Chlamydophila* spp. species. This test was developed through the platform ArrayTube (CLONDIAG® chip technologies) and it obtained specific hybridization patterns for the species in all organisms of the Chlamydiaceae family. Thus, this study proved this test is a viable alternative for the identification of ambiguous *Chlamydia* cell culture, and a possibility for detection of these microorganisms from tissues [163].

Table 4 below summarizes the advantages and disadvantages of some methods for diagno‐ sis of *Chlamydophila psittaci* [20].


The quality and quantity of extracted DNA is essential for the sensitivity of the test [20]. The efficient extraction of DNA samples and removal of PCR inhibitors ensure the proper func‐ tioning of the method [20]. The efficient extraction of DNA is much more difficult in bacte‐ ria, when compared to viruses, since bacteria possessing outer membranes are very resistant

There are several methods of commercial DNA extraction capable of extracting *Chlamydophi‐ la* DNA. The test QIAamp® DNA Mini Kit (Qiagen) was reported as being the one with best results for pharyngeal scrapings, whereas the test High Pure PCR Template Preparation Kit®

These commercial tests are suitable when working with PCR inhibitors [20]. They also con‐ tain a particular reagent for the bacterial and eukaryotic cells lysis. One of these reagents is

The cellular RNA is digested with an RNase and then the lysate is centrifuged in a minicolumn, where the DNA is joined to a solid phase that can be modified by silica hydroxyapatite or special filters membranes. Then, the elution takes place by immersing DNA of high purity and free of PCR inhibitors [20]. Cold storage leads to a rapid loss of DNA from organisms, so samples with low levels of target DNA often become negative. To avoid this problem, DNA stabilizers are used in PCR analysis [161]. Some reagents are available com‐ mercially as the RNA/DNA Stabilization Reagent for Blood/Bone Marrow® from Roche Ap‐

In 2005, a PCR assay proved sensitive enough to detect *Chlamydophila psittaci* in samples from birds [97]. Sachse and colleagues (2005) detected the bacteria based on the amplifica‐ tion, resulting on a specific ompA product for the genus, followed by a second amplification using a specific primer for the genus and a specific primer for the species. At the end, it is

The PCR results are visualized by electrophoresis and the sensitivity of PCR-EIA was set at

These tests are in progress and recently new types of PCR emerged. One such case is the SYBR Green-based real-time PCR that targets the rDNA spacer of *Chlamydophila psittaci* [20]. This test detects 10rDNA copies/ml of extracted DNA and all ompA genotypes of the bacteria [20].

Sachse and colleagues (2005) developed a method to identify *Chlamydia* and *Chlamydophila* spp. species. This test was developed through the platform ArrayTube (CLONDIAG® chip technologies) and it obtained specific hybridization patterns for the species in all organisms of the Chlamydiaceae family. Thus, this study proved this test is a viable alternative for the identification of ambiguous *Chlamydia* cell culture, and a possibility for detection of these

Table 4 below summarizes the advantages and disadvantages of some methods for diagno‐

(Roche) was the top performer for faecal and chloacal samples [20].

created a specific amplicon for *Chlamydophila psittaci* [20, 163].

to destruction [20].

144 Insights from Veterinary Medicine

GIT (Guanidine IsoThiocyanate) [20].

0.1 IFU (infection forming unit) [97].

**h.** DNA microarray-based detection

microorganisms from tissues [163].

sis of *Chlamydophila psittaci* [20].

plied Science [161,162].


**b.** Disinfection

70% isopropyl alcohol and 1% lysol [12, 113].

ty equipment and the use of flow laminar chambers [23].

son, however this process is believed to be uncommon [167-168].

rapid evaluation of the case are vital [12, 123].

[103, 162].

**8. Public health concerns**

*Chlamydophila psittaci* can survive up to 30 days in faeces and cage materials or beds, so regular cleaning of equipment and places where animals are infected is essential [127]. Bacteria of the family *Chlamydiae* are quite sensitive to chemical that affecting the lipid contents or the integrity of the cell walls. Most disinfectants used as detergents inactivate *Cp. psittaci* bacteria, such as a 1 to 1000 ammonium quaternary, 1 to 100 chlorophenol,

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*Chlamydophila psittaci* is a zoonosis, hence it is an organism capable of producing infection in humans [14, 24, 166]. Man is mainly infected by the dispersed bacteria in the inhaled air, af‐ ter faeces, urine or secretions of the respiratory system of infected animals dry [21]. The di‐ rect contact with infected animals may also lead to infection [4], so that the post mortem examination and manipulation of cultures should be done properly, using appropriate safe‐

This disease has particular significance in public health, since parrots are common pets in our houses, even in schools or nursing places [148]. The risk of contracting the disease in‐ creases with the contact with these animals, but also in day-to-day activities, for example mowing the lawn, shrubs or others without a suitable container for this purpose, exposing individuals to the bacteria [16, 167]. Psittacosis can also be transmitted from person to per‐

The incubation period is usually between 5-14 days [12]. In humans, signs of psittaco‐ sis can vary greatly, ranging from cases where they are completely unapparent to sit‐ uations where it can be found signs of systemic disease with severe interstitial pneumonia and encephalitis [12, 170]. Infected humans may develop headaches, chills, discomfort and myalgia [12]. Respiratory involvement is common and a number of cases have been documented [171-174]. This disease is, however, rarely fatal if patients are provided with proper treatment, so an awareness of the dangers of psittacosis and

*Chlamydophila psittaci* was associated with ocular lymphoma [174], however, this subject is still matter of debate and disagreement and conflicting reports have been published [175,176]. The true greatness of this disease is very far from knowing, since cases where psittacosis is diagnosed and individuals have severe signs of disease are only a tiny fraction of the total occurrences. Cases where the disease is less severe, unnoticed or misdiagnosed infections due to similar symptoms with other respiratory pathogens or asymptomatic infections remain in the shadow of *Cp. psittaci* infection in humans

**Table 4.** Advantages and disadvantages of some methods for diagnosis of *Chlamydophila psittaci* (Adapted from [20]).

It is our opinion that there's no better diagnostic test for *Clamidophila psittaci* but a more suit‐ able or adequate to each case instead, for different biologic material sampling and laborato‐ rial conditions. If the goal is to define *Chlamydophila*'s prevalence in a large group of animals, PCR should be the first choice, although more reliable tests imply more expensive tests. If the investment had limitations, an ELISA test can be the most adequate choice for the diagnosis. If the group of animals is just of some units, then cultured cells can be a possi‐ bility if the proper laboratory conditions are available.

#### **6. Treatment**

Treatment of psittacosis is performed with medication, being tetracycline the drug of choice [12, 148]. In cases developing in pregnant women or in children under the age of 9 years, the use of tetracycline or doxycycline is contraindicated, being the use of erythromycin the most suitable. Treatment of the patient should be made for at least 14 consecutive days using the drug of choice [164]. In birds, tetracycline or doxycycline should be given over a period of 7 weeks in the feed or on medicated seed [165].

#### **7. Prevention**

#### **a.** Vaccines

Today there are still no vaccines available against avian chlamydiosis [148]. Attempts on DNA vaccines have reduced signs of disease, injuries and excretion of bacteria. However, a complete protection of individuals remains distant [98]. Currently, treatments and strategies for reducing contamination of these bacteria are the best way to control the disease [12].

#### **b.** Disinfection

**Diagnostic methods Advantages Inconveniences**






diagnosis)

present

non-specific

diagnosis



**Table 4.** Advantages and disadvantages of some methods for diagnosis of *Chlamydophila psittaci* (Adapted from

It is our opinion that there's no better diagnostic test for *Clamidophila psittaci* but a more suit‐ able or adequate to each case instead, for different biologic material sampling and laborato‐ rial conditions. If the goal is to define *Chlamydophila*'s prevalence in a large group of animals, PCR should be the first choice, although more reliable tests imply more expensive tests. If the investment had limitations, an ELISA test can be the most adequate choice for the diagnosis. If the group of animals is just of some units, then cultured cells can be a possi‐

Treatment of psittacosis is performed with medication, being tetracycline the drug of choice [12, 148]. In cases developing in pregnant women or in children under the age of 9 years, the use of tetracycline or doxycycline is contraindicated, being the use of erythromycin the most suitable. Treatment of the patient should be made for at least 14 consecutive days using the drug of choice [164]. In birds, tetracycline or doxycycline should be given over a period of 7

Today there are still no vaccines available against avian chlamydiosis [148]. Attempts on DNA vaccines have reduced signs of disease, injuries and excretion of bacteria. However, a complete protection of individuals remains distant [98]. Currently, treatments and strategies for reducing contamination of these bacteria are the best way to control the


Serology: antibody ELISA

146 Insights from Veterinary Medicine

[20]).

**6. Treatment**

**7. Prevention**

**a.** Vaccines

disease [12].


bility if the proper laboratory conditions are available.

weeks in the feed or on medicated seed [165].

*Chlamydophila psittaci* can survive up to 30 days in faeces and cage materials or beds, so regular cleaning of equipment and places where animals are infected is essential [127]. Bacteria of the family *Chlamydiae* are quite sensitive to chemical that affecting the lipid contents or the integrity of the cell walls. Most disinfectants used as detergents inactivate *Cp. psittaci* bacteria, such as a 1 to 1000 ammonium quaternary, 1 to 100 chlorophenol, 70% isopropyl alcohol and 1% lysol [12, 113].

## **8. Public health concerns**

*Chlamydophila psittaci* is a zoonosis, hence it is an organism capable of producing infection in humans [14, 24, 166]. Man is mainly infected by the dispersed bacteria in the inhaled air, af‐ ter faeces, urine or secretions of the respiratory system of infected animals dry [21]. The di‐ rect contact with infected animals may also lead to infection [4], so that the post mortem examination and manipulation of cultures should be done properly, using appropriate safe‐ ty equipment and the use of flow laminar chambers [23].

This disease has particular significance in public health, since parrots are common pets in our houses, even in schools or nursing places [148]. The risk of contracting the disease in‐ creases with the contact with these animals, but also in day-to-day activities, for example mowing the lawn, shrubs or others without a suitable container for this purpose, exposing individuals to the bacteria [16, 167]. Psittacosis can also be transmitted from person to per‐ son, however this process is believed to be uncommon [167-168].

The incubation period is usually between 5-14 days [12]. In humans, signs of psittaco‐ sis can vary greatly, ranging from cases where they are completely unapparent to sit‐ uations where it can be found signs of systemic disease with severe interstitial pneumonia and encephalitis [12, 170]. Infected humans may develop headaches, chills, discomfort and myalgia [12]. Respiratory involvement is common and a number of cases have been documented [171-174]. This disease is, however, rarely fatal if patients are provided with proper treatment, so an awareness of the dangers of psittacosis and rapid evaluation of the case are vital [12, 123].

*Chlamydophila psittaci* was associated with ocular lymphoma [174], however, this subject is still matter of debate and disagreement and conflicting reports have been published [175,176]. The true greatness of this disease is very far from knowing, since cases where psittacosis is diagnosed and individuals have severe signs of disease are only a tiny fraction of the total occurrences. Cases where the disease is less severe, unnoticed or misdiagnosed infections due to similar symptoms with other respiratory pathogens or asymptomatic infections remain in the shadow of *Cp. psittaci* infection in humans [103, 162].

## **9. Conclusion**

Psittacosis is a disease that is virtually throughout the world and is a zoonosis. This fact must be present in all healthcare professionals, being veterinarians or doctors.

[2] Dickx V, Vanrompay D.*Chlamydia psittaci* in a chicken and turkey hatchery results in zoonotic transmission. Journal of Medical Microbiology 2011;60(Pt 6) 775-779.

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[3] Ciftçi B, Güler ZM, Aydoğdu M, Konur O, Erdoğan Y. Familial outbreak of psittaco‐ sis as the first *Chlamydia psittaci* infection reported from Turkey. Tüberkulöz Toraks

[4] Beeckman DS, Vanrompay DC. Zoonotic *Chlamydophila psittaci* infections from a clin‐

[5] Fraeyman A, Boel A, Van Vaerenbergh K, De Beenhouwer H. Atypical pneumonia due to *Chlamydophila psittaci*: 3 case reports and review of literature. Acta Clinica

[6] Pennycott TW, Dagleish MP, Wood AM, Garcia C. *Chlamydophila psittaci* in wild

[7] Dahlhausen B, Radabaugh CS. Detection of *Chlamydia psittaci* infection in pet birds using a molecular based diagnostic assay. In: Proceedings of the Annual Conference

[8] Maluping RP, Oronan RB, Toledo SU. Detection of *Chlamydophila psittaci* antibodies from captive birds at the Ninoy Aquino Parks and Wildlife Nature Center, Quezon City, Philippines. Annals of Agricultural and Environmental Medicine 2007;14(1)

[9] Voigt A, Schöfl G, Heidrich A, Sachse K, Saluz HP.Full-Length De Novo Sequence of the *Chlamydophila psittaci* Type Strain, 6BC. Journal of Bacteriology 2011;193(10)

[10] Reinhold P, Sachse K, Kaltenboeck B. Chlamydiaceae in cattle: Commensals, trigger

[11] Sprague LD, Schubert E, Hotzel H, Scharf S, Sachse K. The detection of *Chlamydophila psittaci* genotype C infection in dogs. Veterinary Journal 2009;181(3) 274-279.

[12] Smith KA, Bradley KK, Stobierski MG, Tengelsen LA. Compendium of measures to control *Chlamydophila psittaci* (formerly *Chlamydia psittaci*) infection among humans (psittacosis) and pet birds, 2005. Journal of American Veterinary Medical Association

[13] Berk Y, Klaassen CH, Mouton JW, Meis JF. An outbreak of psittacosis at a bird-fan‐ ciers fair in the Netherlands. Nederlands Tijdschrift voor Geneeskdunde 2008;

[14] Gaede W, Reckling KF, Dresenkamp B, Kenklies S, Schubert E, Noack U, Irmscher HM, Ludwig C, Hotzel H, Sachse K. *Chlamydophila psittaci* infections in humans dur‐ ing an outbreak of psittacosis from poultry in Germany. Zoonoses and Public Health

organisms, or pathogens? Veterinary Journal 2011;189(3) 257-267.

of the Association Avian Veterinarians Reno, NV. 1997;p. 191–198; 1997.

ical perspective. Clinical Microbiology and Infection 2009;15(1) 11-17.

birds in the UK. Veterinary Record 2009;164 (5) 157-158.

2008;56(2) 215-220.

191-193.

2662-2663.

2005;226(4) 532-539.

152(34) 1889-1892.

2008; 55(4)184-188.

Belgica 2010;65(3) 192-196.

Psittacines are the most commonly affected animals and this fact must be an alert for veterinarians to pay special caution during its manipulation. Owners and handlers of exotic birds should as well learn how to prevent this disease, as they are a potential risk group for infection.

The number of infected animals by *Chlamydophila psittaci* is underestimated [106] and new cases of outbreaks continue to appear worldwide, some of which often die facing this disease [20].

A more feasible, fast and easy diagnostic method and universally accorded is yet to be implemented.

Altogether, control of this disease should be stricter and information about its maleficence worldwide known.

## **Acknowledgements**

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐ nanced by the Foundation for Science and Technology (FCT).The authors would like to thank to Laboratory of Histology and Anatomical Pathology and to the Laboratory of Medi‐ cal Microbiology of the Dpt. of Veterinary Sciences of the University of Trás-os-Montes-e-Alto-Douro and to staff of the Zoological Parks of Águas e Parque Biológico de Gaia, Zoo of Lourosa, Zoo of Maia.

## **Author details**

João Morais, Ana Cláudia Coelho and Maria dos Anjos Pires

CECAV –Univ. of Trás-os-Montes and Alto Douro, Portugal

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**Acknowledgements**

Lourosa, Zoo of Maia.

**Author details**

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João Morais, Ana Cláudia Coelho and Maria dos Anjos Pires

CECAV –Univ. of Trás-os-Montes and Alto Douro, Portugal

implemented.

Psittacosis is a disease that is virtually throughout the world and is a zoonosis. This fact

Psittacines are the most commonly affected animals and this fact must be an alert for veterinarians to pay special caution during its manipulation. Owners and handlers of exotic birds should as well learn how to prevent this disease, as they are a potential

The number of infected animals by *Chlamydophila psittaci* is underestimated [106] and new cases of outbreaks continue to appear worldwide, some of which often die facing

A more feasible, fast and easy diagnostic method and universally accorded is yet to be

Altogether, control of this disease should be stricter and information about its maleficence

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐ nanced by the Foundation for Science and Technology (FCT).The authors would like to thank to Laboratory of Histology and Anatomical Pathology and to the Laboratory of Medi‐ cal Microbiology of the Dpt. of Veterinary Sciences of the University of Trás-os-Montes-e-Alto-Douro and to staff of the Zoological Parks of Águas e Parque Biológico de Gaia, Zoo of

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[175] Chanudet E, Zhou Y, Bacon CM, Wotherspoon AC, Müller-Hermelink HK, Adam P, Dong HY, de Jong D, Li Y, Wei R, Gong X, Wu Q, Ranaldi R, Goteri G, Pileri SA, Ye H, Hamoudi RA, Liu H, Radford J, Du MQ. *Chlamydia psittaci* is variably associated with ocular adnexal MALT lymphoma in different geographical regions. Journal of Pathology 2006;209(3) 344-351.

**Section 3**

**Current Topics on Hormones and Hormone-**

**Related Diseases**

[176] Zhang GS, Winter JN, Variakojis D, Reich S, Lissner GS, Bryar P, Regner M, Mangold K, Kaul K. Lack of an association between *Chlamydia psittaci* and ocular adnexal lym‐ phoma. Leukemia and Lymphoma 2007;48(3) 577-583.

**Current Topics on Hormones and Hormone-Related Diseases**

[175] Chanudet E, Zhou Y, Bacon CM, Wotherspoon AC, Müller-Hermelink HK, Adam P, Dong HY, de Jong D, Li Y, Wei R, Gong X, Wu Q, Ranaldi R, Goteri G, Pileri SA, Ye H, Hamoudi RA, Liu H, Radford J, Du MQ. *Chlamydia psittaci* is variably associated with ocular adnexal MALT lymphoma in different geographical regions. Journal of

[176] Zhang GS, Winter JN, Variakojis D, Reich S, Lissner GS, Bryar P, Regner M, Mangold K, Kaul K. Lack of an association between *Chlamydia psittaci* and ocular adnexal lym‐

phoma. Leukemia and Lymphoma 2007;48(3) 577-583.

Pathology 2006;209(3) 344-351.

162 Insights from Veterinary Medicine

**Chapter 6**

**The Endocrine Glands in the Dog:**

**From the Cell to Hormone**

Helena Vala, João Rodrigo Mesquita,

Cristina Mega and Carmen Nóbrega

http://dx.doi.org/10.5772/53577

in a slower, but more durable way.

functions and maintaining homeostasis.

pancreas, adrenal (Figure 1), and gonads (ovaries and testes).

– exocrine mechanism.

**1. Introduction**

Fernando Esteves, Carla Santos, Rita Cruz,

Additional information is available at the end of the chapter

The animal body represents one of the more complex and perfect systems of nature. Despite its complexity and its functionality, which is incredibly effective, the control of its basic functions is performed by only two systems: the nervous system and endocrine system. The nervous system is associated with electrical and chemical signals that are transmitted at high speed, resulting in rapid organic activities. The endocrine system acts through the synthesis and release of chemical messengers and is responsible for several functions of the organism,

Endocrinology is the science that studies the internal secretions produced by endocrine glands. Endocrine glands are distributed throughout the body and secrete chemical messengers – hormones, in response to an internal or external stimulus. These hormones are released directly into the bloodstream – endocrine mechanism, in contrast to exocrine glands, which use a ductal system to release their secretions in locations that lead, ultimately, to the exterior of the body

Hormones are transported through the bloodstream to target organs, where they will exert a physiological control, even in low concentrations, coordinating a multiplicity of organic

The main endocrine glands in the animal body include pituitary gland, thyroid, parathyroid,

© 2013 Vala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Vala et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Chapter 6**

## **The Endocrine Glands in the Dog: From the Cell to Hormone**

Helena Vala, João Rodrigo Mesquita, Fernando Esteves, Carla Santos, Rita Cruz, Cristina Mega and Carmen Nóbrega

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53577

## **1. Introduction**

The animal body represents one of the more complex and perfect systems of nature. Despite its complexity and its functionality, which is incredibly effective, the control of its basic functions is performed by only two systems: the nervous system and endocrine system. The nervous system is associated with electrical and chemical signals that are transmitted at high speed, resulting in rapid organic activities. The endocrine system acts through the synthesis and release of chemical messengers and is responsible for several functions of the organism, in a slower, but more durable way.

Endocrinology is the science that studies the internal secretions produced by endocrine glands. Endocrine glands are distributed throughout the body and secrete chemical messengers – hormones, in response to an internal or external stimulus. These hormones are released directly into the bloodstream – endocrine mechanism, in contrast to exocrine glands, which use a ductal system to release their secretions in locations that lead, ultimately, to the exterior of the body – exocrine mechanism.

Hormones are transported through the bloodstream to target organs, where they will exert a physiological control, even in low concentrations, coordinating a multiplicity of organic functions and maintaining homeostasis.

The main endocrine glands in the animal body include pituitary gland, thyroid, parathyroid, pancreas, adrenal (Figure 1), and gonads (ovaries and testes).

© 2013 Vala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Vala et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Schematic location of main endocrine glands. Top right figure represents pituitary gland; top left figure rep‐ resents pancreas; bottom right figure represents thyroid and parathyroid glands; bottom left figure represents adre‐ nal glands.

**Table 1.** Hormones produced by the hypothalamic-pituitary axis.

**Figure 2.** Location of the pituitary gland within the *sella turcica*, next to the optic chiasm.

The pituitary is a small oval shaped gland, lodged in the *sella turcica*, a small depression of the sphenoid bone, which is located at the base of the brain and positioned next to the optic chiasm (Figure 2), being connected to the hypothalamus by a funnel-shaped hypophyseal stalk [1,2].

The Endocrine Glands in the Dog: From the Cell to Hormone

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167

## **2. Hypothalamic-pituitary axis**

The hypothalamus, located at the base of the ventral diencephalon is limited rostrally by the optic chiasm, caudally by the mammillary processes, laterally by the temporal lobes and dorsally by the thalamus. Ventrally, it continues through the infundibulum which connects it to the pituitary gland. Although not considered a real endocrine gland, the hypothalamus coordinates all pituitary activity by the release of a number of peptides and amines that control the secretion of hormones in the pituitary gland (also named hypophysis). Their terminology ends in RH that means releasing hormone (GnRH - gonadotrophin releasing hormone; CRH - Corticotrophin releasing hormone; TRH - Tirotrophin releasing hormone; PRH - Prolactin releasing hormone; GHRH - Growth hormone releasing hormone). Table 1 summarizes the major hormones produced by this hypothalamic-pituitary axis.

**Table 1.** Hormones produced by the hypothalamic-pituitary axis.

**Figure 1.** Schematic location of main endocrine glands. Top right figure represents pituitary gland; top left figure rep‐ resents pancreas; bottom right figure represents thyroid and parathyroid glands; bottom left figure represents adre‐

The hypothalamus, located at the base of the ventral diencephalon is limited rostrally by the optic chiasm, caudally by the mammillary processes, laterally by the temporal lobes and dorsally by the thalamus. Ventrally, it continues through the infundibulum which connects it to the pituitary gland. Although not considered a real endocrine gland, the hypothalamus coordinates all pituitary activity by the release of a number of peptides and amines that control the secretion of hormones in the pituitary gland (also named hypophysis). Their terminology ends in RH that means releasing hormone (GnRH - gonadotrophin releasing hormone; CRH - Corticotrophin releasing hormone; TRH - Tirotrophin releasing hormone; PRH - Prolactin releasing hormone; GHRH - Growth hormone releasing hormone). Table 1 summarizes the

nal glands.

166 Insights from Veterinary Medicine

**2. Hypothalamic-pituitary axis**

major hormones produced by this hypothalamic-pituitary axis.

The pituitary is a small oval shaped gland, lodged in the *sella turcica*, a small depression of the sphenoid bone, which is located at the base of the brain and positioned next to the optic chiasm (Figure 2), being connected to the hypothalamus by a funnel-shaped hypophyseal stalk [1,2].

**Figure 2.** Location of the pituitary gland within the *sella turcica*, next to the optic chiasm.

The pituitary gland is divided into two regions, the adenohypophysis, or anterior pituitary gland, and the neurohypophysis, also called the posterior pituitary gland or pars nervosa.

Thyrotrophs or thyrotropic cells which secrete TSH (basophilic) [14].

also be undifferentiated, nonsecretory cells [4, 15].

**Figure 4.** Adenohypophysis. Pars distalis. HE. Bar = 25 µm.

lactotrophs in the pars distalis [3].

therefore, darkening of skin [10].

pophysis, until a stimulus induces their release [4, 16] (Table 2).

promoting uterine contractions during parturition [10] (Table 2).

The chromophobe cells are also arranged in clusters or cords and their low tinctorial affinity probably indicates that these cells may correspond to depleted cells of any of the above types described or a state of partial degranulation. However, some of the chromophobe cells may

The Endocrine Glands in the Dog: From the Cell to Hormone

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169

**•** The pars tuberalis is composed by cuboidal weakly basophilic cells, arranged in cords, nests or follicles and its function is not yet well established [4]. Studies suggest that one of its primary functions, in seasonal mammals (e.g. sheep), is to mediate photoperiod influences in prolactin secretion variations through an unidentified factor called tuberalin i.e. tuberalin performs hormonal control, both in gene expression and in the release of prolactin from

**•** The pars intermedia is more developed in domestic animals than in man, consisting of nonspecific basophilic cells, arranged mainly in cords and nests, but also in follicles (Figure 5), which secrete MSH [4] that in turn, promotes distribution of melanin granules, and

The neurohypophysis is devoid of endocrine cells, being composed of demielinized neurons (Figure 6) whose bodies are in the supraoptic and paraventricular nuclei of the hypothalamus, constituting the storage of the hypothalamic hormones: antidiuretic hormone (ADH), also called vasopressin, oxytocin and neurophysins. These hormones are produced in the hypo‐ thalamus, transported through the hypothalamic-pituitary axis and stored in the neurohy‐

ADH has several effects on the body, particularly in terms of water saving and in increasing blood pressure. Oxytocin stimulates contraction of the myoepithelial cells of the mammary gland, causing the ejection of milk. It also binds to the smooth muscle cells of the uterus,

The adenohypophysis, in turn, is divided in three sections: pars distalis, pars tuberalis and pars intermedia (Figure 3) [3].

**Figure 3.** Divisions of the pituitary gland in the dog. PT, pars tuberalis; PI, pars intermedia; PD, pars distalis; PN, pars nervosa. HE. Bar = 100 µm.

**•** The parenchyma of the pars distalis is composed by chromophilic cells, acidophilic or basophilic, arranged in cords, nests or follicles (Figure 4) and by small, round endocrine cells, without evident granules - chromophobe cells which means that they have low tinctorial affinity, also known as principal cells, reserve cells or C cells [4]. Chromophilic cells secrete somatotropichormone(alsoknownassomatotropin-STHorgrowthhormone-GH),prolactin (PRL), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteiniz‐ inghormone(LH)(whichisalsocalledinterstitialcell-stimulatinghormoneorICSHinmales), thyrotropinorthyroid-stimulatinghormone(TSH),melanocyte-stimulatinghormone(MSH) and lipotropines [4]. Somatotropin promotes epiphyseal growth [6] and protein produc‐ tion,whereasprolactinleads tomammaryglanddevelopmentandmilksecretion.ACTHacts on the adrenal gland cortex, resulting in an increased release of adrenocortical hormones [7]. FSH and LH are gonadotropic hormones, with different effects in males and females [8,9]. TSH acts on the thyroid gland, stimulating its secretion [10]. Lipotropines have the same precursor molecule as ACTH (propiomelanocortin - POMC) [3,5] and, after cleavage, can originate β-MSH and β-endorfine, a pain blocking agent [3] (Table 2).

Also, considering its secretion, chromophilic cells of pars distalis, may be classified in four types:

Lactotrophs or lactotropic cells which secrete PRL (acidophilic) [11].

Corticotropes or corticotropic cells which secrete ACTH (basophilic) [12].

Gonadotropesorgonadotropic cellswhichsecretegonadotropinsLHandFSH(basophilic)[13].

Thyrotrophs or thyrotropic cells which secrete TSH (basophilic) [14].

The pituitary gland is divided into two regions, the adenohypophysis, or anterior pituitary gland, and the neurohypophysis, also called the posterior pituitary gland or pars nervosa. The adenohypophysis, in turn, is divided in three sections: pars distalis, pars tuberalis and

**PI**

**Figure 3.** Divisions of the pituitary gland in the dog. PT, pars tuberalis; PI, pars intermedia; PD, pars distalis; PN, pars

**•** The parenchyma of the pars distalis is composed by chromophilic cells, acidophilic or basophilic, arranged in cords, nests or follicles (Figure 4) and by small, round endocrine cells, without evident granules - chromophobe cells which means that they have low tinctorial affinity, also known as principal cells, reserve cells or C cells [4]. Chromophilic cells secrete somatotropichormone(alsoknownassomatotropin-STHorgrowthhormone-GH),prolactin (PRL), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteiniz‐ inghormone(LH)(whichisalsocalledinterstitialcell-stimulatinghormoneorICSHinmales), thyrotropinorthyroid-stimulatinghormone(TSH),melanocyte-stimulatinghormone(MSH) and lipotropines [4]. Somatotropin promotes epiphyseal growth [6] and protein produc‐ tion,whereasprolactinleads tomammaryglanddevelopmentandmilksecretion.ACTHacts on the adrenal gland cortex, resulting in an increased release of adrenocortical hormones [7]. FSH and LH are gonadotropic hormones, with different effects in males and females [8,9]. TSH acts on the thyroid gland, stimulating its secretion [10]. Lipotropines have the same precursor molecule as ACTH (propiomelanocortin - POMC) [3,5] and, after cleavage, can

Also, considering its secretion, chromophilic cells of pars distalis, may be classified in four

Gonadotropesorgonadotropic cellswhichsecretegonadotropinsLHandFSH(basophilic)[13].

originate β-MSH and β-endorfine, a pain blocking agent [3] (Table 2).

Corticotropes or corticotropic cells which secrete ACTH (basophilic) [12].

Lactotrophs or lactotropic cells which secrete PRL (acidophilic) [11].

**PT**

**PD**

**PN**

pars intermedia (Figure 3) [3].

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nervosa. HE. Bar = 100 µm.

types:

The chromophobe cells are also arranged in clusters or cords and their low tinctorial affinity probably indicates that these cells may correspond to depleted cells of any of the above types described or a state of partial degranulation. However, some of the chromophobe cells may also be undifferentiated, nonsecretory cells [4, 15].

**Figure 4.** Adenohypophysis. Pars distalis. HE. Bar = 25 µm.


The neurohypophysis is devoid of endocrine cells, being composed of demielinized neurons (Figure 6) whose bodies are in the supraoptic and paraventricular nuclei of the hypothalamus, constituting the storage of the hypothalamic hormones: antidiuretic hormone (ADH), also called vasopressin, oxytocin and neurophysins. These hormones are produced in the hypo‐ thalamus, transported through the hypothalamic-pituitary axis and stored in the neurohy‐ pophysis, until a stimulus induces their release [4, 16] (Table 2).

ADH has several effects on the body, particularly in terms of water saving and in increasing blood pressure. Oxytocin stimulates contraction of the myoepithelial cells of the mammary gland, causing the ejection of milk. It also binds to the smooth muscle cells of the uterus, promoting uterine contractions during parturition [10] (Table 2).

**Figure 5.** Adenohypophysis. Pars intermedia. HE. Bar = 25 µm.

**Figure 6.** Neurohypophysis. HE. Bar = 25 µm.

## **3. Thyroid**

Canine thyroid gland is located at cervical ventral region, lateral and ventral to the 5th-8th tracheal rings (Figure 7), being composed by two separate lobes, occasionally connected by an isthmus [17, 18].

In the lobes, the majority of the epithelial cellular population (90%) ranges in height, from low cuboidal to high columnar epithelial cells, which are organized into follicles – follicular cells (Figure 8) [4, 16].

The follicle is, therefore, the structural and secretory unit of the thyroid gland, whose center is filled with a colloidal secretion (Figure 9), comprising the hormones triiodothyronine (T3) and thyroxine (T4) [4]. In between the follicles, in a parafollicular position, cells with a pale

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**Table 2.** Hormones produced by the pituitary gland and their functions.

**Figure 7.** Thyroid location.

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**Table 2.** Hormones produced by the pituitary gland and their functions.

**Figure 7.** Thyroid location.

**Figure 5.** Adenohypophysis. Pars intermedia. HE. Bar = 25 µm.

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**Figure 6.** Neurohypophysis. HE. Bar = 25 µm.

Canine thyroid gland is located at cervical ventral region, lateral and ventral to the 5th-8th tracheal rings (Figure 7), being composed by two separate lobes, occasionally connected by an

In the lobes, the majority of the epithelial cellular population (90%) ranges in height, from low cuboidal to high columnar epithelial cells, which are organized into follicles – follicular cells

**3. Thyroid**

isthmus [17, 18].

(Figure 8) [4, 16].

The follicle is, therefore, the structural and secretory unit of the thyroid gland, whose center is filled with a colloidal secretion (Figure 9), comprising the hormones triiodothyronine (T3) and thyroxine (T4) [4]. In between the follicles, in a parafollicular position, cells with a pale

**Table 3.** Hormones produced by thyroid gland and their functions.

Parathyroid glands are salmon-coloured and distinct from the thyroid glands. In the dog there are four parathyroid glands, one external and one internal per each thyroid gland [17, 19, 20].

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External parathyroid glands are capsulated and may be found in varied positions, according to the species, which means that they can be placed between a cranial location to the thyroid gland and the entrance of the chest. In dogs, external parathyroid glands are located very close to the thyroid gland, on its cranial pole and externally to its capsule (Figure 10) [17, 19, 20].

The internal parathyroids are unencapsulated glands. They may be absent in some species or may be found included within the thyroid or close to them. In the dog, internal parathyroid glands are within the thyroid capsule, in the caudal and medial aspects of the thyroid [19, 20].

The parenchyma of parathyroid glands is composed of secretory cells, named chief or principal cells, arranged in cords, clusters, chains or rosettes (Figure 11, Figure 12). In some species, such as cattle and humans, it is also composed by oxyphilic cells, organized in small clusters which

**4. Parathyroid**

**Figure 10.** External parathyroid gland location.

**Figure 8.** Thyroid follicles. HE. Bar = 25 µm.

**Figure 9.** Thyroid follicles. Central colloidal eosinophilic material. HE. Bar = 25 µm.

cytoplasm, and a basal nucleus can be found - parafollicular cells or C cells which produce calcitonin [4, 16, 18].

T4 and T3 have very similar effects in regulating cellular metabolism. Calcitonin assists with the regulation of calcium ion concentration in the blood. It is secreted in response to hyper‐ calcemia, decreasing the resorption of calcium from bones and inhibiting the phosphate reabsortion from renal tubules [10] (table 3).

Due to the action of T3 and T4, thyroid is the main gland, acting in the regulation of metabolism.

Thyroid stimulation is performed by TSH, originated in the adenohypophysis, under stimu‐ lation of TRH released by hypothalamus [10].

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**Table 3.** Hormones produced by thyroid gland and their functions.

## **4. Parathyroid**

Parathyroid glands are salmon-coloured and distinct from the thyroid glands. In the dog there are four parathyroid glands, one external and one internal per each thyroid gland [17, 19, 20].

External parathyroid glands are capsulated and may be found in varied positions, according to the species, which means that they can be placed between a cranial location to the thyroid gland and the entrance of the chest. In dogs, external parathyroid glands are located very close to the thyroid gland, on its cranial pole and externally to its capsule (Figure 10) [17, 19, 20].

cytoplasm, and a basal nucleus can be found - parafollicular cells or C cells which produce

T4 and T3 have very similar effects in regulating cellular metabolism. Calcitonin assists with the regulation of calcium ion concentration in the blood. It is secreted in response to hyper‐ calcemia, decreasing the resorption of calcium from bones and inhibiting the phosphate

Due to the action of T3 and T4, thyroid is the main gland, acting in the regulation of metabolism.

Thyroid stimulation is performed by TSH, originated in the adenohypophysis, under stimu‐

calcitonin [4, 16, 18].

**Figure 8.** Thyroid follicles. HE. Bar = 25 µm.

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reabsortion from renal tubules [10] (table 3).

**Figure 9.** Thyroid follicles. Central colloidal eosinophilic material. HE. Bar = 25 µm.

lation of TRH released by hypothalamus [10].

The internal parathyroids are unencapsulated glands. They may be absent in some species or may be found included within the thyroid or close to them. In the dog, internal parathyroid glands are within the thyroid capsule, in the caudal and medial aspects of the thyroid [19, 20].

The parenchyma of parathyroid glands is composed of secretory cells, named chief or principal cells, arranged in cords, clusters, chains or rosettes (Figure 11, Figure 12). In some species, such as cattle and humans, it is also composed by oxyphilic cells, organized in small clusters which have yet no known functions [4]. In dogs, this type of cells were only found in senile animals [21] and were also described in canine parathyroid adenoma [22].

**Table 4.** Hormone production by parathyroid glands.

The pancreas is located in the abdominal cavity. In the dog, it is a V-shaped gland with a body and two lobes. It is acutely flexed with the apex nestling close to the cranial flexure of the duodenum. The slender right lobe runs within the mesoduodenum and the thicker but shorter left lobe extends over the caudal surface of the stomach towards the spleen, within the greater

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The pancreas is one of the mixed glands of the body, since it produces both exocrine (pancreatic juice, important for digestion) and endocrine secretion. The later is produced in the islets of Langerhans, which are randomly scattered in the exocrine parenchyma (Figure 14) [10, 17, 24]. Pancreatic islets are aggregates of endocrine cells required for blood glucose control and diabetes prevention after birth, consisting of polygonal shaped cells, with pale eosinophilic cytoplasm and coarse heterochromatin. Cells are arranged in cords or clusters separated by

**•** α cells, located peripherally and representing 10-20% of islet cells, secrete glucagon, a hyperglycaemic hormone, as well as cholecystokinin, gastric inhibitory protein and ACTH-

sinusoids and are subdivided in the following subtypes (Figure 15) [4, 24, 25]:

**5. Pancreas**

omentum (Figure 13) [4, 17].

**Figure 13.** Localization of the pancreas.

endorphin [4, 10, 24, 26].

**Figure 11.** Parathyroid cellular organization in cords, clusters and rosetes. HE. Bar = 50 µm.

**Figure 12.** Parathyroid chief cells. HE. Bar = 25 µm.

Chief cells synthesize and secrete PTH. This hormone, together with calcitonin and vitamin D, is involved in the regulation of calcium homeostasis. It is released in response to low blood calcium level [23] due to it's ability to enhance the mobilization of calcium ions from the small intestine and from bone resorption, and also by increasing the simultaneous absorption of calcium and excretion of phosphate from distal convoluted tubules of kidneys [10] (Table 4).

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**Table 4.** Hormone production by parathyroid glands.

### **5. Pancreas**

have yet no known functions [4]. In dogs, this type of cells were only found in senile animals

[21] and were also described in canine parathyroid adenoma [22].

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**Figure 11.** Parathyroid cellular organization in cords, clusters and rosetes. HE. Bar = 50 µm.

Chief cells synthesize and secrete PTH. This hormone, together with calcitonin and vitamin D, is involved in the regulation of calcium homeostasis. It is released in response to low blood calcium level [23] due to it's ability to enhance the mobilization of calcium ions from the small intestine and from bone resorption, and also by increasing the simultaneous absorption of calcium and excretion of phosphate from distal convoluted tubules of kidneys [10] (Table 4).

**Figure 12.** Parathyroid chief cells. HE. Bar = 25 µm.

The pancreas is located in the abdominal cavity. In the dog, it is a V-shaped gland with a body and two lobes. It is acutely flexed with the apex nestling close to the cranial flexure of the duodenum. The slender right lobe runs within the mesoduodenum and the thicker but shorter left lobe extends over the caudal surface of the stomach towards the spleen, within the greater omentum (Figure 13) [4, 17].

**Figure 13.** Localization of the pancreas.

The pancreas is one of the mixed glands of the body, since it produces both exocrine (pancreatic juice, important for digestion) and endocrine secretion. The later is produced in the islets of Langerhans, which are randomly scattered in the exocrine parenchyma (Figure 14) [10, 17, 24]. Pancreatic islets are aggregates of endocrine cells required for blood glucose control and diabetes prevention after birth, consisting of polygonal shaped cells, with pale eosinophilic cytoplasm and coarse heterochromatin. Cells are arranged in cords or clusters separated by sinusoids and are subdivided in the following subtypes (Figure 15) [4, 24, 25]:

**•** α cells, located peripherally and representing 10-20% of islet cells, secrete glucagon, a hyperglycaemic hormone, as well as cholecystokinin, gastric inhibitory protein and ACTHendorphin [4, 10, 24, 26].


Besides the above-mentioned cell types, the endocrine pancreas also contain some minor types of cells, that represent around 5% of the total pancreatic cells. These are divided as follows:


So, the actions of the two hormones combined contribute to the control of blood glucose.

cyclase and cAMP production [26].

**Figure 15.** Pancreatic endocrine cells. HE. Bar = 25 µm.

**Table 5.** Hormones produced by the pancreas.

Glucagon secretion by α-cells is highly regulated by multiple factors, being the most important the glucose and insulin levels. Low glucose levels activate specific channels in the brain and in pancreatic α-cells to generate action potentials of sodium and calcium currents, leading to glucagon secretion. Also, somatostatin inhibits glucagon secretion by inhibiting adenylate

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Insulin has several physiologic actions that include stimulation of cellular glucose and po‐ tassium uptake [29]. Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation, amongst other factors, like ATP, glutamate and others [30]. Incre‐ tin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon like pep‐

**Figure 14.** Islets of Langerhans. HE. Bar = 50 µm.

Insulin and glucagon are pancreatic hormones that play pivotal roles in regulating glucose homeostasis and metabolism, which have opposite effects on glycaemia as well as on the metabolism of nutrients [24, 26, 28]. In general, it can be said that:

Insulin – promotes glucose uptake by peripheral tissues, (muscle, liver and adipose tissue) and its accumulation as glycogen and fat, resulting in an anabolic effect [28] and in the removal of glucose from the blood, reducing glycaemia – hypoglycaemic effect [24];

Glucagon – promotes glucose production (catabolic effect) activating liver glycogenolysis and gluconeogenesis, releasing glucose in blood circulation, increasing glucose levels in the blood, promoting glycaemia – hyperglycaemic effect [24, 28].

**Figure 15.** Pancreatic endocrine cells. HE. Bar = 25 µm.

**•** β cells, located in the centre of the islets, are more abundant, representing 60-80% of the

**•** δ cells, responsible for the production of somatostatin, a hormone responsible for preventing drastic variations in both insulin and glucagon [10, 24, 27]. This hormone is also capable of reducing intestinal motility and secretion of digestive juices. These cells represent about

Besides the above-mentioned cell types, the endocrine pancreas also contain some minor types of cells, that represent around 5% of the total pancreatic cells. These are divided as follows: **•** F cells, less abundant (2-3%), secrete pancreatic polypeptide (PP), which affects the produc‐ tion of some digestive enzymes and inhibits biliary and pancreatic secretion, as well as,

**•** D1 cells, that secrete the vasoactive intestinal peptide (VIP) [4] with important functions in the regulation of intestinal motility and intestinal transport of ions and water [10].

**•** EC or enterochromaffin cells, wich secrete secretin, wich stimulates bicarbonate and pancreatic enzyme secretion, and motilin that increases gastric and intestinal motility.

Insulin and glucagon are pancreatic hormones that play pivotal roles in regulating glucose homeostasis and metabolism, which have opposite effects on glycaemia as well as on the

Insulin – promotes glucose uptake by peripheral tissues, (muscle, liver and adipose tissue) and its accumulation as glycogen and fat, resulting in an anabolic effect [28] and in the removal of

Glucagon – promotes glucose production (catabolic effect) activating liver glycogenolysis and gluconeogenesis, releasing glucose in blood circulation, increasing glucose levels in the blood,

metabolism of nutrients [24, 26, 28]. In general, it can be said that:

promoting glycaemia – hyperglycaemic effect [24, 28].

glucose from the blood, reducing glycaemia – hypoglycaemic effect [24];

islets of Langerhans, and secrete insulin, a hypoglycaemic hormone [4, 10, 24].

5-40% of the islets [10, 24].

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intestinal motility [10, 24].

**Figure 14.** Islets of Langerhans. HE. Bar = 50 µm.

So, the actions of the two hormones combined contribute to the control of blood glucose.

Glucagon secretion by α-cells is highly regulated by multiple factors, being the most important the glucose and insulin levels. Low glucose levels activate specific channels in the brain and in pancreatic α-cells to generate action potentials of sodium and calcium currents, leading to glucagon secretion. Also, somatostatin inhibits glucagon secretion by inhibiting adenylate cyclase and cAMP production [26].

**Table 5.** Hormones produced by the pancreas.

Insulin has several physiologic actions that include stimulation of cellular glucose and po‐ tassium uptake [29]. Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation, amongst other factors, like ATP, glutamate and others [30]. Incre‐ tin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon like pep‐ tide-1 (GLP-1), secreted by cells of the gastrointestinal tract in response to meal ingestion, exert important glucorregulatory effects, including the glucose-dependent potentiation of insulin secretion by pancreatic β-cells [31].

Table 5 summarizes hormones of the pancreas and their functions.

## **6. Adrenal glands**

The adrenal glands are paired organs located against the roof of the abdomen near the thoraco‐ lumbar junction, in a position immediately prior to the kidneys and close to their cranial poles (Figure 16). Generally elongated, these glands are often asymmetrical and quite irregular [17].

**Figure 17.** Adrenal cortex. *Zona arcuata*. HE. Bar = 50 µm.

zone produces glucocorticoids [32, 33, 34, 37].

**Figure 18.** Adrenal cortex. *Zona Fasciculata*. HE. Bar = 25 µm.

produced.

The *zona fasciculata* is the middle and thickest zone (corresponding to more than 70% of the cortex) and is composed of parallel columns of secretory cells, one to two cells thick, separated by prominent capillaries. The cells are cuboidal or polyhedral, containing vesicular nucleus, frequently binucleated) and foamy cytoplasm (intracellular lipid droplets) (Figure 18). This

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Glucocorticoids have several functions, including protein catabolism and stimulation of the hepatic gluconeogenesis from amino acids [10, 37]. In dogs, cortisol is the main glucocorticoid

The *zona reticularis* is also composed of polyhedral cells, whose arrangement consists in freely anastomosing cords (Figure 19) [32]. These cells contain less lipid content but have densely granular cytoplasm for which they are called "compact cells" [34]. *The zona reticularis* works

**Figure 16.** Localization of adrenal glands.

Each of these glands is divided into cortex and medulla. The cortex is yellowish and radially striated, while the medulla is more uniform and darker [17].

Both regions, cortex and medulla, correspond to areas specialized in producing different hormones [4, 32, 34]:

**•** The cortex consists of polyhedral secretory cells, arranged into two layered thick cords, which originate radially from the medullar zone [4, 33]. It is composed of 3 distinct zones. The outer zone is the *zona glomerulosa*, a thin region that in dogs and cats is composed by cells arranged in an arched or arcuate pattern, therefore also named as *zona arcuata* (Figure 17) [32, 35, 36].

The *zona glomerulosa* produces mineralocorticoids, particularly the steroid hormone aldoster‐ one [32, 37], which is responsible for increasing sodium retention and water reabsortion in the distal tubules of the kidneys and sodium reabsorption in intestine. Aldosterone also maintains blood concentration and stimulates potassium excretion by the kidneys, thereby, indirectly regulatingextracellularfluidvolume.Adecreaseofmineralocorticoids,bylossofthiszoneorits functional ability, gives rise to water outlet of the blood to the tissues and may result in death, due to retention of high levels of potassium with excess loss of sodium, chloride and water [32].

**Figure 17.** Adrenal cortex. *Zona arcuata*. HE. Bar = 50 µm.

tide-1 (GLP-1), secreted by cells of the gastrointestinal tract in response to meal ingestion, exert important glucorregulatory effects, including the glucose-dependent potentiation of

The adrenal glands are paired organs located against the roof of the abdomen near the thoraco‐ lumbar junction, in a position immediately prior to the kidneys and close to their cranial poles (Figure 16). Generally elongated, these glands are often asymmetrical and quite irregular [17].

Each of these glands is divided into cortex and medulla. The cortex is yellowish and radially

Both regions, cortex and medulla, correspond to areas specialized in producing different

**•** The cortex consists of polyhedral secretory cells, arranged into two layered thick cords, which originate radially from the medullar zone [4, 33]. It is composed of 3 distinct zones. The outer zone is the *zona glomerulosa*, a thin region that in dogs and cats is composed by cells arranged in an arched or arcuate pattern, therefore also named as *zona arcuata* (Figure

The *zona glomerulosa* produces mineralocorticoids, particularly the steroid hormone aldoster‐ one [32, 37], which is responsible for increasing sodium retention and water reabsortion in the distal tubules of the kidneys and sodium reabsorption in intestine. Aldosterone also maintains blood concentration and stimulates potassium excretion by the kidneys, thereby, indirectly regulatingextracellularfluidvolume.Adecreaseofmineralocorticoids,bylossofthiszoneorits functional ability, gives rise to water outlet of the blood to the tissues and may result in death, due to retention of high levels of potassium with excess loss of sodium, chloride and water [32].

insulin secretion by pancreatic β-cells [31].

**6. Adrenal glands**

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**Figure 16.** Localization of adrenal glands.

hormones [4, 32, 34]:

17) [32, 35, 36].

striated, while the medulla is more uniform and darker [17].

Table 5 summarizes hormones of the pancreas and their functions.

The *zona fasciculata* is the middle and thickest zone (corresponding to more than 70% of the cortex) and is composed of parallel columns of secretory cells, one to two cells thick, separated by prominent capillaries. The cells are cuboidal or polyhedral, containing vesicular nucleus, frequently binucleated) and foamy cytoplasm (intracellular lipid droplets) (Figure 18). This zone produces glucocorticoids [32, 33, 34, 37].

Glucocorticoids have several functions, including protein catabolism and stimulation of the hepatic gluconeogenesis from amino acids [10, 37]. In dogs, cortisol is the main glucocorticoid produced.

**Figure 18.** Adrenal cortex. *Zona Fasciculata*. HE. Bar = 25 µm.

The *zona reticularis* is also composed of polyhedral cells, whose arrangement consists in freely anastomosing cords (Figure 19) [32]. These cells contain less lipid content but have densely granular cytoplasm for which they are called "compact cells" [34]. *The zona reticularis* works as a unit together with the *zona fasciculata*, producing androgens (androstenedione) and also glucocorticoids [34, 37].

The adrenal medulla of dogs has a heterogeneous population of cells [38]. In adults, there are 3 types of adrenal medullary cells (1): epinephrine cells (66–75%), norepinephrine cells (25– 33%) and small granule-containing cells (SGC, 1-4%). The adrenal SGC cells of dogs vary from cells with a few granules and a high nucleo-cytoplasmic ratio to cells filled with many granules and a large mass of cytoplasm. Most of the chromaffin granules of these cells are small, ranging from 100 to 200 nm in diameter [39]. The medullary cells produce other peptides in addition to epinephrine and norepinephrine, such as met-enkephalin, substance P, neurotensin, neuropeptide Y, and chromogranin A. The adrenal medulla contains also presynaptic sym‐

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The first step in the synthesis of epinephrine is the enzymatic conversion of tyrosine to dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase. This is the rate-limiting step of hormone synthesis. DOPA is then converted to dopamine within the cytosol by decarboxyla‐ tion. Dopamine can also be converted to norepinephrine by hydroxylation. Norepinephrine then leaves the granule to be converted into epinephrine in the cytosol by phenylethanolamine-N-methyltransferase (PNMT), and epinephrine re-enters the granule for storage in the cell. The activity of PNMT is induced by the high local concentration of glucocorticoids in sinus‐ oidal blood from the adrenal cortex [10, 32]. Acute stress, hypoglycemia or other similar situations, result both in catecholamine secretion and in transsynaptic induction of catechol‐ amine biosynthetic enzymes, including tyrosine hydroxylase. Other environmental influences, including growth factors, extracellular matrix, and a variety of hormonal signals that generate

In table 6, we can see a summary of some of the hormones produced by adrenal glands and

pathetic ganglion cells, randomly scattered [4, 32].

their functions.

**Table 6.** Adrenal glands hormones and functions.

cyclic AMP, also may regulate the function of chromaffin cells [32].

**Figure 19.** Adrenal cortex. *Zona Reticularis*. HE. Bar = 25 µm.

The adrenal cortex is stimulated by ACTH, produced in the adenohypophysis. In response, it produces glucocorticoids (e.g. cortisol), mineralocorticoids (e.g. aldosterone) and minor amounts of sex steroids (androgens and estrogens).

**•** The medulla consists of large columnar or polyhedral secretory cells, randomly distributed, with rich blood supply (Figure 20). The cells have large, vesicular nucleus, basophilic cytoplasm with fine positive chromaffin granules, due to the presence of catecholamines such as epinephrine and norepinephrine, which after exposure to oxidizing agents, such as chromate, yield a brown reaction by the formation of colored polymers [4, 31, 33].

**Figure 20.** Adrenal medulla. HE. Bar = 50 µm.

The adrenal medulla of dogs has a heterogeneous population of cells [38]. In adults, there are 3 types of adrenal medullary cells (1): epinephrine cells (66–75%), norepinephrine cells (25– 33%) and small granule-containing cells (SGC, 1-4%). The adrenal SGC cells of dogs vary from cells with a few granules and a high nucleo-cytoplasmic ratio to cells filled with many granules and a large mass of cytoplasm. Most of the chromaffin granules of these cells are small, ranging from 100 to 200 nm in diameter [39]. The medullary cells produce other peptides in addition to epinephrine and norepinephrine, such as met-enkephalin, substance P, neurotensin, neuropeptide Y, and chromogranin A. The adrenal medulla contains also presynaptic sym‐ pathetic ganglion cells, randomly scattered [4, 32].

The first step in the synthesis of epinephrine is the enzymatic conversion of tyrosine to dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase. This is the rate-limiting step of hormone synthesis. DOPA is then converted to dopamine within the cytosol by decarboxyla‐ tion. Dopamine can also be converted to norepinephrine by hydroxylation. Norepinephrine then leaves the granule to be converted into epinephrine in the cytosol by phenylethanolamine-N-methyltransferase (PNMT), and epinephrine re-enters the granule for storage in the cell. The activity of PNMT is induced by the high local concentration of glucocorticoids in sinus‐ oidal blood from the adrenal cortex [10, 32]. Acute stress, hypoglycemia or other similar situations, result both in catecholamine secretion and in transsynaptic induction of catechol‐ amine biosynthetic enzymes, including tyrosine hydroxylase. Other environmental influences, including growth factors, extracellular matrix, and a variety of hormonal signals that generate cyclic AMP, also may regulate the function of chromaffin cells [32].

In table 6, we can see a summary of some of the hormones produced by adrenal glands and their functions.

**Table 6.** Adrenal glands hormones and functions.

as a unit together with the *zona fasciculata*, producing androgens (androstenedione) and also

The adrenal cortex is stimulated by ACTH, produced in the adenohypophysis. In response, it produces glucocorticoids (e.g. cortisol), mineralocorticoids (e.g. aldosterone) and minor

**•** The medulla consists of large columnar or polyhedral secretory cells, randomly distributed, with rich blood supply (Figure 20). The cells have large, vesicular nucleus, basophilic cytoplasm with fine positive chromaffin granules, due to the presence of catecholamines such as epinephrine and norepinephrine, which after exposure to oxidizing agents, such as

chromate, yield a brown reaction by the formation of colored polymers [4, 31, 33].

glucocorticoids [34, 37].

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**Figure 19.** Adrenal cortex. *Zona Reticularis*. HE. Bar = 25 µm.

amounts of sex steroids (androgens and estrogens).

**Figure 20.** Adrenal medulla. HE. Bar = 50 µm.

## **7. Clinical significance**

Endocrine diseases, associated to altered functions of endocrine glands, are frequently seen in veterinary practice. The endocrine glands described may be targeted by several conditions summarized in table 7.

**•** Hypothyroidism

**•** Diabetes mellitus

be present [45].

**•** Hyperadrenocorticism

Hypothyroidism may result from destruction of the thyroid gland (primary hypothyroidism), decreased hypothalamic secretion of TRH or inadequate pituitary TSH secretion. Over 95% of canine hypothyroidism cases are primary [40, 41]. Two histologic forms of primary hypothyr‐ oidism predominate in the dog: lymphocytic thyroiditis and idiopathic atrophy. Regardless of the aetiology, the end result is the same: progressive destruction of the thyroid gland and

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Hypothyroidism occurs mainly in middle aged, purebred dogs. Doberman Pinschers and Golden Retrievers are the two breeds most frequently described [42]. Clinical signs associated with hypothyroidism are varied, and behavioural changes are associated with a reduced metabolic rate, dermatological signs, cardiovascular and neuromuscular abnormalities. Obesity, in different grades is always present. Lethargy and cold intolerance are also frequently observed. Seborrhoea, bilateral and symmetrical alopecia, and pyoderma are also common dermatological symptoms. These signs can be accompanied by bradycardia, low voltage ECG

Diabetes mellitus is the result of any situation that affects insulin production, insulin transport or the sensitivity of target tissues to insulin [45]. Many factors are known to contribute to the development of diabetes and its complications [46, 47]. These include genetics, diet, sedentary

Canine diabetes mellitus is generally classified as insulin dependent or non-insulin dependent based on the need for insulin treatment [40, 48]. Most diabetic dogs are thought to have a disease like human type I diabetes mellitus and are insulin dependent [40, 49, 50]. Unlike human type I diabetes mellitus, which occurs mainly in young human patients, in dogs it is more likely to occur later in life [40, 45, 48]. Dogs do not appear to progress from obesityinduced insulin resistance to type 2 diabetes mellitus, probably because pancreatic beta cells in dogs are either not sensitive to toxicity due mild hyperglycemia or lack other components of the pathophysiology of beta cell failure in type 2 diabetes mellitus [51]. Certain breeds, including Australian Terrier, Keeshounds, Alaskan Malamutes, Finnish Spitzes, Standard and Miniature Schnauzers, Miniature Poodles, and English Springer Spaniels, seem to be at increased risk to develop diabetes. Others, such as Boxers, German Shepherd dogs, Cocker Spaniels and Collies seemed to be at decreased risk [40, 52]. Typical clinical signs are polyuria, polydipsia, polyphagia and weight loss. Exercise intolerance or decreased activity, ketonic breath, recurrent infections (urinary tract, conjunctivitis), cataracts and hepatomegaly can also

Hyperadrenocorticism can be spontaneous or iatrogenic. Spontaneous hyperadrenocorticism is associated with inappropriate secretion of ACTH by the pituitary (pituitary-dependent hyperadrenocorticism) or with a primary adrenal disorder (adrenal-dependent hyperadreno‐ corticism) [40, 53, 54]. Over 80% of dogs with spontaneous hyperdarenocorticism suffer from

lifestyle, perinatal factors, age, obesity and inflammatory causes [47, 48].

consequent deficiency of circulating thyroid hormones [40, 41].

complexes and weakness [40, 41, 43, 44].


**Table 7.** Pathologic conditions affecting endocrine glands.

In dogs, the most common endocrine diseases are summarized in table 8.

**Table 8.** Most common endocrine diseases in dogs.

### **•** Hypothyroidism

**7. Clinical significance**

**Table 7.** Pathologic conditions affecting endocrine glands.

**Table 8.** Most common endocrine diseases in dogs.

In dogs, the most common endocrine diseases are summarized in table 8.

summarized in table 7.

182 Insights from Veterinary Medicine

Endocrine diseases, associated to altered functions of endocrine glands, are frequently seen in veterinary practice. The endocrine glands described may be targeted by several conditions

Hypothyroidism may result from destruction of the thyroid gland (primary hypothyroidism), decreased hypothalamic secretion of TRH or inadequate pituitary TSH secretion. Over 95% of canine hypothyroidism cases are primary [40, 41]. Two histologic forms of primary hypothyr‐ oidism predominate in the dog: lymphocytic thyroiditis and idiopathic atrophy. Regardless of the aetiology, the end result is the same: progressive destruction of the thyroid gland and consequent deficiency of circulating thyroid hormones [40, 41].

Hypothyroidism occurs mainly in middle aged, purebred dogs. Doberman Pinschers and Golden Retrievers are the two breeds most frequently described [42]. Clinical signs associated with hypothyroidism are varied, and behavioural changes are associated with a reduced metabolic rate, dermatological signs, cardiovascular and neuromuscular abnormalities. Obesity, in different grades is always present. Lethargy and cold intolerance are also frequently observed. Seborrhoea, bilateral and symmetrical alopecia, and pyoderma are also common dermatological symptoms. These signs can be accompanied by bradycardia, low voltage ECG complexes and weakness [40, 41, 43, 44].

**•** Diabetes mellitus

Diabetes mellitus is the result of any situation that affects insulin production, insulin transport or the sensitivity of target tissues to insulin [45]. Many factors are known to contribute to the development of diabetes and its complications [46, 47]. These include genetics, diet, sedentary lifestyle, perinatal factors, age, obesity and inflammatory causes [47, 48].

Canine diabetes mellitus is generally classified as insulin dependent or non-insulin dependent based on the need for insulin treatment [40, 48]. Most diabetic dogs are thought to have a disease like human type I diabetes mellitus and are insulin dependent [40, 49, 50]. Unlike human type I diabetes mellitus, which occurs mainly in young human patients, in dogs it is more likely to occur later in life [40, 45, 48]. Dogs do not appear to progress from obesityinduced insulin resistance to type 2 diabetes mellitus, probably because pancreatic beta cells in dogs are either not sensitive to toxicity due mild hyperglycemia or lack other components of the pathophysiology of beta cell failure in type 2 diabetes mellitus [51]. Certain breeds, including Australian Terrier, Keeshounds, Alaskan Malamutes, Finnish Spitzes, Standard and Miniature Schnauzers, Miniature Poodles, and English Springer Spaniels, seem to be at increased risk to develop diabetes. Others, such as Boxers, German Shepherd dogs, Cocker Spaniels and Collies seemed to be at decreased risk [40, 52]. Typical clinical signs are polyuria, polydipsia, polyphagia and weight loss. Exercise intolerance or decreased activity, ketonic breath, recurrent infections (urinary tract, conjunctivitis), cataracts and hepatomegaly can also be present [45].

**•** Hyperadrenocorticism

Hyperadrenocorticism can be spontaneous or iatrogenic. Spontaneous hyperadrenocorticism is associated with inappropriate secretion of ACTH by the pituitary (pituitary-dependent hyperadrenocorticism) or with a primary adrenal disorder (adrenal-dependent hyperadreno‐ corticism) [40, 53, 54]. Over 80% of dogs with spontaneous hyperdarenocorticism suffer from pituitary dependent hypercorticism, resulting in an over secretion of ACTH [53, 54]. This results in bilateral adrenocortical hyperplasia and excess cortisol production. The negative feedback loop between cortisol and ACTH is lost. Adrenal-dependent hyperadrenocorticism represent the remaining 20% of spontaneous hyperadrenocorticism and is generally associated with unilateral (or occasionally bilateral) adrenal tumours. This is a typical disease of middle age to old animals. Dogs showing pituitary-dependent hyperadrenocorticism exhibit a mean age of 7-9 years old, and those with adrenal-dependent hyperadrenocorticism, a mean age of 11-12 years old. There are some breeds that are most frequently associated with hyperadre‐ nocorticism, but any breed can develop it. Poodles, Daschunds and small Terriers seem to be more predisposed to this disease. Most common clinical signs include polydipsia and polyuria, polyphagia, abdominal distension, liver enlargement, muscle weakness, lethargy, skin changes, alopecia, persistent anoestrus or testicular atrophy, *calcinosis cutis* and neurological signs [54, 55].

professional with technical and scientific knowledge, enabled with the exact notion of the involved etiopathogenic mechanisms and capable of making a diagnostic and therapeutical difference. This concerted attitude would certainly contribute to improve veterinary medical

This work was supported by Portuguese Foundation for Science and Technology and Center

, Fernando Esteves2

1 Center for Studies in Education, and Health Technologies. CI&DETS. Agrarian School of

[1] Done S., Goody P., Evans S., Stickland N., editors. Color Atlas of Veterinary Anato‐

[2] Ningying X., Xiaoling J. Molecular Characterization of Hypothalamo-pituitary-thy‐ roid genes in pig (Sus scrofa). In Perez-Marin C. (ed.) A bird's-eye view of Veterinary Medicine. Rijeka: InTech; 2012. p545-56 http://www.intechopen.com/books/a-bird-seye-view-of-veterinary-medicine/molecular-characterization-of-thyroid-peroxidase-

[3] Morgan P., Williams L. The pars tuberalis of the pituitary: a gateway for neuroendo‐

[4] Banks W.J., editor. Applied Veterinary Histology. St. Louis: Mosby; 1993. p 408-28.

[5] Pankov I., Chekhranova M., Karpova S., Iatsyshina S.M., Sazina E. Molecular and ge‐ netic study of the role of hormones receptors, and enzymes in regulation of repro‐ duction, lipid metabolism, and other human physiological functions. Vestnik

2 Agrarian Superior School of Viseu, Polytechnic Institute of Viseu. Viseu, Portugal

my. The dog & Cat. Edinburgh: Mosby; 2004. p2.62, 2.66, 2.67, 2.79.

gene-in-porcine-sus-scrofa- (accessed the 8 June 2012).

crine output. Reviews of Reproduction 1996;(1) 153–61.

Rossiiskoi Akademii Meditsinskikh Nauk 2005;(9) 6-13.

, Carla Santos2

The Endocrine Glands in the Dog: From the Cell to Hormone

http://dx.doi.org/10.5772/53577

185

, Rita Cruz2

,

care to a level of excellence in the care of animal patients.

for Studies in Education, Technologies and Health.

and Carmen Nóbrega2

Viseu, Polytechnic Institute of Viseu, Viseu, Portugal

Helena Vala1,2, João Rodrigo Mesquita2

**Acknowledgements**

**Author details**

Cristina Mega2

**References**

#### **•** Hypoadrenocorticism

Hypoadrenocorticism is a syndrome associated to a deficient production of mineralocorticoid and/or glucocorticoid secretion by the adrenal glands. This can arise from a destruction of more than 90% of both adrenal cortices, causing inability to produce corticosteroids (Primary hypoadrenocorticism or Addison's disease), or from a deficiency in ACTH production by the pituitary (Secondary hypoadrenocorticism) [56, 57]. Besides different pathophysiology, primary and secondary hypoadrenocorticism also exhibit different clinical signs. In dogs, primary hypoadrenocorticism is more representative. It is often associated with idiopathic adrenocortical atrophy, or with some therapeutic or surgical procedures, like mitotane treatment for hyperadrenocorticism or bilateral adrenalectomy. This condition generally affects young and middle age dogs with a median age of 4 years [57]. Great Danes, Portuguese Water Dogs, Rottweilers, Standard Poodles, West Highland White Terrier and Soft-coated Wheaten Terriers are the breeds in greater risk to develop hypoadrenocorticism [56]. Females, especially intact females, are in higher risk. Clinical signs may vary, from mild to severe, regarding the evolution of the disease. Acute hypoadrenocorticism can be life threatening and represents a medical emergency. The animal can be in a hypovolaemic shock, and generally is found in a state of collapse, or collapses when stressed. Weak pulse, bradycardia, abdominal pain, vomiting, diarrhoea, dehydration and hypothermia can also be present. Chronic presentation is more common than the acute disease. Animals with chronic hypoadrecocorti‐ cism may present anorexia, vomiting, lethargy, depression and weakness [57].

### **8. Conclusions**

Malfunction of endocrine glands leads to severe multivariate syndromes, requiring a special‐ ized medical approach and appropriate nursing care. For this reason, professionals engaged in clinical veterinary practice need to know animal organic structures and how they function as a whole, including the role of the endocrine system, its glands and their hormones. This systematic approach to endocrine disorders promotes not only a trained professional but a professional with technical and scientific knowledge, enabled with the exact notion of the involved etiopathogenic mechanisms and capable of making a diagnostic and therapeutical difference. This concerted attitude would certainly contribute to improve veterinary medical care to a level of excellence in the care of animal patients.

## **Acknowledgements**

pituitary dependent hypercorticism, resulting in an over secretion of ACTH [53, 54]. This results in bilateral adrenocortical hyperplasia and excess cortisol production. The negative feedback loop between cortisol and ACTH is lost. Adrenal-dependent hyperadrenocorticism represent the remaining 20% of spontaneous hyperadrenocorticism and is generally associated with unilateral (or occasionally bilateral) adrenal tumours. This is a typical disease of middle age to old animals. Dogs showing pituitary-dependent hyperadrenocorticism exhibit a mean age of 7-9 years old, and those with adrenal-dependent hyperadrenocorticism, a mean age of 11-12 years old. There are some breeds that are most frequently associated with hyperadre‐ nocorticism, but any breed can develop it. Poodles, Daschunds and small Terriers seem to be more predisposed to this disease. Most common clinical signs include polydipsia and polyuria, polyphagia, abdominal distension, liver enlargement, muscle weakness, lethargy, skin changes, alopecia, persistent anoestrus or testicular atrophy, *calcinosis cutis* and neurological

Hypoadrenocorticism is a syndrome associated to a deficient production of mineralocorticoid and/or glucocorticoid secretion by the adrenal glands. This can arise from a destruction of more than 90% of both adrenal cortices, causing inability to produce corticosteroids (Primary hypoadrenocorticism or Addison's disease), or from a deficiency in ACTH production by the pituitary (Secondary hypoadrenocorticism) [56, 57]. Besides different pathophysiology, primary and secondary hypoadrenocorticism also exhibit different clinical signs. In dogs, primary hypoadrenocorticism is more representative. It is often associated with idiopathic adrenocortical atrophy, or with some therapeutic or surgical procedures, like mitotane treatment for hyperadrenocorticism or bilateral adrenalectomy. This condition generally affects young and middle age dogs with a median age of 4 years [57]. Great Danes, Portuguese Water Dogs, Rottweilers, Standard Poodles, West Highland White Terrier and Soft-coated Wheaten Terriers are the breeds in greater risk to develop hypoadrenocorticism [56]. Females, especially intact females, are in higher risk. Clinical signs may vary, from mild to severe, regarding the evolution of the disease. Acute hypoadrenocorticism can be life threatening and represents a medical emergency. The animal can be in a hypovolaemic shock, and generally is found in a state of collapse, or collapses when stressed. Weak pulse, bradycardia, abdominal pain, vomiting, diarrhoea, dehydration and hypothermia can also be present. Chronic presentation is more common than the acute disease. Animals with chronic hypoadrecocorti‐

cism may present anorexia, vomiting, lethargy, depression and weakness [57].

Malfunction of endocrine glands leads to severe multivariate syndromes, requiring a special‐ ized medical approach and appropriate nursing care. For this reason, professionals engaged in clinical veterinary practice need to know animal organic structures and how they function as a whole, including the role of the endocrine system, its glands and their hormones. This systematic approach to endocrine disorders promotes not only a trained professional but a

signs [54, 55].

**•** Hypoadrenocorticism

184 Insights from Veterinary Medicine

**8. Conclusions**

This work was supported by Portuguese Foundation for Science and Technology and Center for Studies in Education, Technologies and Health.

## **Author details**

Helena Vala1,2, João Rodrigo Mesquita2 , Fernando Esteves2 , Carla Santos2 , Rita Cruz2 , Cristina Mega2 and Carmen Nóbrega2

1 Center for Studies in Education, and Health Technologies. CI&DETS. Agrarian School of Viseu, Polytechnic Institute of Viseu, Viseu, Portugal

2 Agrarian Superior School of Viseu, Polytechnic Institute of Viseu. Viseu, Portugal

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315-26.


**Chapter 7**

**Sex Steroid Hormones and**

Raquel Sánchez-Céspedes and

Juana Martín de las Mulas

http://dx.doi.org/10.5772/54324

**1. Introduction**

**Tumors in Domestic Animals**

Yolanda Millán, Silvia Guil-Luna, Carlos Reymundo,

Sex steroid hormones play a role in the development and control of animal tumours, partic‐ ularly in those arising in their target organs. Due to their incidence and prevalence, mamma‐ ry tumours of female dogs and cats are among the most frequently studied with focus on the role of ovarian oestrogen and progesterone. In these tumours, sex steroid hormones have been shown to act during the three steps of the carcinogenesis cascade: initiation, pro‐ motion and progression. Experimental data have shown the mutagenic effect of oestrogens [1] while epidemiologic and clinical studies highlighted the role of ovarian hormones as pro‐ moters on mammary tumours in both the dog and the cat [2-9]. Finally, oestrogens and pro‐ gesterone further act during tumour progression. Their role in the last two steps of

Studies on the role of ovarian hormones during tumour progression depend on the capabili‐ ty of demonstrating the presence of oestrogen receptors (ER) and progesterone receptors (PR) in tumours. Earlier studies on the field were published in the seventies of the 20th cen‐ tury, all related to mammary gland tumours. Some of these studies revealed the presence of unoccupied hormone receptors in tissue homogenates from mammary tumours of dogs and cats [10-13]. However, actual knowledge comes from the late nineties, when different groups of researchers standardized immunohistochemical (IHC) methods of analysis for ER and PR in feline and canine tissues [14-19]. Once the IHC analysis revealed the presence of sex steroid hormones receptors in mammary tumours, several studied analyzed their value as favourable prognostic indicators [18, 20-25] adding new data to the well-known similari‐

> © 2013 Millán et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Millán et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

carcinogenesis makes it possible to control the evolution of the disease.

Additional information is available at the end of the chapter

## **Chapter 7**

## **Sex Steroid Hormones and Tumors in Domestic Animals**

Yolanda Millán, Silvia Guil-Luna, Carlos Reymundo, Raquel Sánchez-Céspedes and Juana Martín de las Mulas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54324

## **1. Introduction**

Sex steroid hormones play a role in the development and control of animal tumours, partic‐ ularly in those arising in their target organs. Due to their incidence and prevalence, mamma‐ ry tumours of female dogs and cats are among the most frequently studied with focus on the role of ovarian oestrogen and progesterone. In these tumours, sex steroid hormones have been shown to act during the three steps of the carcinogenesis cascade: initiation, pro‐ motion and progression. Experimental data have shown the mutagenic effect of oestrogens [1] while epidemiologic and clinical studies highlighted the role of ovarian hormones as pro‐ moters on mammary tumours in both the dog and the cat [2-9]. Finally, oestrogens and pro‐ gesterone further act during tumour progression. Their role in the last two steps of carcinogenesis makes it possible to control the evolution of the disease.

Studies on the role of ovarian hormones during tumour progression depend on the capabili‐ ty of demonstrating the presence of oestrogen receptors (ER) and progesterone receptors (PR) in tumours. Earlier studies on the field were published in the seventies of the 20th cen‐ tury, all related to mammary gland tumours. Some of these studies revealed the presence of unoccupied hormone receptors in tissue homogenates from mammary tumours of dogs and cats [10-13]. However, actual knowledge comes from the late nineties, when different groups of researchers standardized immunohistochemical (IHC) methods of analysis for ER and PR in feline and canine tissues [14-19]. Once the IHC analysis revealed the presence of sex steroid hormones receptors in mammary tumours, several studied analyzed their value as favourable prognostic indicators [18, 20-25] adding new data to the well-known similari‐

© 2013 Millán et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Millán et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ties between and human and animal mammary tumours [26]. However, the important role of ER and PR as predictive factors of response to endocrine treatment of breast cancer has been rarely analyzed in animal tumours although recent studies based on the blockade of PR in canine mammary carcinomas and reproductive tract tumours of female and male dogs and cats support their value in the control of these diseases [27-30].

**1.** They could be applied to tissue samples routinely processed for histopathology and could be performed in the pathology laboratory, rendering them both more accessible

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**2.** They provided information regarding the specific location of ER and PR, enabling anal‐ ysis of the relationship between receptor expression and tissue structures in normal and/or tumoural mammary glands, thus avoiding the false-positive findings common

**3.** They furnished information on all the receptors present in tissue samples, irrespective

The process of standardizing IHC methods of ER and PR detection in animal tissues in‐ volved the use of commercially available antibodies raised against human antigens first as antibodies raised against canine and feline antigens were not produced. Then, these IHC methods for detecting ER and PR in formalin-fixed, paraffin wax-embedded tissue samples needed to be validated before they could be routinely implemented, as stipulated by the "National Institutes of Health Consensus Conference on Estrogen Receptors in Breast Can‐ cer" (NIHCC), held in 1979. The first step in the validation procedure was the comparison of ER and PR detection using both biochemical and IHC techniques and the correlation of IHC results with those of the gold-standard radioligand-binding assays in order to evaluate their specificity and sensitivity. In 1999, Graham et al. [15] analysed ERα expression in formalinfixed, paraffin-embedded tissue samples from canine mammary tumours using biochemical and IHC methods, reporting good correlation between the two. In 2000 and 2002, Martín de las Mulas et al. [17, 19] validated the IHC method for the detection of ERα and PR detection, respectively, in formalin-fixed, paraffin-embedded tissue samples from feline mammary tu‐ mours using the avidin-biotin-peroxidase complex (ABC) method, and compared their find‐ ings with those obtained using the DCC biochemical method; reported agreement between the two methods was 72.7% for ERα and 95.6% for PR. The IHC technique used demonstrat‐

ed good specificity (true-negatives) and sensibility (true-positives) (Table 1).

**DCC versus IHC ERα PR SPECIFICITY** 95.6% 89.4% **SENSIBILITY** 47.6% 87.5%

**Table 1.** Specificity and sensibility of an IHC method for detecting ER and PR, versus a biochemical method (DCC) in

Second, the NIHCC (1979) also recommended the clinical validation of technically validated IHC methods since the presence of ER and/or PR in human breast cancer was known to be a favourable prognostic indicator as well as a predictive marker of the response to adjuvant hormone therapy [41, 42]. The prognostic value of the IHC expression of ERα and PR ex‐ pression in canine mammary carcinoma has been demonstrated in the dog and the cat [18,

and less costly.

in biochemical examinations.

of the occupation status.

feline mammary carcinomas [17, 19].

Mammary gland tumours are the most frequent tumours in female dogs and the third in the cat. However, they are not the only tumours known to be sex steroid-dependent. Tumours of the reproductive tract of female and male dogs and cats and some skin appendages can be defined as hormone-dependent on the basis of the IHC expression of ERα and PR [31, 32]. In addition, epidemiological and clinical studies support the role of steroid hormones as tumour promoters [28]. The most common tumours in the genital tract of the bitch are be‐ nign smooth muscle tumours of the vagina, vulva and perineal skin. Their hormone-de‐ pendence is similar to that of mammary gland tumours as the majority of canine genital tract leiomyomas express PR and also respond to neoadjuvant treatments with the PR antag‐ onist aglepristone with a reduction in size [28].

In the dog, and to a lesser extent in the cat, benign and malignant sebaceous gland neopla‐ sias are among the most common skin tumours. Studies concerning the role of sex steroid hormones during tumour promotion and progression are scarce but do show there is ER and PR expression in sebaceous gland tumours of dogs. Therefore, not only androgens but also progesterone and estrogens may regulate hormone-related physiology. In fact, preclini‐ cal studies indicate partial clinical remission after chemical castration in old dogs with hepa‐ toid gland tumours and heart failure [30].

## **2. Methods for studying steroid hormone receptors**

Immunohistochemical (IHC) methods based on antigen-antibody reactions are widely used today to detect steroid hormone receptors in tumours. New specific antibodies have been developed against a range of steroid hormone receptors, enabling reliable detection by ordi‐ nary light microscopy. Traditionally, however, steroid hormone receptors were detected by biochemical assay based on the binding of radiolabelled ligands to unoccupied receptors. Among the most commonly used biochemical techniques was the dextran-coated charcoal (DCC) method [33], which – until the late nineties – provided all the data available on ste‐ roid hormone receptor expression in mammary carcinomas in female domestic animals. Bio‐ chemical techniques, however, were not without drawbacks: they had to be applied to frozen tissue samples, were very expensive, needed specialised equipment and were not widely accessible. For this reason, published research on the use of these techniques was scanty. The development of monoclonal antibodies highly specific to oestrogen receptor (ERα) and progesterone receptor (PR) proteins enabled the development of immunohisto‐ chemical techniques based on the binding of receptors to specific antibodies [34-37]. These techniques offered several major advantages over biochemical methods [38-40]:

**1.** They could be applied to tissue samples routinely processed for histopathology and could be performed in the pathology laboratory, rendering them both more accessible and less costly.

ties between and human and animal mammary tumours [26]. However, the important role of ER and PR as predictive factors of response to endocrine treatment of breast cancer has been rarely analyzed in animal tumours although recent studies based on the blockade of PR in canine mammary carcinomas and reproductive tract tumours of female and male dogs

Mammary gland tumours are the most frequent tumours in female dogs and the third in the cat. However, they are not the only tumours known to be sex steroid-dependent. Tumours of the reproductive tract of female and male dogs and cats and some skin appendages can be defined as hormone-dependent on the basis of the IHC expression of ERα and PR [31, 32]. In addition, epidemiological and clinical studies support the role of steroid hormones as tumour promoters [28]. The most common tumours in the genital tract of the bitch are be‐ nign smooth muscle tumours of the vagina, vulva and perineal skin. Their hormone-de‐ pendence is similar to that of mammary gland tumours as the majority of canine genital tract leiomyomas express PR and also respond to neoadjuvant treatments with the PR antag‐

In the dog, and to a lesser extent in the cat, benign and malignant sebaceous gland neopla‐ sias are among the most common skin tumours. Studies concerning the role of sex steroid hormones during tumour promotion and progression are scarce but do show there is ER and PR expression in sebaceous gland tumours of dogs. Therefore, not only androgens but also progesterone and estrogens may regulate hormone-related physiology. In fact, preclini‐ cal studies indicate partial clinical remission after chemical castration in old dogs with hepa‐

Immunohistochemical (IHC) methods based on antigen-antibody reactions are widely used today to detect steroid hormone receptors in tumours. New specific antibodies have been developed against a range of steroid hormone receptors, enabling reliable detection by ordi‐ nary light microscopy. Traditionally, however, steroid hormone receptors were detected by biochemical assay based on the binding of radiolabelled ligands to unoccupied receptors. Among the most commonly used biochemical techniques was the dextran-coated charcoal (DCC) method [33], which – until the late nineties – provided all the data available on ste‐ roid hormone receptor expression in mammary carcinomas in female domestic animals. Bio‐ chemical techniques, however, were not without drawbacks: they had to be applied to frozen tissue samples, were very expensive, needed specialised equipment and were not widely accessible. For this reason, published research on the use of these techniques was scanty. The development of monoclonal antibodies highly specific to oestrogen receptor (ERα) and progesterone receptor (PR) proteins enabled the development of immunohisto‐ chemical techniques based on the binding of receptors to specific antibodies [34-37]. These

techniques offered several major advantages over biochemical methods [38-40]:

and cats support their value in the control of these diseases [27-30].

onist aglepristone with a reduction in size [28].

192 Insights from Veterinary Medicine

toid gland tumours and heart failure [30].

**2. Methods for studying steroid hormone receptors**


The process of standardizing IHC methods of ER and PR detection in animal tissues in‐ volved the use of commercially available antibodies raised against human antigens first as antibodies raised against canine and feline antigens were not produced. Then, these IHC methods for detecting ER and PR in formalin-fixed, paraffin wax-embedded tissue samples needed to be validated before they could be routinely implemented, as stipulated by the "National Institutes of Health Consensus Conference on Estrogen Receptors in Breast Can‐ cer" (NIHCC), held in 1979. The first step in the validation procedure was the comparison of ER and PR detection using both biochemical and IHC techniques and the correlation of IHC results with those of the gold-standard radioligand-binding assays in order to evaluate their specificity and sensitivity. In 1999, Graham et al. [15] analysed ERα expression in formalinfixed, paraffin-embedded tissue samples from canine mammary tumours using biochemical and IHC methods, reporting good correlation between the two. In 2000 and 2002, Martín de las Mulas et al. [17, 19] validated the IHC method for the detection of ERα and PR detection, respectively, in formalin-fixed, paraffin-embedded tissue samples from feline mammary tu‐ mours using the avidin-biotin-peroxidase complex (ABC) method, and compared their find‐ ings with those obtained using the DCC biochemical method; reported agreement between the two methods was 72.7% for ERα and 95.6% for PR. The IHC technique used demonstrat‐ ed good specificity (true-negatives) and sensibility (true-positives) (Table 1).


**Table 1.** Specificity and sensibility of an IHC method for detecting ER and PR, versus a biochemical method (DCC) in feline mammary carcinomas [17, 19].

Second, the NIHCC (1979) also recommended the clinical validation of technically validated IHC methods since the presence of ER and/or PR in human breast cancer was known to be a favourable prognostic indicator as well as a predictive marker of the response to adjuvant hormone therapy [41, 42]. The prognostic value of the IHC expression of ERα and PR ex‐ pression in canine mammary carcinoma has been demonstrated in the dog and the cat [18, 20-25]. Finally, preclinical and clinical studies of our group have shown that the PR antago‐ nist Aglepristone produces partial clinical remission of canine mammary carcinoma [29].

While prolonged administration of oestrogens has not been shown to increase the incidence of mammary tumours [55,56], administration of medroxyprogesterone acetate and proges‐ tins over a prolonged period to young female dogs increases the risk of mainly benign mam‐ mary tumours; the intensity of that risk depends on the dosage received and on the regularity or irregularity of treatment [6]. A number of authors, however, also report an in‐ creased incidence of malignant tumours [7]. The risk of developing a malignant tumour ris‐ es following long-term experimental administration of oestrogens combined with high-dose progestins as shown by experimental studies to analyze the effects of the pill [55, 57], and following the administration of drugs with combined progestagenic-oestrogenic activity to control the signs of oestrus [6-9]. Thus, prolonged administration of high-dose (125 x human dose) of a progestational compound (lynestrenol) prompted the development of breast can‐ cer in 40% of intact treated dogs [6]. By contrast, a combined regime of low-dose progesta‐ gen+oestrogen appears to afford some protection [6]. Cats treated with synthetic progestins or oestrogen-progestin combinations displayed a threefold higher risk of developing benign

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**Figure 1.** Details of a canine simple carcinoma showing expression of ERα in the nucleus of luminal epithelial cells

The role played by ovarian steroid hormones during tumour progression (i.e., once the tu‐ mours are detected clinically) has been studied through their expression of ERα and PR. Early studies using biochemical methods of ERα and PR analysis showed that between 65% and 92% of mammary tumours contain hormone receptors [17, 19]. With IHC methods, dif‐ ferential expression between benign and malignant tumours has been observed in dogs and cats as the expression was higher in the former. However, data concerning ERα expression in mammary carcinomas are very different among different laboratories. Thus, in the dog, 7% [59], 10% [16], 11% [14], 22% [20], 24% [15], 59% [60, 61], 87.5% [18] and 92.6% [62] of canine mammary carcinomas have been shown to express ERα (Figure 1). Concerning PR expression, reported data are the following: 14% [63], 33% [14], 42%-52% [62, 64], 60% [16] and 66% [20](Figure 2). In the cat, 7% and 17% of in situ carcinomas expressed ERα and PR, respectively [59] while invasive carcinomas expression figures range from 10% [65] to 25% [17] for ERα and from 38.5% and 43% for PR [19, 65]. Many of these studies point to proges‐

and malignant mammary tumours than untreated cats [58].

(brown colour). ABC. 40X.

At present, IHC methods are routinely used in a number of veterinary laboratories for the detection of ER and PR. However, no consensus has yet been reached regarding tis‐ sue preparation, the antibodies and techniques to be used, and the evaluation of results. New image-analysis systems may help to objectively evaluate receptor expression and to standardise the results obtained by different pathologists [43]. The guidelines issued by the American Society of Clinical Oncology/College of American Pathologists to improve the accuracy of immunohistochemical ERα and PR testing in breast cancer and their utili‐ ty as predictive markers of the response to hormone therapy may serve as a model for veterinary pathologists [44].

#### **3. Mammary gland tumours in dogs and cats**

Mammary gland tumours are the most common neoplasms in female dogs and the third most common in female cats, but are rare in other domestic animals. Between 41% and 53% of mammary tumours in dogs and between 85% and 93% in cats are malignant. Most au‐ thors agree that these tumours account for around 50% and 17% of all neoplasms in dogs and cats, respectively, with an incidence of 205 and 25.4 per 100.000 dogs and cats at risk. Median age at tumour presentation is 10-12 years in both species [45]. Tumours develop al‐ most exclusively in females, appearing only rarely in males [46-48]. Breed is also a risk fac‐ tor: mammary tumours are more common in pure-bred than in mixed-breed animals, and some breeds – including Poodles, Boxers, Beagles, a number of Spaniel breeds, and Siamese cats – display a higher incidence than others [45, 49-51]. Other risk factors are more contro‐ versial, or have been the subject of less research: diet, exposure to radiation, family back‐ ground and individual history of benign and malignant mammary lesions. The action of ovarian hormones – oestrogens and progesterone – on mammary gland tissue during differ‐ ent stages of development is also a risk factor associated with the development of mammary tumours, while breeding-related factors such as parity, age at first gestation, number of pregnancies, pseudopregnancy and changes in the oestrous cycle, do not appear to influence the risk of developing mammary tumours, although not all authors agree on this [45].

The development of mammary tumours in female dogs and cats is clearly hormone-depend‐ ent, and they offer a good spontaneous model of human breast cancer. The preventive ef‐ fects of castration on the development of mammary tumours in both species have been reported [52-54]. Early ovariectomy in dogs and cats has a protective effect against both be‐ nign and malignant mammary tumours, the intensity of that effect depending on the num‐ ber of cycles before spaying. In dogs spayed before the first oestrus, the risk of developing a mammary tumour is 0.05 %, compared to 8% after the first oestrus and 26% after the second oestrus; the protective effect of spaying disappears after the age of 2.5 years. In cats, spaying before 6 months of age reduces the risk of mammary carcinoma by 91% with respect to un‐ spayed cats, while spaying before 1 year of age cuts the risk by 86%.

While prolonged administration of oestrogens has not been shown to increase the incidence of mammary tumours [55,56], administration of medroxyprogesterone acetate and proges‐ tins over a prolonged period to young female dogs increases the risk of mainly benign mam‐ mary tumours; the intensity of that risk depends on the dosage received and on the regularity or irregularity of treatment [6]. A number of authors, however, also report an in‐ creased incidence of malignant tumours [7]. The risk of developing a malignant tumour ris‐ es following long-term experimental administration of oestrogens combined with high-dose progestins as shown by experimental studies to analyze the effects of the pill [55, 57], and following the administration of drugs with combined progestagenic-oestrogenic activity to control the signs of oestrus [6-9]. Thus, prolonged administration of high-dose (125 x human dose) of a progestational compound (lynestrenol) prompted the development of breast can‐ cer in 40% of intact treated dogs [6]. By contrast, a combined regime of low-dose progesta‐ gen+oestrogen appears to afford some protection [6]. Cats treated with synthetic progestins or oestrogen-progestin combinations displayed a threefold higher risk of developing benign and malignant mammary tumours than untreated cats [58].

20-25]. Finally, preclinical and clinical studies of our group have shown that the PR antago‐ nist Aglepristone produces partial clinical remission of canine mammary carcinoma [29].

At present, IHC methods are routinely used in a number of veterinary laboratories for the detection of ER and PR. However, no consensus has yet been reached regarding tis‐ sue preparation, the antibodies and techniques to be used, and the evaluation of results. New image-analysis systems may help to objectively evaluate receptor expression and to standardise the results obtained by different pathologists [43]. The guidelines issued by the American Society of Clinical Oncology/College of American Pathologists to improve the accuracy of immunohistochemical ERα and PR testing in breast cancer and their utili‐ ty as predictive markers of the response to hormone therapy may serve as a model for

Mammary gland tumours are the most common neoplasms in female dogs and the third most common in female cats, but are rare in other domestic animals. Between 41% and 53% of mammary tumours in dogs and between 85% and 93% in cats are malignant. Most au‐ thors agree that these tumours account for around 50% and 17% of all neoplasms in dogs and cats, respectively, with an incidence of 205 and 25.4 per 100.000 dogs and cats at risk. Median age at tumour presentation is 10-12 years in both species [45]. Tumours develop al‐ most exclusively in females, appearing only rarely in males [46-48]. Breed is also a risk fac‐ tor: mammary tumours are more common in pure-bred than in mixed-breed animals, and some breeds – including Poodles, Boxers, Beagles, a number of Spaniel breeds, and Siamese cats – display a higher incidence than others [45, 49-51]. Other risk factors are more contro‐ versial, or have been the subject of less research: diet, exposure to radiation, family back‐ ground and individual history of benign and malignant mammary lesions. The action of ovarian hormones – oestrogens and progesterone – on mammary gland tissue during differ‐ ent stages of development is also a risk factor associated with the development of mammary tumours, while breeding-related factors such as parity, age at first gestation, number of pregnancies, pseudopregnancy and changes in the oestrous cycle, do not appear to influence the risk of developing mammary tumours, although not all authors agree on this [45].

The development of mammary tumours in female dogs and cats is clearly hormone-depend‐ ent, and they offer a good spontaneous model of human breast cancer. The preventive ef‐ fects of castration on the development of mammary tumours in both species have been reported [52-54]. Early ovariectomy in dogs and cats has a protective effect against both be‐ nign and malignant mammary tumours, the intensity of that effect depending on the num‐ ber of cycles before spaying. In dogs spayed before the first oestrus, the risk of developing a mammary tumour is 0.05 %, compared to 8% after the first oestrus and 26% after the second oestrus; the protective effect of spaying disappears after the age of 2.5 years. In cats, spaying before 6 months of age reduces the risk of mammary carcinoma by 91% with respect to un‐

spayed cats, while spaying before 1 year of age cuts the risk by 86%.

veterinary pathologists [44].

194 Insights from Veterinary Medicine

**3. Mammary gland tumours in dogs and cats**

**Figure 1.** Details of a canine simple carcinoma showing expression of ERα in the nucleus of luminal epithelial cells (brown colour). ABC. 40X.

The role played by ovarian steroid hormones during tumour progression (i.e., once the tu‐ mours are detected clinically) has been studied through their expression of ERα and PR. Early studies using biochemical methods of ERα and PR analysis showed that between 65% and 92% of mammary tumours contain hormone receptors [17, 19]. With IHC methods, dif‐ ferential expression between benign and malignant tumours has been observed in dogs and cats as the expression was higher in the former. However, data concerning ERα expression in mammary carcinomas are very different among different laboratories. Thus, in the dog, 7% [59], 10% [16], 11% [14], 22% [20], 24% [15], 59% [60, 61], 87.5% [18] and 92.6% [62] of canine mammary carcinomas have been shown to express ERα (Figure 1). Concerning PR expression, reported data are the following: 14% [63], 33% [14], 42%-52% [62, 64], 60% [16] and 66% [20](Figure 2). In the cat, 7% and 17% of in situ carcinomas expressed ERα and PR, respectively [59] while invasive carcinomas expression figures range from 10% [65] to 25% [17] for ERα and from 38.5% and 43% for PR [19, 65]. Many of these studies point to proges‐ terone, and not oestrogens, as the sex steroid hormone driving proliferation in mammary gland tumours as all benign tumours of the canine mammary gland and 2/3 of malignant tumours express PR [14, 16, 20, 22].

228 tumours (155 malignant and 73 benign) from 100 bitches, Martín de las Mulas et al. [20] found that a total of 76% of tumours (96% benign and 66% malignant) expressed ERα and/or PR. In seven cases with lymph node metastasis, both the primary tumours and their meta‐ stases were ERα and PR negative, indicating a loss of hormone influence and thus greater aggressiveness. Expression of PR alone in 66% of malignant tumours suggested that PR

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Surgery is the treatment of choice for feline and canine mammary tumours. Treatment with drugs such as tamoxifen has been tested in dogs with mammary carcinoma, but is not recommended due to their considerable side effects; these drugs are therefore not currently in use [70]. Ovariohysterectomy (OHE) performed at the time of surgery does not appear to produce any clear benefit in dogs with mammary carcinoma [71, 72]. The only adjuvant treatment administered at present is chemotherapy, and results are not

In 1998, Cappelletti et al. [73] reported different ERα and PR counts in tumours analysed be‐ fore and after treatment with a range of drugs including tamoxifen, concluding that malig‐ nant mammary neoplasms were sensitive to steroid hormone treatment. A feline dysplasia of the mammary gland, the feline fibroadenomatous change, is highly sensitive to Alizin®, a PR antagonist [74, 75]. The fact that all benign tumours and two thirds of malignant tumours in dogs express mainly PR [16, 17, 22] points to the potential use of progesterone receptor antagonists as a neoadjuvant and/or adjuvant treatment for these tumours. A recent study performed by our group [29] has shown that administration of the PR receptor antagonist Aglepristone (Alizin®, Virbac, France) as neoadjuvant treatment in female dogs inhibits the proliferation of PR-expressing mammary carcinomas. Twenty-seven non-spayed bitches with mammary tumours were treated with Alizin® before surgical tumour removal. Tu‐ mour tissue samples were analysed before and after treatment, and PR expression was re‐ duced following treatment (36.4% versus 59.1% prior to treatment). The proliferative index (PI) was also analysed before and after treatment, using the avidin-biotin-peroxidase com‐ plex technique and a proliferative marker (Ki67). A significant decrease in the PI was record‐ ed in tumours expressing PR, while no change was observed in those not expressing PR, suggesting that the PR antagonist Aglepristone inhibited tumour proliferation in PR-posi‐

**4. Female and male reproductive-tract tumours in dogs and cats**

The actual incidence of female reproductive-tract tumours is difficult to ascertain, presuma‐ bly because a significant percentage of dogs and cats are neutered [76]. These tumours may arise in the vagina, vulva, uterus or ovaries; vulvar and vaginal tumours are the most com‐ mon (after mammary gland tumours), accounting for 2.4% to 3% of all canine neoplasms [77, 78]. No incidence rates are available for vulvar and vaginal tumours in cats. Uterine tu‐ mours are rare in both dogs and cats, accounting for 0.3%-0.4% and 0.2%-1.5% of all canine

played a more important role than ERα in tumour proliferation.

wholly satisfactory [45].

tive tumours by blocking the PR.

**4.1. Female reproductive tract**

**Figure 2.** Canine simple carcinoma showing PR expression in the nucleus of neoplastic epithelial cells. (brown colour). ABC. 20X.

After the cloning of a second ER in rats and humans, designated ERβ [66, 67], it became known that currently reported data correlated with the isoform α of ER (ERα). Our group was the first to publish data on the expression of the second isoform of the ER (ERβ) in mammary tissues of dogs [68], and one more study has been performed up to date [69]. ERβ expression was observed in the ductal and acinar epithelium of normal mammary glands and in one third of mammary tumours. Expression was greater in benign than in malignant tumours [68]. Among malignant tumours, ERβ expression was greater in complex and mixed tumours than in simple carcinomas, indicating that ERβ may also be a prognostic fac‐ tor of these tumours (Figure 3).

**Figure 3.** ERβ immunoreaction in the nucleus of neoplastic epithelial cells of canine mammary carcinoma. ABC. 20X.

The prognostic value of steroid hormone receptor activity in malignant tumours (lower risk of relapse and metastasis, together with greater survival time) is now widely accepted after several univariate and multivariate prognostic studies using IHC methods for detection of ER and PR [18, 20-25]. In a study using IHC techniques to detect ER and PR expression in 228 tumours (155 malignant and 73 benign) from 100 bitches, Martín de las Mulas et al. [20] found that a total of 76% of tumours (96% benign and 66% malignant) expressed ERα and/or PR. In seven cases with lymph node metastasis, both the primary tumours and their meta‐ stases were ERα and PR negative, indicating a loss of hormone influence and thus greater aggressiveness. Expression of PR alone in 66% of malignant tumours suggested that PR played a more important role than ERα in tumour proliferation.

Surgery is the treatment of choice for feline and canine mammary tumours. Treatment with drugs such as tamoxifen has been tested in dogs with mammary carcinoma, but is not recommended due to their considerable side effects; these drugs are therefore not currently in use [70]. Ovariohysterectomy (OHE) performed at the time of surgery does not appear to produce any clear benefit in dogs with mammary carcinoma [71, 72]. The only adjuvant treatment administered at present is chemotherapy, and results are not wholly satisfactory [45].

In 1998, Cappelletti et al. [73] reported different ERα and PR counts in tumours analysed be‐ fore and after treatment with a range of drugs including tamoxifen, concluding that malig‐ nant mammary neoplasms were sensitive to steroid hormone treatment. A feline dysplasia of the mammary gland, the feline fibroadenomatous change, is highly sensitive to Alizin®, a PR antagonist [74, 75]. The fact that all benign tumours and two thirds of malignant tumours in dogs express mainly PR [16, 17, 22] points to the potential use of progesterone receptor antagonists as a neoadjuvant and/or adjuvant treatment for these tumours. A recent study performed by our group [29] has shown that administration of the PR receptor antagonist Aglepristone (Alizin®, Virbac, France) as neoadjuvant treatment in female dogs inhibits the proliferation of PR-expressing mammary carcinomas. Twenty-seven non-spayed bitches with mammary tumours were treated with Alizin® before surgical tumour removal. Tu‐ mour tissue samples were analysed before and after treatment, and PR expression was re‐ duced following treatment (36.4% versus 59.1% prior to treatment). The proliferative index (PI) was also analysed before and after treatment, using the avidin-biotin-peroxidase com‐ plex technique and a proliferative marker (Ki67). A significant decrease in the PI was record‐ ed in tumours expressing PR, while no change was observed in those not expressing PR, suggesting that the PR antagonist Aglepristone inhibited tumour proliferation in PR-posi‐ tive tumours by blocking the PR.

## **4. Female and male reproductive-tract tumours in dogs and cats**

#### **4.1. Female reproductive tract**

terone, and not oestrogens, as the sex steroid hormone driving proliferation in mammary gland tumours as all benign tumours of the canine mammary gland and 2/3 of malignant

**Figure 2.** Canine simple carcinoma showing PR expression in the nucleus of neoplastic epithelial cells. (brown colour).

After the cloning of a second ER in rats and humans, designated ERβ [66, 67], it became known that currently reported data correlated with the isoform α of ER (ERα). Our group was the first to publish data on the expression of the second isoform of the ER (ERβ) in mammary tissues of dogs [68], and one more study has been performed up to date [69]. ERβ expression was observed in the ductal and acinar epithelium of normal mammary glands and in one third of mammary tumours. Expression was greater in benign than in malignant tumours [68]. Among malignant tumours, ERβ expression was greater in complex and mixed tumours than in simple carcinomas, indicating that ERβ may also be a prognostic fac‐

**Figure 3.** ERβ immunoreaction in the nucleus of neoplastic epithelial cells of canine mammary carcinoma. ABC. 20X.

The prognostic value of steroid hormone receptor activity in malignant tumours (lower risk of relapse and metastasis, together with greater survival time) is now widely accepted after several univariate and multivariate prognostic studies using IHC methods for detection of ER and PR [18, 20-25]. In a study using IHC techniques to detect ER and PR expression in

tumours express PR [14, 16, 20, 22].

196 Insights from Veterinary Medicine

tor of these tumours (Figure 3).

ABC. 20X.

The actual incidence of female reproductive-tract tumours is difficult to ascertain, presuma‐ bly because a significant percentage of dogs and cats are neutered [76]. These tumours may arise in the vagina, vulva, uterus or ovaries; vulvar and vaginal tumours are the most com‐ mon (after mammary gland tumours), accounting for 2.4% to 3% of all canine neoplasms [77, 78]. No incidence rates are available for vulvar and vaginal tumours in cats. Uterine tu‐ mours are rare in both dogs and cats, accounting for 0.3%-0.4% and 0.2%-1.5% of all canine and feline tumours, respectively [77, 79-84]. Ovarian tumours are also uncommon in dogs and cats; although the true incidence is unknown, the reported incidence in intact bitches is 6.25%, thus representing 0.5% to 1.2% of all canine tumours [85, 86], while reported inci‐ dence in cats ranges from 0.7% to 3.6% [87]. The most common neoplasms in the canine fe‐ male reproductive tract are benign tumours of mesenchymal origin, classified as leiomyomas, fibroleiomyomas, fibromas and polyps depending on the amount of connective tissue present [88,89]. The use of markers for smooth-muscle differentiation (e.g. desmin, calponin, smooth muscle actin) is valuable for the accurate identification of smooth muscle involvement in tumour growth [31, 90, 91] (Figure 4).

tion marker (Ki67), recording similar low values at days 15 and 25 of treatment, suggesting that Aglepristone did not reduce tumour size by reducing the tumour cell proliferation rate but rather through increasing apoptosis. It was therefore concluded that the size of PR-ex‐ pressing benign tumours of the canine vagina could be reduced by palliative or neoadjuvant therapy with the PR antagonist Aglepristone. These findings regarding steroid hormone re‐ ceptor expression in canine tumours of the female reproductive tract highlight the potential of hormone therapy in selected cases. In cats, leiomyomas located in the mammary gland and in the perineal region are reported to express ERα and PR [27]; however, no research has yet focussed on the expression of steroid hormone receptors in feline leiomyomas locat‐

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**Figure 5.** Immunohistochemical detection of ERα in a leiomyoma. Most of the tumour cells show immunoreactive

**Figure 6.** A) Female dog with a perineal mass measuring 9.1 x 5.4 cm. (B) The appearance of the tumour mass 28 days

Male reproductive-tract tumours in dogs and cats may arise in the testes, prostate, penis or foreskin. Testicular tumours are the second most common cancer in intact dogs, accounting

products to ER antibody in their nucleus (brown colour)(arrow). ABC. 40X.

after treatment with aglepristone. The size is reduced to 6.4 x 4.7 cm [28].

**4.2. Male reproductive tract**

ed in the vagina, vulva or uterus.

**Figure 4.** Immunohistochemical detection of the smooth muscle protein calponin in a leiomymoma. Most of the tu‐ mour cells show immunoreactive products to calponin antibody in their cytoplasms (brown colour). The vascular smooth muscle cells are also reactive (arrow) while endothelial cells are unreactive (arrowhead). ABC. 20X.

Leiomyoma is among the most common tumours of the female reproductive tract in many domestic species. It is located primarily in the vulva and vagina, followed by the uterus and its incidence is greater in older non-neutered bitches [77, 78]. In two different studies, all dogs diagnosed with leiomyoma were non-neutered bitches [78, 93] and the recurrence rate was 15% in bitches left unneutered after local excision. On the contrary, there was no recur‐ rence in any animal when ovariohysterectomy was performed at the same time as excision. In another study [77], no leiomyomas were diagnosed in ovariectomised bitches under two years old. These epidemiological and clinical findings support the hormone-dependent na‐ ture of leiomyomas [88]. In addition, the role of ovarian steroid hormone receptors in the progression of female reproductive tract leiomyomas in the dog has also been demonstrat‐ ed. Thus, Millán et al. [31] performed the first study on canine leiomyomas from the repro‐ ductive tract (uterus, vagina and vulva) demonstrating the IHC expression of ERα and PR in tumour tissue samples. Half of the leiomyomas (50%) expressed ERα and more than three quarters (82.1%) expressed PR (Figure 5).

Finally, a pioneer study of our group reported that the expression of PR in a canine vaginal fibroma was a predictive factor of favourable response to hormone therapy. Aglepritone at a dose of 10 mg/kg injected at days 1, 2, 8, 15, 28 and 35 prompted a progressive reduction in the size of the mass, which measured 9.1 x 5.4 cm on day 1 and 6.4 x 4.7 cm on day 45 [28] (Figure 6). The authors evaluated the proliferation index in the same study using a prolifera‐ tion marker (Ki67), recording similar low values at days 15 and 25 of treatment, suggesting that Aglepristone did not reduce tumour size by reducing the tumour cell proliferation rate but rather through increasing apoptosis. It was therefore concluded that the size of PR-ex‐ pressing benign tumours of the canine vagina could be reduced by palliative or neoadjuvant therapy with the PR antagonist Aglepristone. These findings regarding steroid hormone re‐ ceptor expression in canine tumours of the female reproductive tract highlight the potential of hormone therapy in selected cases. In cats, leiomyomas located in the mammary gland and in the perineal region are reported to express ERα and PR [27]; however, no research has yet focussed on the expression of steroid hormone receptors in feline leiomyomas locat‐ ed in the vagina, vulva or uterus.

**Figure 5.** Immunohistochemical detection of ERα in a leiomyoma. Most of the tumour cells show immunoreactive products to ER antibody in their nucleus (brown colour)(arrow). ABC. 40X.

**Figure 6.** A) Female dog with a perineal mass measuring 9.1 x 5.4 cm. (B) The appearance of the tumour mass 28 days after treatment with aglepristone. The size is reduced to 6.4 x 4.7 cm [28].

#### **4.2. Male reproductive tract**

and feline tumours, respectively [77, 79-84]. Ovarian tumours are also uncommon in dogs and cats; although the true incidence is unknown, the reported incidence in intact bitches is 6.25%, thus representing 0.5% to 1.2% of all canine tumours [85, 86], while reported inci‐ dence in cats ranges from 0.7% to 3.6% [87]. The most common neoplasms in the canine fe‐ male reproductive tract are benign tumours of mesenchymal origin, classified as leiomyomas, fibroleiomyomas, fibromas and polyps depending on the amount of connective tissue present [88,89]. The use of markers for smooth-muscle differentiation (e.g. desmin, calponin, smooth muscle actin) is valuable for the accurate identification of smooth muscle

**Figure 4.** Immunohistochemical detection of the smooth muscle protein calponin in a leiomymoma. Most of the tu‐ mour cells show immunoreactive products to calponin antibody in their cytoplasms (brown colour). The vascular

Leiomyoma is among the most common tumours of the female reproductive tract in many domestic species. It is located primarily in the vulva and vagina, followed by the uterus and its incidence is greater in older non-neutered bitches [77, 78]. In two different studies, all dogs diagnosed with leiomyoma were non-neutered bitches [78, 93] and the recurrence rate was 15% in bitches left unneutered after local excision. On the contrary, there was no recur‐ rence in any animal when ovariohysterectomy was performed at the same time as excision. In another study [77], no leiomyomas were diagnosed in ovariectomised bitches under two years old. These epidemiological and clinical findings support the hormone-dependent na‐ ture of leiomyomas [88]. In addition, the role of ovarian steroid hormone receptors in the progression of female reproductive tract leiomyomas in the dog has also been demonstrat‐ ed. Thus, Millán et al. [31] performed the first study on canine leiomyomas from the repro‐ ductive tract (uterus, vagina and vulva) demonstrating the IHC expression of ERα and PR in tumour tissue samples. Half of the leiomyomas (50%) expressed ERα and more than three

Finally, a pioneer study of our group reported that the expression of PR in a canine vaginal fibroma was a predictive factor of favourable response to hormone therapy. Aglepritone at a dose of 10 mg/kg injected at days 1, 2, 8, 15, 28 and 35 prompted a progressive reduction in the size of the mass, which measured 9.1 x 5.4 cm on day 1 and 6.4 x 4.7 cm on day 45 [28] (Figure 6). The authors evaluated the proliferation index in the same study using a prolifera‐

smooth muscle cells are also reactive (arrow) while endothelial cells are unreactive (arrowhead). ABC. 20X.

involvement in tumour growth [31, 90, 91] (Figure 4).

198 Insights from Veterinary Medicine

quarters (82.1%) expressed PR (Figure 5).

Male reproductive-tract tumours in dogs and cats may arise in the testes, prostate, penis or foreskin. Testicular tumours are the second most common cancer in intact dogs, accounting for roughly 90% of all reproductive-tract tumours [96]. In cats, however, testicular tumours are rare [97]. Prostate tumours are uncommon in dogs, with a reported incidence of between 0.2% to 0.6% [98, 99], and equally rare in cats [97, 100-102]. Many penis and foreskin tu‐ mours affect the epithelial surface of these structures, the most common being the transmis‐ sible venereal tumour of the penis in dogs [103].

important role in the pathogenesis of these tumours, as they do in mammary tumours. It is well known that oestrogens, progesterone and androgens not only help regulate skin development and function, such as the development and/or physiology of sebaceous glands and hair follicles, but are also involved in the development and biological behav‐

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201

To date, a number of studies have demonstrated a possible relationship between sex steroid hormones and sebaceous, perianal and hepatoid gland tumours and mast-cell tumours in domestic animals. To better understand this relationship, recent research has focussed on pinpointing the site of action of these hormones and on locating their receptors in both nor‐ mal and tumour tissues. As a result, biochemical and immunohistochemical studies carried out over the last few years have detected androgen receptors (AR), oestrogen receptor α (ERα) and progesterone receptors (PR) in epidermis, hair follicles and fundamentally in the sebaceous glands of canine skin [113-117]. These results suggest that not only androgens and oestrogens, but also progesterone, may play a major role in the regulation of normal skin

Sebaceous gland tumours are among the most common skin tumours in the dog. They can be divided into four groups based on histological appearance: sebaceous hyperplasia, seba‐ ceous epitheliomas, sebaceous adenomas and sebaceous adenocarcinomas. They account for between 6.8% to 7.9% of all skin tumours in dogs, and between 2.3% to 4.4% in cats [118]. Canine benign and malignant sebaceous gland neoplasias may provide a suitable experi‐ mental model for the study of hormone influences on the development of glandular tu‐ mours [114]. As in human skin, specific staining for ERα and PR is seen mainly in the basal cells of normal sebaceous glands [115-117]. However, unlike in human medicine, no data are available regarding the possible involvement of these steroid sex hormones in the pathology of canine and feline skin and the development of cutaneous neoplasms. A single study by the present authors analysed ERα and PR expression in canine sebaceous gland hyperpla‐ sias, adenomas/epitheliomas and carcinomas [117], revealing that canine sebaceous glands express both ERα and PR (Figure 7 y 8). Moreover, differences were recorded between types of lesion in the number of cells expressing ERα and PR. Compared with normal sebaceous glands, ERα expression was significantly lower in sebaceous gland epitheliomas and adeno‐ carcinomas, suggesting that ERα plays a key physiological role in the maintenance of nor‐ mal sebaceous glands, and that a decrease in levels influences the development of both benign and malignant neoplasms. A number of studies indicate that this drop in ERα could be secondary to changes in androgen or oestrogen production, but further research is re‐ quired to confirm this hypothesis in canine sebaceous glands. The cited study also found that PR expression in adenocarcinomas was significantly lower than in normal and hyper‐ plastic sebaceous glands, suggesting that tumour growth may become less hormone-de‐ pendent during the progression phase of carcinogenesis, as reported in certain human mammary tumours. Unlike ERα, the proportion of PR-positive cells did not differ signifi‐ cantly from that found in normal sebaceous glands, a finding also reported in humans [112]. This may indicate that progesterone does not necessarily influence the growth of this type of tumours. Conversely, sebaceous gland carcinomas display a significant loss in PR staining

appendage function and in the pathogenesis and development of neoplasms.

iour of certain skin neoplasms [112].

Currently, the only male reproductive-tract tumour in domestic species for which published data is available regarding the possible influence of steroid hormones on tumour develop‐ ment is canine prostate carcinoma. The dog is the only non-primate species that develops spontaneous prostate cancer [104]. Most tumours in this location are of epithelial origin [98, 105] and mainly affect older dogs (average age 10 years), prostate carcinoma being common [98]. The prostate is an androgen-dependent organ [106], and androgens achieve their effect through activation of androgen receptors (AR). However, there is a good deal of controversy concerning the influence of androgens on the development and biological behaviour of ma‐ lignant prostate tumours. While some studies have found no evidence that castration has a protective effect against prostate carcinoma [99], others argue that castration before sexual maturity reduces the risk of this malignancy [105]; still others suggest that castration may actually increase the incidence and aggressive behaviour of canine prostate carcinomas [98]. The expression of steroid hormone receptors in tumour tissue samples of canine prostate carcinoma has been rarely studied. The more complete study up to date showed that expres‐ sion of AR, ERα and ERβ was lower in malignant tumour epithelial cells than in normal prostate tissue and benign lesions, suggesting that oestrogen actions in the prostate are com‐ plex and may play a dual role in the aetiology of prostate cancer [32, 107]. This study also demonstrated for the first time that PR expression in canine prostate tumours is greater than in normal prostate tissue [32], although the effect of progesterone at this location still re‐ mains to be demonstrated, as does the potential for hormone therapy.

### **5. Cutaneous tumours in dogs and cats**

Tumours of the skin and subcutaneous tissue are the most common neoplasms affecting dogs and the second most common in cats [108]. Incidence rates have been estimated at around 450 per 100,000 dogs and 120 per 100,000 cats [108]. In dogs, most skin tumours are benign, the most frequent being histiocytomas and sebaceous gland adenomas. In cats, how‐ ever, approximately 50% to 65% of skin tumours are histologically malignant, the most fre‐ quently-reported being squamous cell carcinomas [109].

Epidemiological research has identified breed and age as major risk factors for these tu‐ mours. A number of authors note a linear increase in risk by a factor of 1.1 per year of increasing age; additionally, pure-bred dogs appear to be twice as likely to develop a malignancy as cross-breeds. When all types of tumours are considered together, no sig‐ nificant sex predilection is apparent [110]. A number of etiological factors – physical, vi‐ ral, genetic and molecular – have been reported for some skin tumours [111]. Over the last few years, it has become increasingly evident that steroid sex hormones may play an important role in the pathogenesis of these tumours, as they do in mammary tumours. It is well known that oestrogens, progesterone and androgens not only help regulate skin development and function, such as the development and/or physiology of sebaceous glands and hair follicles, but are also involved in the development and biological behav‐ iour of certain skin neoplasms [112].

for roughly 90% of all reproductive-tract tumours [96]. In cats, however, testicular tumours are rare [97]. Prostate tumours are uncommon in dogs, with a reported incidence of between 0.2% to 0.6% [98, 99], and equally rare in cats [97, 100-102]. Many penis and foreskin tu‐ mours affect the epithelial surface of these structures, the most common being the transmis‐

Currently, the only male reproductive-tract tumour in domestic species for which published data is available regarding the possible influence of steroid hormones on tumour develop‐ ment is canine prostate carcinoma. The dog is the only non-primate species that develops spontaneous prostate cancer [104]. Most tumours in this location are of epithelial origin [98, 105] and mainly affect older dogs (average age 10 years), prostate carcinoma being common [98]. The prostate is an androgen-dependent organ [106], and androgens achieve their effect through activation of androgen receptors (AR). However, there is a good deal of controversy concerning the influence of androgens on the development and biological behaviour of ma‐ lignant prostate tumours. While some studies have found no evidence that castration has a protective effect against prostate carcinoma [99], others argue that castration before sexual maturity reduces the risk of this malignancy [105]; still others suggest that castration may actually increase the incidence and aggressive behaviour of canine prostate carcinomas [98]. The expression of steroid hormone receptors in tumour tissue samples of canine prostate carcinoma has been rarely studied. The more complete study up to date showed that expres‐ sion of AR, ERα and ERβ was lower in malignant tumour epithelial cells than in normal prostate tissue and benign lesions, suggesting that oestrogen actions in the prostate are com‐ plex and may play a dual role in the aetiology of prostate cancer [32, 107]. This study also demonstrated for the first time that PR expression in canine prostate tumours is greater than in normal prostate tissue [32], although the effect of progesterone at this location still re‐

mains to be demonstrated, as does the potential for hormone therapy.

Tumours of the skin and subcutaneous tissue are the most common neoplasms affecting dogs and the second most common in cats [108]. Incidence rates have been estimated at around 450 per 100,000 dogs and 120 per 100,000 cats [108]. In dogs, most skin tumours are benign, the most frequent being histiocytomas and sebaceous gland adenomas. In cats, how‐ ever, approximately 50% to 65% of skin tumours are histologically malignant, the most fre‐

Epidemiological research has identified breed and age as major risk factors for these tu‐ mours. A number of authors note a linear increase in risk by a factor of 1.1 per year of increasing age; additionally, pure-bred dogs appear to be twice as likely to develop a malignancy as cross-breeds. When all types of tumours are considered together, no sig‐ nificant sex predilection is apparent [110]. A number of etiological factors – physical, vi‐ ral, genetic and molecular – have been reported for some skin tumours [111]. Over the last few years, it has become increasingly evident that steroid sex hormones may play an

**5. Cutaneous tumours in dogs and cats**

quently-reported being squamous cell carcinomas [109].

sible venereal tumour of the penis in dogs [103].

200 Insights from Veterinary Medicine

To date, a number of studies have demonstrated a possible relationship between sex steroid hormones and sebaceous, perianal and hepatoid gland tumours and mast-cell tumours in domestic animals. To better understand this relationship, recent research has focussed on pinpointing the site of action of these hormones and on locating their receptors in both nor‐ mal and tumour tissues. As a result, biochemical and immunohistochemical studies carried out over the last few years have detected androgen receptors (AR), oestrogen receptor α (ERα) and progesterone receptors (PR) in epidermis, hair follicles and fundamentally in the sebaceous glands of canine skin [113-117]. These results suggest that not only androgens and oestrogens, but also progesterone, may play a major role in the regulation of normal skin appendage function and in the pathogenesis and development of neoplasms.

Sebaceous gland tumours are among the most common skin tumours in the dog. They can be divided into four groups based on histological appearance: sebaceous hyperplasia, seba‐ ceous epitheliomas, sebaceous adenomas and sebaceous adenocarcinomas. They account for between 6.8% to 7.9% of all skin tumours in dogs, and between 2.3% to 4.4% in cats [118]. Canine benign and malignant sebaceous gland neoplasias may provide a suitable experi‐ mental model for the study of hormone influences on the development of glandular tu‐ mours [114]. As in human skin, specific staining for ERα and PR is seen mainly in the basal cells of normal sebaceous glands [115-117]. However, unlike in human medicine, no data are available regarding the possible involvement of these steroid sex hormones in the pathology of canine and feline skin and the development of cutaneous neoplasms. A single study by the present authors analysed ERα and PR expression in canine sebaceous gland hyperpla‐ sias, adenomas/epitheliomas and carcinomas [117], revealing that canine sebaceous glands express both ERα and PR (Figure 7 y 8). Moreover, differences were recorded between types of lesion in the number of cells expressing ERα and PR. Compared with normal sebaceous glands, ERα expression was significantly lower in sebaceous gland epitheliomas and adeno‐ carcinomas, suggesting that ERα plays a key physiological role in the maintenance of nor‐ mal sebaceous glands, and that a decrease in levels influences the development of both benign and malignant neoplasms. A number of studies indicate that this drop in ERα could be secondary to changes in androgen or oestrogen production, but further research is re‐ quired to confirm this hypothesis in canine sebaceous glands. The cited study also found that PR expression in adenocarcinomas was significantly lower than in normal and hyper‐ plastic sebaceous glands, suggesting that tumour growth may become less hormone-de‐ pendent during the progression phase of carcinogenesis, as reported in certain human mammary tumours. Unlike ERα, the proportion of PR-positive cells did not differ signifi‐ cantly from that found in normal sebaceous glands, a finding also reported in humans [112]. This may indicate that progesterone does not necessarily influence the growth of this type of tumours. Conversely, sebaceous gland carcinomas display a significant loss in PR staining intensity; PR loss may be one factor involved in the pathogenesis of canine sebaceous gland neoplasms.

ianal neoplasms [121]. Although found in both male and female dogs, the highest incidence is reported in intact males of mean age 10 or more [122]. This evidence has for years been interpreted as proof of a clear stimulation of tumour development by sex ste‐ roid hormones, particularly androgens [123]. Adenomas have been found to be hormoneresponsive; a full or partial regression has been observed following castration or oestrogen treatment [113, 120, 124]. In females, perianal adenomas occur almost exclu‐ sively in ovariohysterectomised animals whose low oestrogen levels fail to suppress tu‐ mour growth. Similarly, testicular interstitial cell tumours – which clinical observation suggests are associated with an increase in systemic androgen levels – occur more fre‐ quently in association with perianal tumours [125]. While hormone dependence has been clearly demonstrated in the case of perianal adenomas, there appears to be no link be‐ tween perianal adenocarcinomas and steroid sex hormones. Perianal gland carcinomas do not regress following castration and are not responsive to hormone therapy with oes‐ trogens [121, 124]. However, receptors for these hormones have been found in normal, hyperplastic and neoplastic perianal glands in dogs (Figure 9). An early report identified androgen-binding sites in perianal adenomas [124]. Research by Pisani *et al.* [126] detect‐ ed AR expression in all normal and abnormal glands, although in hyperplastic tissues the proportion of positive nuclei was significantly greater than in normal tissue. A simi‐ lar increase in the percentage of positive-staining nuclei was also observed in perianal epitheliomas, while in adenomas the increase with respect to normal tissue was only slight. In adenocarcinomas, the proportion of AR-positive cells was similar to that ob‐

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203

**Figure 9.** Immunohistochemical localization of ERα in a hepatoid gland adenoma. Nuclear staining is observed in bas‐

All these observations support the view that therapy based on antagonists of these hor‐ mones could prove beneficial in the treatment of these tumours. The present authors have carried out a preliminary clinical trial in order to evaluate the effect of Deslorelin (Suprelor‐ inTM Virbac), a GnRH antagonist, on the clinical response of perianal gland adenomas [127]. This antagonist suppresses pituitary production of the hormones LH and FSH and of steroid sex hormones; its effect has been compared to that of surgical castration. The trial found that

al cells (arrow) and differentiated hepatoid cells (arrowhead). ABC. 20X.

served in benign tumours.

**Figure 7.** Immunohistochemical localization of PR in sebaceous gland hyperplasia. Nuclear staining is observed in bas‐ al cells (arrow) and differentiated sebocytes (arrowhead) [117]. ABC. 40X.

The influence of female sex steroids on their receptors in skin has been highlighted by sever‐ al studies. When an ovariectomised bitch received a local estradiol (E2) implant, ER levels in the affected skin were found to be six times higher than in control skin [119]. However, de‐ spite the considerable amount of work done to date in this field, further research is still re‐ quired to demonstrate the presence of AR and examine of the effects of these hormones and their pharmacological antagonists on tumour development.

Other tumours considered clearly hormone-dependent include those arising in the peria‐ nal or hepatoid glands, modified sebaceous glands located in the perianal dermis. The most frequently observed tumours of this region in dogs are perianal adenoma, perianal adenocarcinoma and apocrine gland adenocarcinoma of the anal sac. Perianal adenomas account for between 58% and 96% of all canine perianal tumours [120]. Unlike adeno‐ mas, perianal gland carcinomas are rare, accounting for between 3% and 17% of all per‐ ianal neoplasms [121]. Although found in both male and female dogs, the highest incidence is reported in intact males of mean age 10 or more [122]. This evidence has for years been interpreted as proof of a clear stimulation of tumour development by sex ste‐ roid hormones, particularly androgens [123]. Adenomas have been found to be hormoneresponsive; a full or partial regression has been observed following castration or oestrogen treatment [113, 120, 124]. In females, perianal adenomas occur almost exclu‐ sively in ovariohysterectomised animals whose low oestrogen levels fail to suppress tu‐ mour growth. Similarly, testicular interstitial cell tumours – which clinical observation suggests are associated with an increase in systemic androgen levels – occur more fre‐ quently in association with perianal tumours [125]. While hormone dependence has been clearly demonstrated in the case of perianal adenomas, there appears to be no link be‐ tween perianal adenocarcinomas and steroid sex hormones. Perianal gland carcinomas do not regress following castration and are not responsive to hormone therapy with oes‐ trogens [121, 124]. However, receptors for these hormones have been found in normal, hyperplastic and neoplastic perianal glands in dogs (Figure 9). An early report identified androgen-binding sites in perianal adenomas [124]. Research by Pisani *et al.* [126] detect‐ ed AR expression in all normal and abnormal glands, although in hyperplastic tissues the proportion of positive nuclei was significantly greater than in normal tissue. A simi‐ lar increase in the percentage of positive-staining nuclei was also observed in perianal epitheliomas, while in adenomas the increase with respect to normal tissue was only slight. In adenocarcinomas, the proportion of AR-positive cells was similar to that ob‐ served in benign tumours.

intensity; PR loss may be one factor involved in the pathogenesis of canine sebaceous gland

**Figure 7.** Immunohistochemical localization of PR in sebaceous gland hyperplasia. Nuclear staining is observed in bas‐

**Figure 8.** Immunohistochemical localization of PR in a sebaceous gland epithelioma. Nuclear staining is observed in

The influence of female sex steroids on their receptors in skin has been highlighted by sever‐ al studies. When an ovariectomised bitch received a local estradiol (E2) implant, ER levels in the affected skin were found to be six times higher than in control skin [119]. However, de‐ spite the considerable amount of work done to date in this field, further research is still re‐ quired to demonstrate the presence of AR and examine of the effects of these hormones and

Other tumours considered clearly hormone-dependent include those arising in the peria‐ nal or hepatoid glands, modified sebaceous glands located in the perianal dermis. The most frequently observed tumours of this region in dogs are perianal adenoma, perianal adenocarcinoma and apocrine gland adenocarcinoma of the anal sac. Perianal adenomas account for between 58% and 96% of all canine perianal tumours [120]. Unlike adeno‐ mas, perianal gland carcinomas are rare, accounting for between 3% and 17% of all per‐

al cells (arrow) and differentiated sebocytes (arrowhead) [117]. ABC. 40X.

basal cells (arrow) and differentiated sebocytes (arrowhead) [117]. ABC. 40X.

their pharmacological antagonists on tumour development.

neoplasms.

202 Insights from Veterinary Medicine

**Figure 9.** Immunohistochemical localization of ERα in a hepatoid gland adenoma. Nuclear staining is observed in bas‐ al cells (arrow) and differentiated hepatoid cells (arrowhead). ABC. 20X.

All these observations support the view that therapy based on antagonists of these hor‐ mones could prove beneficial in the treatment of these tumours. The present authors have carried out a preliminary clinical trial in order to evaluate the effect of Deslorelin (Suprelor‐ inTM Virbac), a GnRH antagonist, on the clinical response of perianal gland adenomas [127]. This antagonist suppresses pituitary production of the hormones LH and FSH and of steroid sex hormones; its effect has been compared to that of surgical castration. The trial found that subcutaneous deslorelin implants induced complete remission in at least 50% of dogs and a partial response to treatment in a further 29%. This antagonist could thus be considered as a new option for the treatment of perianal gland adenomas.

[4] Kwapien RP, Giles RC, Geil RG, Casey HW: Malignant mammary tumors in beagle dogs dosed with investigational oral contraceptive steroids. Journal of the National

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205

[5] Giles RC, Kwapien RP, Geil RG, Casey HW. Mammary nodules in beagle dogs ad‐ ministered investigational oral contraceptive steroids. Journal of the National Cancer

[6] Misdorp W. Progestagens and mammary tumours in dogs and cats. Acta Endocrino‐

[7] Stovring M, Moe L, Glattre E. A population-based case-control study of canine mam‐ mary tumors and clinical use of medroxiprogesterone acetate. Acta Pathologica, mi‐

[9] Loretti AP, Ilha MR, Ordás J, Martín de las Mulas J. Clinical, pathological and immu‐ nohistochemical study of feline mammary fibroepithelial hyperplasia following a single injection of depot medroxyprogesterone acetate. Journal of Feline Medicine

[10] Mialot JP, Andre F, Martin PH, Cotard MP, Raynaud JP. Etude des recepteurs des hormones steroids dans les tummeurs mammaires de la chienne. I: mise en evidence, caracterisation et relation avec le type histologique. Recueil de Medicine Veterinaire

[11] Parodi AL, Misdorp W, Mialot JP, Mialot M, Hart AA, Hurtrel M, Salomon JC. Intra‐ tumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy. Cancer Immunology Immunotherapy 1983;15(3) 172-7.

[12] Martin PM, Cotard M, Mialot JP, André F, Raynaud JP. Animal models for hormonedependent human breast cancer. Relationship between steroid receptor profiles in ca‐ nine and feline mammary tumors and survival rate. Cancer Chemotherapy and

[13] Sartin EA, Barnes S, Kwapien RP, Wolfe LG. Estrogen and progesterone receptor sta‐ tus of mammary carcinomas and correlation with clinical outcome in dogs. American

[14] Manzel O, Wurm S, Ueberschär S, Hoppen H-O: Estrogen and Progesterone recep‐ tors in histologic sections of canine mammary tumours in correlation to histologic type: conference proceedings, September 28, 1995, Edinburgh, Unite Kindong, ECVP;

[15] Graham JC, O'Keefe DA, Gelberg HB. Immnunohistochemical assay for detecting es‐ trogen receptors in canine mammary tumors. Am J Vet Res 1999;60(5) 627-630.

crobiologica, et immunologica Scandinavica (APMIS) 1997;105 590-596.

[8] Allen HL. Feline mammary hypertrophy. Veterinary Pathology 1973;10 501-508.

Cancer Institute 1980; 65 137-144.

Institute 1978;60:1351-1364.

logica (Copenh) 1991;125 27-31.

and Surgery 2005;7(1) 43-52.

Pharmacolgy 1984;12(1) 13-17.

journal of Veterinary Researche 1992;53 2196-2200.

1982;158; 215-221.

1995.

Finally, the association between mast-cell tumours (MCTs) and steroid sex hormones re‐ mains controversial. The role of these hormones in tumour pathogenesis is poorly under‐ stood, although some evidence is now available: one study has detected cytosolic receptors for oestrogen and progesterone in canine MCTs [127], while another found that 6 out of 9 MCTs contained no ERα and 3 were questionable [128].

## **6. Conclusion**

Sex steroid hormones are involved in the development of animal tumours with high epide‐ miological and clinical impact. Research in the field has shown the potential benefits of en‐ docrine manipulations to control tumour progression in a neoadjuvant or adjuvant setting. From a comparative point of view, steroid hormone dependent animal tumors represent an accessible and natural model of human disease.

## **Author details**

Yolanda Millán\* , Silvia Guil-Luna, Carlos Reymundo, Raquel Sánchez-Céspedes and Juana Martín de las Mulas

\*Address all correspondence to: an2mirum@uco.es

Department of Comparative Pathology, University of Córdoba, Córdoba, Spain

Yolanda Millán and Silvia Guil-Luna are equal first authors

## **References**


[4] Kwapien RP, Giles RC, Geil RG, Casey HW: Malignant mammary tumors in beagle dogs dosed with investigational oral contraceptive steroids. Journal of the National Cancer Institute 1980; 65 137-144.

subcutaneous deslorelin implants induced complete remission in at least 50% of dogs and a partial response to treatment in a further 29%. This antagonist could thus be considered as a

Finally, the association between mast-cell tumours (MCTs) and steroid sex hormones re‐ mains controversial. The role of these hormones in tumour pathogenesis is poorly under‐ stood, although some evidence is now available: one study has detected cytosolic receptors for oestrogen and progesterone in canine MCTs [127], while another found that 6 out of 9

Sex steroid hormones are involved in the development of animal tumours with high epide‐ miological and clinical impact. Research in the field has shown the potential benefits of en‐ docrine manipulations to control tumour progression in a neoadjuvant or adjuvant setting. From a comparative point of view, steroid hormone dependent animal tumors represent an

, Silvia Guil-Luna, Carlos Reymundo, Raquel Sánchez-Céspedes and

[1] Ahern TE, Bird RC, Church Bird AE, Wolfe LG: Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines. American Journal of Vet‐

[2] Dorn CR, Don T, Scheneider R, Hibbard HH. Survey of animal neoplasms in Alame‐ da and Contra Costa Counties, Calif.:II. Cancer morbidity in dogs and cats from Ala‐

[3] Schneider R, Dorn CR, Taylor D. Factors influencing canine mammary tumor devel‐ opment and postsurgical survival. Journal of the National Cancer Institute 1969;43

meda County. Journal of the National Cancer Institute 1968;40 307-318.

Department of Comparative Pathology, University of Córdoba, Córdoba, Spain

new option for the treatment of perianal gland adenomas.

MCTs contained no ERα and 3 were questionable [128].

accessible and natural model of human disease.

\*Address all correspondence to: an2mirum@uco.es

erinary Research 1996;57 693-696.

Yolanda Millán and Silvia Guil-Luna are equal first authors

**6. Conclusion**

204 Insights from Veterinary Medicine

**Author details**

Yolanda Millán\*

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Juana Martín de las Mulas


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**Chapter 8**

**Feline Mammary Fibroepithelial Hyperplasia:**

Feline mammary masses are frequently suspected of being mammary tumours. Immediate attention is required as over 80% of mammary tumours in cats are malignant [1,2], albeit mammary masses in cats are less common than in dogs. However, prevalence of mammary tumours is highly variable with the geographic region, as it tends to be lower in areas where most cats are neutered at a young age. Due to the negative prognosis generally attributed to feline mammary tumours, little attention has been paid to benign mammary growths and mastectomy is still often performed to deal with feline mammary fibroepithelial hyperplasia.

Feline mammary fibroepithelial hyperplasia represents a benign, progesterone-associated fibroglandular proliferation of one or more mammary glands that may occur in both the female and male cat [3,4]. It is also named feline hypertrophy, fibroadenomatous changes, mammary

Feline mammary fibroepithelial hyperplasia (FEH) is characterized by the sudden onset of mammary swollen within a short period of 2 to 5 weeks, frequently concerning several mammary glands. When exuberant it is often at the origin of the consultation [6-9]. Ulceration and abscessation of the mammary gland may occur due to gland enlargement and trauma, in

Feline mammary fibroepithelial hyperplasia is considered to be a benign condition, yet its behaviour and gross appearance is similar to mammary neoplasic lesions, in particular when solitary ulcerated or violaceous lesions are present. Although it rapid growth may cause concern, fibroepithelial mammary lesions are reversible, and the volume of the mammary masses tend to decrease after luteolysis or at the end of exogenous progesta‐

> © 2013 Payan-Carreira; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Payan-Carreira; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**A Clinical Approach**

Additional information is available at the end of the chapter

hyperplasia or fibroadenoma complex [3-5].

chronic situations [9,10].

gen activity [4,5,11].

Rita Payan-Carreira

**1. Introduction**

http://dx.doi.org/10.5772/55550


## **Feline Mammary Fibroepithelial Hyperplasia: A Clinical Approach**

Rita Payan-Carreira

[119] Eigenmann JE, Poortman J, Koeman JP. Estrogen induced flank alopecia in the fe‐ male dog, evidence for local rather than systemic hyperoestrogenism. Journal of the

[120] Nielsen SW, Aftosmis J. Canine Perianal Gland Tumors. Journal of American Veteri‐

[121] Vail DM, Withrow SJ, Schwarz PD, Powers BE. Perianal adenocarcinomas in the ca‐ nine male: a restrospective study of 41 cases. Journal of American Animal Hopital

[122] Turek, MM., & Withrow, SJ. (2001). Perineal tumors, In: Vail DM.,Withrow SJ (eds.) Small Animal Clinical Oncology. Philadelphia, WB Saunders; 2001. P503-510.

[123] Isitor GN, Weinman DE. Origin and early development of canine circumanal glands.

[124] Wilson GP, Hayes HM. Castration for treatment of perianal gland neoplasms in the dog. Journal of American Veterinary Medicine Association 1979; 174 (12) 1301-1303.

[125] Hayes HM, Wilson GP. Hormone-dependent neoplasms of the canine perianal gland.

[126] Pisani G, Millanta F, Lorenzi D, Vannozzi I, Poli A. Androgen receptor expression in normal, hyperplastic and neoplastic hepatoid glands in the dog. Research in Veteri‐

[127] Elling H, Ungemach FR. Sexual hormone receptors in canine mast cell tumour cyto‐

[128] Larsen AE, Grier RL. Evaluation of canine mast cell tumors for presence of estrogen

receptors. American Journal of Veterinary Research 1989; 50 1779-1780.

American Animal Hospital Association 1984; 20 (1) 621-624.

American Journal of Veterinary Research 1979; 40 (4) 487-492.

sol. Journal of Comparative Pathology 1982; 92 629-630.

nary Medical Association 1964; 144(15) 127-135.

Association 1990; 26(3) 329-334.

214 Insights from Veterinary Medicine

Cancer Research 1977; 37 (1) 2068-2071.

nary Science 2006; 81 231-236.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55550

## **1. Introduction**

Feline mammary masses are frequently suspected of being mammary tumours. Immediate attention is required as over 80% of mammary tumours in cats are malignant [1,2], albeit mammary masses in cats are less common than in dogs. However, prevalence of mammary tumours is highly variable with the geographic region, as it tends to be lower in areas where most cats are neutered at a young age. Due to the negative prognosis generally attributed to feline mammary tumours, little attention has been paid to benign mammary growths and mastectomy is still often performed to deal with feline mammary fibroepithelial hyperplasia.

Feline mammary fibroepithelial hyperplasia represents a benign, progesterone-associated fibroglandular proliferation of one or more mammary glands that may occur in both the female and male cat [3,4]. It is also named feline hypertrophy, fibroadenomatous changes, mammary hyperplasia or fibroadenoma complex [3-5].

Feline mammary fibroepithelial hyperplasia (FEH) is characterized by the sudden onset of mammary swollen within a short period of 2 to 5 weeks, frequently concerning several mammary glands. When exuberant it is often at the origin of the consultation [6-9]. Ulceration and abscessation of the mammary gland may occur due to gland enlargement and trauma, in chronic situations [9,10].

Feline mammary fibroepithelial hyperplasia is considered to be a benign condition, yet its behaviour and gross appearance is similar to mammary neoplasic lesions, in particular when solitary ulcerated or violaceous lesions are present. Although it rapid growth may cause concern, fibroepithelial mammary lesions are reversible, and the volume of the mammary masses tend to decrease after luteolysis or at the end of exogenous progesta‐ gen activity [4,5,11].

© 2013 Payan-Carreira; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Payan-Carreira; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Tentative diagnosis of mammary fibroepithelial hyperplasia should be based on the gross appearance of the lesions and on the history despite that most frequently, historic information is limited or incomplete, as a previous occurring estrus is seldom detected. Thus, diagnosis of feline mammary fibroepithelial hyperplasia is a clinical issue, and is not difficult to be established when all the mammary glands show a rapid enlargement, independently of the size of the swollen mammary gland [4,9]. This diagnosis may be further supported by the raised blood progesterone levels or by reported recent proges‐ tin treatment. However, when fibroepithelial lesions develop in only one mammary gland, distinguish between hyperplasia and mammary tumour may become more challenging. Biopsies or excision of the mammary lesions are frequently performed. Nevertheless, differential diagnosis with mammary carcinomas has to be carefully established, as around 85% of all mammary neoplasias are malignant [9].

at presentation in case of mammary neoplasia, which is middle-aged queens, since the risk for

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217

Moreover, few reports exist on the occurrence of FEH in males under treatment with antian‐ drogenic drugs, such as delmadinon acetate (Meisl et al., cited in [8]) and cyproterone acetate [12], frequently used by cat fanciers for eliminating the urine spraying in intact adult tomcats. Infrequently, descriptions of FEH in spayed queens or male cats supposedly not submitted to steroid treatment have been published [11,23], but the doubt remained on the absence of an

It is generally accepted that the incidence of this disturbance may reach up to 20% of the mammary masses detected in cats, its prevalence varying with the country or the region, which reflect cultural differences in the reproductive management of domestic and freeroaming felids. In a study developed in the north of Portugal, based on the excisional material sent for histopathology analysis, the mammary fibroepithelial hyperplasia reach 13% of the feline mammary masses [21]. Nevertheless, according to our experience, incidence of FEH seems to be in regression among the group of animals submitted to progesterone-based contraception, may be due to the fact that most contraceptive treat‐ ments are now based in oral, veterinary drugs (such as Megescat®) instead of human design depot products (like Depo-Provera®). Nevertheless, the medroxyprogesterone acetate and the megestrol acetate are the most frequently reported progestin associated to

Mammary enlargement is usually observed within 1-2 weeks after estrus or within 2-6 weeks

Apparently, no breed predisposition has been suggested for FEH. Even so, the majority of the cases were described in domestic shorthaired cats, which could simply be due to the fact that

The exact pathogenesis of FEH remains unclear, although sex steroid involvement has been acknowledged for long. Progesterone or its synthetic analogues have being recognized as being

The interaction between the activity of the mammary gland and the sex steroids is recognised for long. In brief, development and growth of the mammary gland is under the control of progesterone, which effects are mainly mediated through the progesterone receptor (PR) on stromal and epithelial cells [25]. Local activation of PR triggers a cascade of specific and sequential series of molecules, specific for each glandular element, which stimulates mammary gland proliferation. In physiological conditions, the cyclic changes between estrogens and progesterone stimulate or repress the cyclic activation of such PR-mediated pathways [25]. A decrease in PR levels is associated to a reduction of progesterone activity. Progesterone has

**3. Pathogenesis of the feline mammary fibroepithelial hyperplasia**

mammary tumour increase with the cat age, particularly at 10-12 years [1,22].

FEH, in particular when the drug is injected [13,14,18,24].

it may constitute the majority of the population worldwide.

at the origin of most of the FEH situations described.

involuntary hormonal treatment.

after hormone treatment.

As FEH is a non-neoplastic progesterone-associated disorder, it is possible for most situations to apply medical treatment. Administration of antiprogestins remains the elective medical treatment, and its schedule and duration is usually related to the severity of the problem at presentation. Complete excision of the mammary chain, under the supposition of a mammary tumour, may become a really aggressive surgery. Nowadays, with the available medical options mastectomy should be avoided in case of FEH.

Recurrence of the situation, although possible, remains controversial [6,11-13]. Nevertheless it is an important issue when discussing the therapeutic approach with owners. Ovariohys‐ terectomy remains an option in animals not intent to reproduction and in animals submitted to progesterone-based contraception, even if postponed until mammary glands regress into the normal size.

The objective of this work was to present and discuss the clinical approaches available to establish the diagnosis and the therapeutic options for feline mammary fibroepithelial hyperplasia. The final purpose in the diagnosis and treatment of such disease is not only to confirm that the clinical situation was correctly identified, but also to select the most suitable therapeutic approach to each patient, and also avoiding precipitate mastectomy and other complications of the surgical act, with the minimum repercussions on patients' welfare.

## **2. Epidemiology of the feline mammary fibroepithelial hyperplasia**

Feline mammary fibroepithelial hyperplasia occurs in intact queens of any age, in pregnant females and in female or male cats under progestin treatment [6-8,14]. It predominantly affects younger intact female cats, a segment of the population that also presents an increased ratio of spontaneous ovulation [15,16]. The reported age range for FEH is 6 months to 13 years [17-20]. Not so frequently, the condition may also be seen in aged females, associated or not with a contraceptive treatment, and sporadically in hormonally treated male tomcats. In a local study, the age range for FEH was 10 months to 10 years (the median for the age was 3 years), and the condition was exclusively diagnosed in queens [21]. This contrast with the usual age at presentation in case of mammary neoplasia, which is middle-aged queens, since the risk for mammary tumour increase with the cat age, particularly at 10-12 years [1,22].

Tentative diagnosis of mammary fibroepithelial hyperplasia should be based on the gross appearance of the lesions and on the history despite that most frequently, historic information is limited or incomplete, as a previous occurring estrus is seldom detected. Thus, diagnosis of feline mammary fibroepithelial hyperplasia is a clinical issue, and is not difficult to be established when all the mammary glands show a rapid enlargement, independently of the size of the swollen mammary gland [4,9]. This diagnosis may be further supported by the raised blood progesterone levels or by reported recent proges‐ tin treatment. However, when fibroepithelial lesions develop in only one mammary gland, distinguish between hyperplasia and mammary tumour may become more challenging. Biopsies or excision of the mammary lesions are frequently performed. Nevertheless, differential diagnosis with mammary carcinomas has to be carefully established, as around

As FEH is a non-neoplastic progesterone-associated disorder, it is possible for most situations to apply medical treatment. Administration of antiprogestins remains the elective medical treatment, and its schedule and duration is usually related to the severity of the problem at presentation. Complete excision of the mammary chain, under the supposition of a mammary tumour, may become a really aggressive surgery. Nowadays, with the available medical

Recurrence of the situation, although possible, remains controversial [6,11-13]. Nevertheless it is an important issue when discussing the therapeutic approach with owners. Ovariohys‐ terectomy remains an option in animals not intent to reproduction and in animals submitted to progesterone-based contraception, even if postponed until mammary glands regress into

The objective of this work was to present and discuss the clinical approaches available to establish the diagnosis and the therapeutic options for feline mammary fibroepithelial hyperplasia. The final purpose in the diagnosis and treatment of such disease is not only to confirm that the clinical situation was correctly identified, but also to select the most suitable therapeutic approach to each patient, and also avoiding precipitate mastectomy and other complications of the surgical act, with the minimum repercussions on patients' welfare.

**2. Epidemiology of the feline mammary fibroepithelial hyperplasia**

Feline mammary fibroepithelial hyperplasia occurs in intact queens of any age, in pregnant females and in female or male cats under progestin treatment [6-8,14]. It predominantly affects younger intact female cats, a segment of the population that also presents an increased ratio of spontaneous ovulation [15,16]. The reported age range for FEH is 6 months to 13 years [17-20]. Not so frequently, the condition may also be seen in aged females, associated or not with a contraceptive treatment, and sporadically in hormonally treated male tomcats. In a local study, the age range for FEH was 10 months to 10 years (the median for the age was 3 years), and the condition was exclusively diagnosed in queens [21]. This contrast with the usual age

85% of all mammary neoplasias are malignant [9].

options mastectomy should be avoided in case of FEH.

the normal size.

216 Insights from Veterinary Medicine

Moreover, few reports exist on the occurrence of FEH in males under treatment with antian‐ drogenic drugs, such as delmadinon acetate (Meisl et al., cited in [8]) and cyproterone acetate [12], frequently used by cat fanciers for eliminating the urine spraying in intact adult tomcats. Infrequently, descriptions of FEH in spayed queens or male cats supposedly not submitted to steroid treatment have been published [11,23], but the doubt remained on the absence of an involuntary hormonal treatment.

It is generally accepted that the incidence of this disturbance may reach up to 20% of the mammary masses detected in cats, its prevalence varying with the country or the region, which reflect cultural differences in the reproductive management of domestic and freeroaming felids. In a study developed in the north of Portugal, based on the excisional material sent for histopathology analysis, the mammary fibroepithelial hyperplasia reach 13% of the feline mammary masses [21]. Nevertheless, according to our experience, incidence of FEH seems to be in regression among the group of animals submitted to progesterone-based contraception, may be due to the fact that most contraceptive treat‐ ments are now based in oral, veterinary drugs (such as Megescat®) instead of human design depot products (like Depo-Provera®). Nevertheless, the medroxyprogesterone acetate and the megestrol acetate are the most frequently reported progestin associated to FEH, in particular when the drug is injected [13,14,18,24].

Mammary enlargement is usually observed within 1-2 weeks after estrus or within 2-6 weeks after hormone treatment.

Apparently, no breed predisposition has been suggested for FEH. Even so, the majority of the cases were described in domestic shorthaired cats, which could simply be due to the fact that it may constitute the majority of the population worldwide.

## **3. Pathogenesis of the feline mammary fibroepithelial hyperplasia**

The exact pathogenesis of FEH remains unclear, although sex steroid involvement has been acknowledged for long. Progesterone or its synthetic analogues have being recognized as being at the origin of most of the FEH situations described.

The interaction between the activity of the mammary gland and the sex steroids is recognised for long. In brief, development and growth of the mammary gland is under the control of progesterone, which effects are mainly mediated through the progesterone receptor (PR) on stromal and epithelial cells [25]. Local activation of PR triggers a cascade of specific and sequential series of molecules, specific for each glandular element, which stimulates mammary gland proliferation. In physiological conditions, the cyclic changes between estrogens and progesterone stimulate or repress the cyclic activation of such PR-mediated pathways [25]. A decrease in PR levels is associated to a reduction of progesterone activity. Progesterone has been reported as having a major role in mammary ductal branching [26,27], while estrogens acting via the ER have been associated to ductal elongation and bifurcation [27].

proliferation, with mean values similar to those of carcinomas in accordance with the results

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219

Despite that increase expression of PR has been found in the FEH lesions in comparison to normal diestrous mammary tissue samples, the blood levels for progesterone are usually within the normal levels for the species [6], although cats may present considerable variation in their progesterone blood levels [36]. This is suggestive that the disease would correspond

**4. Morphological and pathological features of mammary fibroepithelial**

Macroscopically, FEH lesions appear as firm, well-circumscribed but unencapsulated masses, that may present two types of macroscopic patterns: the solid type, of smooth-surfaced tissue with scant fluid; and the parenchymal, intraductal pattern, with fluid-filled spaces [18]. The two patterns can be combined in the same lesion, or one of them can predominate over the other. The cut surface is solid, diffusely white or grey-white and homogeneous [9,37,38]. Areas containing gelatinous material may be found, disposed as cleft-like spaces created by the enlarged ducts [21,37] (Figure 1). Although necrosis or ulceration are rare [17], they can be found in long lasting situations or whenever the reduction of the mammary gland swelling

**Figure 1.** Gross appearance of feline mammary fibroepithelial hyperplasia lesions. On the left, the cut surface of a for‐ malin-fixed lesion showing a solid pattern. On the right, the cut surface of a fresh lesion showing several cleft-like

Microscopically, the two patterns are similar [18]: the diseased mammary gland is character‐ ized by the proliferation of glandular fibroepithelial elements. The lesions correspond to well-

to a disturbed, exaggerated response of the tissue to the circulating hormones.

of a previous investigation [35].

**hyperplasia lesions**

was attempted by progestin administration.

areas (arrow), typical of the intraductal pattern.

An aberrant regulation or those pathways may be at the origin of the disturbed response to the progesterone stimulation and contribute to the development of mammary gland hyper‐ plastic or neoplastic growth. It is possible that such response may be associated to two factors: the extreme sensitivity of the feline mammary gland to sex steroids action, as referred in older studies (Bässler, cited in [17]); and the fact that the mammary gland is usually very thin when non-pregnant or non-lactating [28].

In a recent study, the two progesterone receptors (PR) isoforms (A and B) have been evidenced in tissue samples from fibroepithelial hyperplasia lesions, with predominant expression in the ductal epithelium. It was also reported a higher expression of PR in the stroma of FEH lesions in comparison to those found in stroma from mammary carcinomas [29]. The presence of estrogen receptors (ER) in FEH lesions remains a subject of controversy, as the number of cases where ER has been detected varies along the reports [20,30,31]. Nevertheless, a slight reduction in ER expression seems to accompany the process. Expression of PR in a progesterone-target tissue is dependant of the previous stimulation by estrogens via ER, while progesterone effects also include the down-regulation of estrogen receptors. So it is also possible that the length of progesterone dominance or the circulating levels of progesterone may influence the amount of ER found in mammary tissue in the available studies.

The potential role of estrogens in the development of fibroepithelial hyperplasia needs clarification, as it may influence the relationship between the progesterone and estrogen receptors in mammary gland tissue. Further, in one recent study the concentrations of estradiol in animals suffering from FEH were higher than values typical for the luteal phase, both in case of the first appearance of fibroepithelial hyperplasia and in recurrences [13].

Progestagens (progesterone and synthetic progestins) influences on feline mammary glands result in the stimulation of the cellular proliferation through PR stimulation. It was proposed that binding of progestagens to PR would enhancement the local GH expression [29,32]. The GH presents a mitogenic action, which is mediated by insulin-like growth factor-1 (IGF-1) [32,33], a molecule shown to possess a strong mitogenic and anti-apoptotic effect on the mammary epithelial cells. The increased expression of GH, GH receptor and IGF-I was demonstrated in FEH lesions [29].

For fibroadenomatous hyperplasia associated to the cyproterone acetate administration it was found that this drug may present a "gestagenic" effect, which it was suggested to contribute to the development of fibroepithelial hyperplasia of mammary glands [12].

Comparative studies of the proliferative index (measured by Ki67/MIB1 expression) in feline fibroepithelial hyperplasia and other mammary tumours showed that, in spite of being a benign disturbance, it shows a very high proliferative index similar to the one observed in invasive mammary carcinomas [34,35]. Fibroepithelial hyperplasia, which exhibits unique morphological and biological features, is characterised by rapid proliferation of epithelium and stroma [35]. Regardless its classification as a hyperplastic lesion, with a favourable biological behaviour, all cases of fibroepithelial hyperplasia exhibited high rates of cell proliferation, with mean values similar to those of carcinomas in accordance with the results of a previous investigation [35].

been reported as having a major role in mammary ductal branching [26,27], while estrogens

An aberrant regulation or those pathways may be at the origin of the disturbed response to the progesterone stimulation and contribute to the development of mammary gland hyper‐ plastic or neoplastic growth. It is possible that such response may be associated to two factors: the extreme sensitivity of the feline mammary gland to sex steroids action, as referred in older studies (Bässler, cited in [17]); and the fact that the mammary gland is usually very thin when

In a recent study, the two progesterone receptors (PR) isoforms (A and B) have been evidenced in tissue samples from fibroepithelial hyperplasia lesions, with predominant expression in the ductal epithelium. It was also reported a higher expression of PR in the stroma of FEH lesions in comparison to those found in stroma from mammary carcinomas [29]. The presence of estrogen receptors (ER) in FEH lesions remains a subject of controversy, as the number of cases where ER has been detected varies along the reports [20,30,31]. Nevertheless, a slight reduction in ER expression seems to accompany the process. Expression of PR in a progesterone-target tissue is dependant of the previous stimulation by estrogens via ER, while progesterone effects also include the down-regulation of estrogen receptors. So it is also possible that the length of progesterone dominance or the circulating levels of progesterone may influence the amount

The potential role of estrogens in the development of fibroepithelial hyperplasia needs clarification, as it may influence the relationship between the progesterone and estrogen receptors in mammary gland tissue. Further, in one recent study the concentrations of estradiol in animals suffering from FEH were higher than values typical for the luteal phase, both in

Progestagens (progesterone and synthetic progestins) influences on feline mammary glands result in the stimulation of the cellular proliferation through PR stimulation. It was proposed that binding of progestagens to PR would enhancement the local GH expression [29,32]. The GH presents a mitogenic action, which is mediated by insulin-like growth factor-1 (IGF-1) [32,33], a molecule shown to possess a strong mitogenic and anti-apoptotic effect on the mammary epithelial cells. The increased expression of GH, GH receptor and IGF-I was

For fibroadenomatous hyperplasia associated to the cyproterone acetate administration it was found that this drug may present a "gestagenic" effect, which it was suggested to contribute

Comparative studies of the proliferative index (measured by Ki67/MIB1 expression) in feline fibroepithelial hyperplasia and other mammary tumours showed that, in spite of being a benign disturbance, it shows a very high proliferative index similar to the one observed in invasive mammary carcinomas [34,35]. Fibroepithelial hyperplasia, which exhibits unique morphological and biological features, is characterised by rapid proliferation of epithelium and stroma [35]. Regardless its classification as a hyperplastic lesion, with a favourable biological behaviour, all cases of fibroepithelial hyperplasia exhibited high rates of cell

case of the first appearance of fibroepithelial hyperplasia and in recurrences [13].

to the development of fibroepithelial hyperplasia of mammary glands [12].

acting via the ER have been associated to ductal elongation and bifurcation [27].

non-pregnant or non-lactating [28].

218 Insights from Veterinary Medicine

demonstrated in FEH lesions [29].

of ER found in mammary tissue in the available studies.

Despite that increase expression of PR has been found in the FEH lesions in comparison to normal diestrous mammary tissue samples, the blood levels for progesterone are usually within the normal levels for the species [6], although cats may present considerable variation in their progesterone blood levels [36]. This is suggestive that the disease would correspond to a disturbed, exaggerated response of the tissue to the circulating hormones.

## **4. Morphological and pathological features of mammary fibroepithelial hyperplasia lesions**

Macroscopically, FEH lesions appear as firm, well-circumscribed but unencapsulated masses, that may present two types of macroscopic patterns: the solid type, of smooth-surfaced tissue with scant fluid; and the parenchymal, intraductal pattern, with fluid-filled spaces [18]. The two patterns can be combined in the same lesion, or one of them can predominate over the other. The cut surface is solid, diffusely white or grey-white and homogeneous [9,37,38]. Areas containing gelatinous material may be found, disposed as cleft-like spaces created by the enlarged ducts [21,37] (Figure 1). Although necrosis or ulceration are rare [17], they can be found in long lasting situations or whenever the reduction of the mammary gland swelling was attempted by progestin administration.

**Figure 1.** Gross appearance of feline mammary fibroepithelial hyperplasia lesions. On the left, the cut surface of a for‐ malin-fixed lesion showing a solid pattern. On the right, the cut surface of a fresh lesion showing several cleft-like areas (arrow), typical of the intraductal pattern.

Microscopically, the two patterns are similar [18]: the diseased mammary gland is character‐ ized by the proliferation of glandular fibroepithelial elements. The lesions correspond to welldemarcated, non-encapsulated growths within the mammary gland [17,31], with the ducts forming pseudo-acinar or cystic structures, encircled by a loose, myxoid stroma [21]. Although the proportions of epithelial and connective tissue are variable with the lesion and distinct from the one found in the normal gland, the branched ducts and stroma retains its organization in lobular-like units. The branching ductal structures are lined by several layers of epithelial cells and surrounded by markedly proliferating and oedematous connective tissue. Loose periductal connective tissue, that gives higher prominence to the mammary stroma, is loosetextured and merged in the periphery to the more dense collagenous tissue that separates the mammary lobules [9,17,31,37,38]. Mitotic figures are commonly found both in the epithelium and the stroma [17,31], and apocrine differentiation is frequently found within the epithelial component. Further, it is often observed that the cells in the intralobular stroma lack polarity and show indistinct borders [17], and also some degree of cytological atypia, which is reactive [21]. Thereby, a falsely malignant appearance is created, which could be patent in the results for a fine needle aspiration biopsy. An inflammatory infiltrate is seldom found, and when present it is mostly of the lymphoplasmocitary type [21].

may induce some distress with movement or reluctance to walk and a reduction of appetite.

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221

Whatever the dimensions of the mammary glands, when FEH develops in pregnant females, no milk is produced in the diseased glands [23]. Consequently, after parturition, kittens are unable to nurse satisfactorily and usually the owners refer to litter vocalisation, restless and

In some severe or prolonged situations, the primary FEH may co-exist with mastitis or ulceration (Figure 2). In our clinic, mastitis is more frequently found in lactating females suffering from FEH. However, ulceration may develop secondary to perfusion problems derived from skin overstretching, with local ischemia, leading to abscessation, or also due to excessive grooming. Ulceration predisposes the diseased gland to mastitis or abscessation and subsequently to systemic illness [10-12]. In such situations, depending on the severity of the process, the skin may be wet, exudative, haemorrhagic and abnormal glandular discharge, with necrotic debris or purulent. Involvement of the regional lymph nodes is possible [10]. Then, the animal may be presented to consultation with fever, lethargy, anorexia, pale mucous

Little information is available on the haematological and blood biochemistry changes in animals suffering from FEH. Nevertheless, in animals suffering from non-complicated mammary fibroepithelial hyperplasia, most parameters analysed (blood haematology and biochemistry) were within the normal range values for the species [11,14]. In animals with FEH co-existing with mastitis and ulceration, it can be found anaemia [10,39], normal to increase packed-cell volume [12,13,39] and the leukocyte count near the maximum normal limit or increased [10,12,39]. All these changes have been associated to inflammation and/or seques‐

On what concerns the blood biochemistry, for most cases the values for blood urea and creatinine, or for the hepatic enzymes (such as the alkaline phosphatase, the alanine amino‐ transferase and the aspartate aminotransferase) were within the normal limits for cats [11-13],

Diagnosis of feline mammary fibroepithelial hyperplasia is always a clinical issue, and should be based on the symptoms, the patient signalment and on history [39,40]. Differential diagno‐ ses should include the mammary tumours (adenocarcinoma or carcinoma, mammary adeno‐

Usually it is not difficult to establish a diagnosis when multiple glands are enlarged. An important criterion is the rapid onset of the mammary swelling, independently of the size of the swollen mammary gland. Also the age of the female may be suggestive of FEH, as it is

ma, or mammary sarcoma) and the mammary fibroepithelial hyperplasia.

tration of fluid within the distended mammary tissue or to patient dehydration.

Less severe lesions usually evolve in the absence of an inflammatory reaction.

fading, with offspring death over a short-time period in postpartum.

membranes and dehydration.

or slightly decreased [10].

**6. Diagnostic evaluation**

**6.1. Reaching a tentative diagnosis**

## **5. Clinical presentation**

Usually, the main complaint for FEH is the existence of excessive mammary enlargement that evolved rapidly. This in fact characterises the disease.

Feline mammary glands thickness is minimal in cycling females, and also it does not change much until close to parturition [28]. Consequently, for most cases an increase of the volume of the mammary glands, either isolated or multiple, in otherwise clinically healthy animals, draws the attention of the cat owner. Time since the beginning of the mammary enlargement till the animal presentation seems to vary with the form of the FEH. It tends to be shorter in cases of more notorious swelling of multiple glands and may be longer in cases of solitary and smaller lesions.

The major clinical sign is the swollen, firm mammary gland tissue, that can be detected in as multiple, bilateral enlargement of the mammary chains, or develop as a solitary, unique lesion that may develop from any of the mammary glands (Figure 2). The size of the enlarged glands isquitevariable,rangingfrom1.5to18cm[21].Inourexperience,whenmultiplelesionsdevelop, asymmetrical lesions are more frequently found in non-pregnant females, while females being pregnant tend to develop more homogeneous swellings of the mammary glands (Figure 2).

At the visual inspection, the skin covering the diseased mammary glands may be tense and erythematous, in particular in larger lesions. The nipples can be difficult to find due to the size of the gland. At palpation, the lesions are presented as diffuse, firm and consistent masses, or in some cases they present a soft and more gelatinous, floating consistency. If notorious swelling develops, the diseased mammary glands may become pendulous. In uncomplicated situations and unless the masses are too swollen, the lesions are not painful, although some distress may be elicit during mammary manipulation during the clinical examination. Further, when severe swelling of the mammary glands developed, locomotory problems may arise that may induce some distress with movement or reluctance to walk and a reduction of appetite. Less severe lesions usually evolve in the absence of an inflammatory reaction.

Whatever the dimensions of the mammary glands, when FEH develops in pregnant females, no milk is produced in the diseased glands [23]. Consequently, after parturition, kittens are unable to nurse satisfactorily and usually the owners refer to litter vocalisation, restless and fading, with offspring death over a short-time period in postpartum.

In some severe or prolonged situations, the primary FEH may co-exist with mastitis or ulceration (Figure 2). In our clinic, mastitis is more frequently found in lactating females suffering from FEH. However, ulceration may develop secondary to perfusion problems derived from skin overstretching, with local ischemia, leading to abscessation, or also due to excessive grooming. Ulceration predisposes the diseased gland to mastitis or abscessation and subsequently to systemic illness [10-12]. In such situations, depending on the severity of the process, the skin may be wet, exudative, haemorrhagic and abnormal glandular discharge, with necrotic debris or purulent. Involvement of the regional lymph nodes is possible [10]. Then, the animal may be presented to consultation with fever, lethargy, anorexia, pale mucous membranes and dehydration.

Little information is available on the haematological and blood biochemistry changes in animals suffering from FEH. Nevertheless, in animals suffering from non-complicated mammary fibroepithelial hyperplasia, most parameters analysed (blood haematology and biochemistry) were within the normal range values for the species [11,14]. In animals with FEH co-existing with mastitis and ulceration, it can be found anaemia [10,39], normal to increase packed-cell volume [12,13,39] and the leukocyte count near the maximum normal limit or increased [10,12,39]. All these changes have been associated to inflammation and/or seques‐ tration of fluid within the distended mammary tissue or to patient dehydration.

On what concerns the blood biochemistry, for most cases the values for blood urea and creatinine, or for the hepatic enzymes (such as the alkaline phosphatase, the alanine amino‐ transferase and the aspartate aminotransferase) were within the normal limits for cats [11-13], or slightly decreased [10].

## **6. Diagnostic evaluation**

demarcated, non-encapsulated growths within the mammary gland [17,31], with the ducts forming pseudo-acinar or cystic structures, encircled by a loose, myxoid stroma [21]. Although the proportions of epithelial and connective tissue are variable with the lesion and distinct from the one found in the normal gland, the branched ducts and stroma retains its organization in lobular-like units. The branching ductal structures are lined by several layers of epithelial cells and surrounded by markedly proliferating and oedematous connective tissue. Loose periductal connective tissue, that gives higher prominence to the mammary stroma, is loosetextured and merged in the periphery to the more dense collagenous tissue that separates the mammary lobules [9,17,31,37,38]. Mitotic figures are commonly found both in the epithelium and the stroma [17,31], and apocrine differentiation is frequently found within the epithelial component. Further, it is often observed that the cells in the intralobular stroma lack polarity and show indistinct borders [17], and also some degree of cytological atypia, which is reactive [21]. Thereby, a falsely malignant appearance is created, which could be patent in the results for a fine needle aspiration biopsy. An inflammatory infiltrate is seldom found, and when

Usually, the main complaint for FEH is the existence of excessive mammary enlargement that

Feline mammary glands thickness is minimal in cycling females, and also it does not change much until close to parturition [28]. Consequently, for most cases an increase of the volume of the mammary glands, either isolated or multiple, in otherwise clinically healthy animals, draws the attention of the cat owner. Time since the beginning of the mammary enlargement till the animal presentation seems to vary with the form of the FEH. It tends to be shorter in cases of more notorious swelling of multiple glands and may be longer in cases of solitary and

The major clinical sign is the swollen, firm mammary gland tissue, that can be detected in as multiple, bilateral enlargement of the mammary chains, or develop as a solitary, unique lesion that may develop from any of the mammary glands (Figure 2). The size of the enlarged glands isquitevariable,rangingfrom1.5to18cm[21].Inourexperience,whenmultiplelesionsdevelop, asymmetrical lesions are more frequently found in non-pregnant females, while females being pregnant tend to develop more homogeneous swellings of the mammary glands (Figure 2).

At the visual inspection, the skin covering the diseased mammary glands may be tense and erythematous, in particular in larger lesions. The nipples can be difficult to find due to the size of the gland. At palpation, the lesions are presented as diffuse, firm and consistent masses, or in some cases they present a soft and more gelatinous, floating consistency. If notorious swelling develops, the diseased mammary glands may become pendulous. In uncomplicated situations and unless the masses are too swollen, the lesions are not painful, although some distress may be elicit during mammary manipulation during the clinical examination. Further, when severe swelling of the mammary glands developed, locomotory problems may arise that

present it is mostly of the lymphoplasmocitary type [21].

evolved rapidly. This in fact characterises the disease.

**5. Clinical presentation**

220 Insights from Veterinary Medicine

smaller lesions.

#### **6.1. Reaching a tentative diagnosis**

Diagnosis of feline mammary fibroepithelial hyperplasia is always a clinical issue, and should be based on the symptoms, the patient signalment and on history [39,40]. Differential diagno‐ ses should include the mammary tumours (adenocarcinoma or carcinoma, mammary adeno‐ ma, or mammary sarcoma) and the mammary fibroepithelial hyperplasia.

Usually it is not difficult to establish a diagnosis when multiple glands are enlarged. An important criterion is the rapid onset of the mammary swelling, independently of the size of the swollen mammary gland. Also the age of the female may be suggestive of FEH, as it is

Although it should not be considered as a rule, frequently FEH solitary lesions reach larger

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Discoloration of the skin underlying FEH lesions was once reported [41], but I was not able to

Occurrence of FEH indicates that the animal ovulated and endogenous progesterone is raised or that it was treated with progestins. Hence, the mammary fibroepithelial hyperplasia diagnosis may be further supported by determination of blood progesterone levels. However, one should be aware that progesterone levels may be low when the underlying cause are exogenous progestins, because nowadays progesterone analysis are quite specific and may not cross-label with the used progestin molecule. Thus, it is also of utmost importance to determine

Biopsies are often referred as being the most acceptable form to confirm the diagnosis of mammary fibroepithelial hyperplasia. However, cytological differentiation between benign and malignant mammary lesions is difficult. The accuracy of cytological differentiation is low, and its specificity has not yet been attributable. Further, the cytological analysis should be interpreted together with the symptoms and the sudden onset of the clinical signs [11].

It should be remembered that mammary fibroepithelial hyperplasia lesions are highly proliferative [34,35] and that some degree of cytological atypia [21] are often described, which along with the described loss of cell polarity [17] and the occurrence of mitosis [17,31], can create a falsely malignant appearance that could biased the diagnosis. Consequently, if a histopathological diagnosis is wanted, an excisional biopsy is preferable to a fine needle

Diagnosis of FEH in cytological specimens should meet the following criteria: Two different cells (one of uniform epithelial cells and one of spindle-shaped mesenchymal cells) should coexist, and may display a moderate anisocytosis and anisokaryosis, with only minimal nuclear criteria of malignancy. A large amount of eosinophilic extracellular matrix is expectably found

Mammary ultrasonography may also be helpful on the diagnosis of feline mammary fibroe‐ pithelial hyperplasia. Furthermore it is a rapid and easily performed method for assessment of the mammary gland structure. Generally, the ultrasonographic mammary echogenicity is higher in FEH lesions when compared to normal and lactational feline mammary glands (Figure 3). On ultrasound images, FEH lesions present mainly as a well-circumscribed solid mass of granular, slightly hyperechoic texture, with regularly delimited margins. It is also common to found small cleft-like structures, appearing as irregular anechoic areas, without acoustic enhancement, and small hyperechoic foci scattered within the glands image, which are independent of the form of FEH (multiple or solitary form). The presence of clefts in mammary fibroepithelial lesions provided a more heterogeneous appearance to the ultra‐ sound images. In our practice clefts are more frequently found in animals under progestin treatment. The ultrasound pattern is more homogeneous in solid lesions, whilst when the intraductal pattern dominates, anechoic areas corresponding to clefts of different shapes are

volumes than those referred to feline mammary tumours [22], and are softener.

confirm that association in my practice.

the existence of a recent progestin treatment.

aspiration, despite being more expensive.

in close proximity to the cells (Mesher, cited in [11]).

found within the mammary gland parenchyma (Figure 4).

**6.2. Diagnostic endorsements**

**Figure 2.** Diverse aspects of feline mammary fibroepithelial hyperplasia. A – A solitary lesion in a female cat submitted to megestrol acetate treatment. B - Multiple lesions showing asymmetric distribution of the diseased mammary glands, which also presented different dimensions, in a young spontaneous-ovulatory queen. C – In larger lesions, the skin around the nipple may be stretched, moist and violet, due to excessive grooming. D – FEH complicated with mas‐ titis and ulceration in a female at post-partum day 6. E – FEH in a peri-partum young female that also showed skin erosion around the nipples of the caudal mammae.

more frequently found in young females. The sex of the animal should not be an exclusion criterion, as FEH also develop in males submitted to hormonal treatment for urine spraying or skin conditions. Neither it should be the reproductive status of the cat, as FEH can develop in neutered cats with pyoderma or miliary dermatitis following hormonal treatment with progestins. Moreover, a rapid enlargement of the mammary chains in a early or mid-pregnant females should lead to the suspicion of FEH, as the mammary glands shows little development until near parturition in cats. Unless an excessive swelling of the mammary exits, FEH is painless.

Yet, when fibroepithelial lesions develop in a single mammary gland, distinguishing between hyperplasia and mammary tumour may become more challenging, particularly in mature or older animals. As in other FEH conditions, the lesion develops at a very rapid rate, is frequently painless and although firm it is also turgid with a regular oedematous texture at palpation. Although it should not be considered as a rule, frequently FEH solitary lesions reach larger volumes than those referred to feline mammary tumours [22], and are softener.

Discoloration of the skin underlying FEH lesions was once reported [41], but I was not able to confirm that association in my practice.

Occurrence of FEH indicates that the animal ovulated and endogenous progesterone is raised or that it was treated with progestins. Hence, the mammary fibroepithelial hyperplasia diagnosis may be further supported by determination of blood progesterone levels. However, one should be aware that progesterone levels may be low when the underlying cause are exogenous progestins, because nowadays progesterone analysis are quite specific and may not cross-label with the used progestin molecule. Thus, it is also of utmost importance to determine the existence of a recent progestin treatment.

#### **6.2. Diagnostic endorsements**

more frequently found in young females. The sex of the animal should not be an exclusion criterion, as FEH also develop in males submitted to hormonal treatment for urine spraying or skin conditions. Neither it should be the reproductive status of the cat, as FEH can develop in neutered cats with pyoderma or miliary dermatitis following hormonal treatment with progestins. Moreover, a rapid enlargement of the mammary chains in a early or mid-pregnant females should lead to the suspicion of FEH, as the mammary glands shows little development until near parturition in cats. Unless an excessive swelling of the mammary exits, FEH is

**Figure 2.** Diverse aspects of feline mammary fibroepithelial hyperplasia. A – A solitary lesion in a female cat submitted to megestrol acetate treatment. B - Multiple lesions showing asymmetric distribution of the diseased mammary glands, which also presented different dimensions, in a young spontaneous-ovulatory queen. C – In larger lesions, the skin around the nipple may be stretched, moist and violet, due to excessive grooming. D – FEH complicated with mas‐ titis and ulceration in a female at post-partum day 6. E – FEH in a peri-partum young female that also showed skin

Yet, when fibroepithelial lesions develop in a single mammary gland, distinguishing between hyperplasia and mammary tumour may become more challenging, particularly in mature or older animals. As in other FEH conditions, the lesion develops at a very rapid rate, is frequently painless and although firm it is also turgid with a regular oedematous texture at palpation.

painless.

erosion around the nipples of the caudal mammae.

222 Insights from Veterinary Medicine

Biopsies are often referred as being the most acceptable form to confirm the diagnosis of mammary fibroepithelial hyperplasia. However, cytological differentiation between benign and malignant mammary lesions is difficult. The accuracy of cytological differentiation is low, and its specificity has not yet been attributable. Further, the cytological analysis should be interpreted together with the symptoms and the sudden onset of the clinical signs [11].

It should be remembered that mammary fibroepithelial hyperplasia lesions are highly proliferative [34,35] and that some degree of cytological atypia [21] are often described, which along with the described loss of cell polarity [17] and the occurrence of mitosis [17,31], can create a falsely malignant appearance that could biased the diagnosis. Consequently, if a histopathological diagnosis is wanted, an excisional biopsy is preferable to a fine needle aspiration, despite being more expensive.

Diagnosis of FEH in cytological specimens should meet the following criteria: Two different cells (one of uniform epithelial cells and one of spindle-shaped mesenchymal cells) should coexist, and may display a moderate anisocytosis and anisokaryosis, with only minimal nuclear criteria of malignancy. A large amount of eosinophilic extracellular matrix is expectably found in close proximity to the cells (Mesher, cited in [11]).

Mammary ultrasonography may also be helpful on the diagnosis of feline mammary fibroe‐ pithelial hyperplasia. Furthermore it is a rapid and easily performed method for assessment of the mammary gland structure. Generally, the ultrasonographic mammary echogenicity is higher in FEH lesions when compared to normal and lactational feline mammary glands (Figure 3). On ultrasound images, FEH lesions present mainly as a well-circumscribed solid mass of granular, slightly hyperechoic texture, with regularly delimited margins. It is also common to found small cleft-like structures, appearing as irregular anechoic areas, without acoustic enhancement, and small hyperechoic foci scattered within the glands image, which are independent of the form of FEH (multiple or solitary form). The presence of clefts in mammary fibroepithelial lesions provided a more heterogeneous appearance to the ultra‐ sound images. In our practice clefts are more frequently found in animals under progestin treatment. The ultrasound pattern is more homogeneous in solid lesions, whilst when the intraductal pattern dominates, anechoic areas corresponding to clefts of different shapes are found within the mammary gland parenchyma (Figure 4).

Radiology is of little interest in cases of FEH, as for most situations lateral abdominal surveys only shows the enlargement of the mammary glands, an intact body wall and sporadically homogeneous fluid opacity in the diseased mammary glands [10]. In comparison, ultrasonog‐ raphy can bring you more information through the assessment of the lesion echogenicity and pattern. However, when attempting to establish a differential diagnosis with mammary carcinoma, thoracic and abdominal radiographs are advised to screen for possible metastases and calcification.

response with a reduction of the mammary swelling and improvement of the clinical condition, which can be obtained around day 3 post-administration (for the doses and schedule, please

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In most animals diagnosed with FEH, the extent of the swelling of the mammary glands and the possibility of necrosis and infection warrant treatment, though this is generally considered as a benign disturbance [23]. Even so, seldom sporadic recovery is observed [6], and when

The feline mammary fibroepithelial hyperplasia being a progesterone-associated disturbance, the therapeutic approach should focus on the removal of the progesterone influences in order to revert the symptoms. Thus, discontinuing of any ongoing hormone therapy is mandatory. Available approaches should be discussed with the cat owner, including a prevision of the costs for the treatment, the time to full recovery and the possibility for the occurrence of a relapse. For most FEH situations 21-24 days may be needed to fully reversion of the mammary

In addition to the treatment directed to feline mammary fibroepithelial hyperplasia, situations complicated with mastitis and skin ulceration or abscessation or systemic illness, additional treatment targeting the recovery of the inflammatory condition and the stabilization of the patient may be needed. Adequate broad-spectrum antimicrobial treatment (with Amoxicillin-Clavulanic acid or Cephradine for example), or fluid replacement may be needed. Also, when pain or discomfort exists, short-time treatment with nonsteroidal anti-inflammatory drugs (such as Meloxicam, Ketoprofen or Carprofen) may be used to alleviate the symptoms.

Until the late 90´s decade, ovariectomy or ovariohysterectomy were considered the most suitable treatments [19]. The lateral surgical approach was preferable to the ventral to avoid the trauma of the mammary tissue. Excision of the ovaries usually leads to regression of the mammary tissue within three to four weeks, but in some situations regression was not

Mastectomy is discouraged as a first approach to the feline mammary fibroepithelial hyper‐ plasia. Only in animals not responding to spaying or to the medical treatment, partial or total mastectomy may be considered, but the surgery is difficult to perform because of the exten‐ siveness of the mammary glands. A radical mastectomy often leads to complications and is

Nowadays, medical therapeutic approaches are available in most countries. Economic constraints may influence the drug of choice, and this may influence the recovery time. Also,

only to be recommended when other options have failed.

gland enlargement, but it may vary with the selected therapeutic approach.

see next section).

**7. Therapeutic approaches**

**7.1. Surgical approaches**

**7.2. Medical approaches**

achieved [11,23].

described it usually take several weeks to months.

**Figure 3.** Ultrasound images of normal feline mammary gland in non-pregnant, late pregnant and lactating females (from left to right).

**Figure 4.** Ultrasound images of feline mammary fibroepithelial hyperplasia lesions. On the left, images from a solid pattern lesion. On the right, images from lesions presenting cleft-like anechoic areas, characteristics of the intraductal pattern.

Finally, confirmation of the tentative diagnosis can also be achieved through the response to Aglepristone treatment. Aglepristone as an antiprogesterone drug can elicit a positive response with a reduction of the mammary swelling and improvement of the clinical condition, which can be obtained around day 3 post-administration (for the doses and schedule, please see next section).

## **7. Therapeutic approaches**

Radiology is of little interest in cases of FEH, as for most situations lateral abdominal surveys only shows the enlargement of the mammary glands, an intact body wall and sporadically homogeneous fluid opacity in the diseased mammary glands [10]. In comparison, ultrasonog‐ raphy can bring you more information through the assessment of the lesion echogenicity and pattern. However, when attempting to establish a differential diagnosis with mammary carcinoma, thoracic and abdominal radiographs are advised to screen for possible metastases

**Figure 3.** Ultrasound images of normal feline mammary gland in non-pregnant, late pregnant and lactating females

**Figure 4.** Ultrasound images of feline mammary fibroepithelial hyperplasia lesions. On the left, images from a solid pattern lesion. On the right, images from lesions presenting cleft-like anechoic areas, characteristics of the intraductal pattern.

Finally, confirmation of the tentative diagnosis can also be achieved through the response to Aglepristone treatment. Aglepristone as an antiprogesterone drug can elicit a positive

and calcification.

224 Insights from Veterinary Medicine

(from left to right).

In most animals diagnosed with FEH, the extent of the swelling of the mammary glands and the possibility of necrosis and infection warrant treatment, though this is generally considered as a benign disturbance [23]. Even so, seldom sporadic recovery is observed [6], and when described it usually take several weeks to months.

The feline mammary fibroepithelial hyperplasia being a progesterone-associated disturbance, the therapeutic approach should focus on the removal of the progesterone influences in order to revert the symptoms. Thus, discontinuing of any ongoing hormone therapy is mandatory.

Available approaches should be discussed with the cat owner, including a prevision of the costs for the treatment, the time to full recovery and the possibility for the occurrence of a relapse. For most FEH situations 21-24 days may be needed to fully reversion of the mammary gland enlargement, but it may vary with the selected therapeutic approach.

In addition to the treatment directed to feline mammary fibroepithelial hyperplasia, situations complicated with mastitis and skin ulceration or abscessation or systemic illness, additional treatment targeting the recovery of the inflammatory condition and the stabilization of the patient may be needed. Adequate broad-spectrum antimicrobial treatment (with Amoxicillin-Clavulanic acid or Cephradine for example), or fluid replacement may be needed. Also, when pain or discomfort exists, short-time treatment with nonsteroidal anti-inflammatory drugs (such as Meloxicam, Ketoprofen or Carprofen) may be used to alleviate the symptoms.

### **7.1. Surgical approaches**

Until the late 90´s decade, ovariectomy or ovariohysterectomy were considered the most suitable treatments [19]. The lateral surgical approach was preferable to the ventral to avoid the trauma of the mammary tissue. Excision of the ovaries usually leads to regression of the mammary tissue within three to four weeks, but in some situations regression was not achieved [11,23].

Mastectomy is discouraged as a first approach to the feline mammary fibroepithelial hyper‐ plasia. Only in animals not responding to spaying or to the medical treatment, partial or total mastectomy may be considered, but the surgery is difficult to perform because of the exten‐ siveness of the mammary glands. A radical mastectomy often leads to complications and is only to be recommended when other options have failed.

#### **7.2. Medical approaches**

Nowadays, medical therapeutic approaches are available in most countries. Economic constraints may influence the drug of choice, and this may influence the recovery time. Also, when predicting the recovery time, one should be aware that FEH secondary to exogenous progestin would take longer to regress if antiprogesterone drugs are not selected.

Several studies demonstrated that the progesterone receptor blocker Aglepristone (Alizine®, Virbac, France) can successfully revert FEH [8,11,13,19,23]. Aglepristone is a molecule that competitively binds to the progesterone receptor without activating the hormone response cascade in target tissues. This drug binds to with a 9-fold affinity to progesterone receptor, and according to the manufacturer its residence time in the organism is of 6 days, if administered once in the dose of 20mg/kg or twice at 10mg/kg [23]. Although not licenced to be used in cats, this drug is commonly used to induce abortion or to treat pyometra in this species. By consequence, its application in cats is under the veterinarian responsibility.

Before starting the treatment with Aglepristone it is important to exclude pregnancy, as this drug may elicit abortion of a premature birth. When FEH develops in pregnant females it is mandatory that the therapeutic approaches are discussed with the cat owner in detail, and is important to mention that despite the mammary enlargement, the diseased glands will not produce milk and also that the kittens attempts to nurse may predispose to complications such as mastitis and ulceration, which will worsen the evolution of the primary condition.

are negative to prolactin [43]. Nevertheless, such drugs may be helpful when it is need to

**Table 1.** Administration regimens proposed for Aglepristone (Alizine®, Virbac, France) treatments in feline mammary

**Alizine® doses Treatment schedule References**

2 doses, 24h apart, for 4 consecutive weeks

0,66ml/kg/d corresponding to [23]

10mg/kg/d On days 1, 2 and 7 [40] 0,5ml/kg/d corresponding to

20mg/kg/d Once a week, for 4 consecutive weeks

Repeat at week intervals to full recovery [13] 4 to 5 consecutive days [19] 4 to 5 consecutive days and again on day 7 [8] On days 1, 2, 7, 14 and 21 [42]

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2 doses, 24h apart;

Cabergoline (Galastop®, Ceva Santé Animale, France) is a dopaminergic agonist that produces a selective and long-lasting inhibitory effect on prolactin secretion, which in turn may be helpful to supress lactation. In dogs and cats it also induces luteolysis, and consequently it may induces abortion. Cabergoline is used for interrupt lactation at a dose of 5µg/kg body weight, *per os* (PO), once daily for 5-7 consecutive days depending on the severity of the situation. It is also used for mastitis treatment. Its use was described in association with castration in a tomcat [11], or in association with aglepristone in an assumed pregnant young

Bromocriptine (Parlodel® is the most frequently used pharmacological presentation) is used in the veterinary practice less often than Cabergoline, as it was found to induce abnormal behavioural effects, such as limb flicks, head/body shakes, and hallucinatory-like behaviour as well as excessive grooming [45]. This drug can be used at the dose of 0.25mg/cat/day, PO,

Generally, the prognosis for uncomplicated feline mammary fibroepithelial hyperplasia is good. The co-existence of mastitis or ulceration may induce some concern, particularly when the situation was left untreated for a long period. In rare situations, abscess formation and

Spontaneous regression of the enlarged mammary glands after removal of the progesterone influences may occur, but it may take up to 11 months. Nevertheless, ovariohysterectomy or

for 5 to 7 days. Its use on FEH situations enrols the same concerns as for Cabergoline.

discontinue the queen lactation, in cases where FEH develops in lactating females.

queen [44].

0,33ml/kg/d corresponding to

0,33ml/kg/d corresponding to

10mg/kg/d

15mg/kg/day

fibroepithelial hyperplasia.

**8. Prognosis**

systemic illness worsen the prognosis.

Several therapeutic schedules have been described in the literature for Aglepristone in FEH (Table 1). Personally, I prefer to inject 10mg/kg of Aglepristone (Alizine®) on days 1 and 3, subcutaneously (SC), and to re-evaluate the situation a week later. If necessary, a second administration is performed following the same schedule. Rarely (only one situation in 25 cases treated with Alisine®) I needed to perform a third administration (again two doses 48h apart), in a female that was submitted to oral progestin treatment that started during estrus. Reduction of the mammary volume, in particular the mammary thickness, is the major parameter for assessment of the response to treatment. Mammary thickness can be assessed by ultrasonog‐ raphy. By using this schedule, it can be observed a slight reduction in the thickness of the disease mammary glands between days 1 and 3, which is predictive of the expected length of the treatment. For most cases, FEH recovery was obtained in 3 to 4 weeks, with only one situation (the one above mentioned) taking 6 weeks to obtain full regression of the mammary condition.

Varying with the reports, the a mean of 4 to 5 treatments (Table 1) are needed to recover from FEH [23,42], and full recovery was obtained in varying periods that last for 3 to 11 weeks [23].

Occasionally, short-term skin irritation at the site of injection has been reported [23], but it seldom originates a problem.

In some case descriptions, dopamine agonists such as Cabergoline and Bromocriptine were also used for FEH treatment [40]. Though these products were not licenced for cats in some countries, they are commonly used in the feline practice. Vomiting or anorexia are described as side effects in a small proportion of cases, as well as a slight depression of the blood pressure, although these symptoms tend to disappear with continued treatment. Nonetheless, its usefulness in the treatment of feline mammary fibroepithelial hyperplasia remain uncertain, as prolactin has not been described as one of the players in FEH pathogenesis and FEH lesions


**Table 1.** Administration regimens proposed for Aglepristone (Alizine®, Virbac, France) treatments in feline mammary fibroepithelial hyperplasia.

are negative to prolactin [43]. Nevertheless, such drugs may be helpful when it is need to discontinue the queen lactation, in cases where FEH develops in lactating females.

Cabergoline (Galastop®, Ceva Santé Animale, France) is a dopaminergic agonist that produces a selective and long-lasting inhibitory effect on prolactin secretion, which in turn may be helpful to supress lactation. In dogs and cats it also induces luteolysis, and consequently it may induces abortion. Cabergoline is used for interrupt lactation at a dose of 5µg/kg body weight, *per os* (PO), once daily for 5-7 consecutive days depending on the severity of the situation. It is also used for mastitis treatment. Its use was described in association with castration in a tomcat [11], or in association with aglepristone in an assumed pregnant young queen [44].

Bromocriptine (Parlodel® is the most frequently used pharmacological presentation) is used in the veterinary practice less often than Cabergoline, as it was found to induce abnormal behavioural effects, such as limb flicks, head/body shakes, and hallucinatory-like behaviour as well as excessive grooming [45]. This drug can be used at the dose of 0.25mg/cat/day, PO, for 5 to 7 days. Its use on FEH situations enrols the same concerns as for Cabergoline.

### **8. Prognosis**

when predicting the recovery time, one should be aware that FEH secondary to exogenous

Several studies demonstrated that the progesterone receptor blocker Aglepristone (Alizine®, Virbac, France) can successfully revert FEH [8,11,13,19,23]. Aglepristone is a molecule that competitively binds to the progesterone receptor without activating the hormone response cascade in target tissues. This drug binds to with a 9-fold affinity to progesterone receptor, and according to the manufacturer its residence time in the organism is of 6 days, if administered once in the dose of 20mg/kg or twice at 10mg/kg [23]. Although not licenced to be used in cats, this drug is commonly used to induce abortion or to treat pyometra in this species. By

Before starting the treatment with Aglepristone it is important to exclude pregnancy, as this drug may elicit abortion of a premature birth. When FEH develops in pregnant females it is mandatory that the therapeutic approaches are discussed with the cat owner in detail, and is important to mention that despite the mammary enlargement, the diseased glands will not produce milk and also that the kittens attempts to nurse may predispose to complications such

Several therapeutic schedules have been described in the literature for Aglepristone in FEH (Table 1). Personally, I prefer to inject 10mg/kg of Aglepristone (Alizine®) on days 1 and 3, subcutaneously (SC), and to re-evaluate the situation a week later. If necessary, a second administration is performed following the same schedule. Rarely (only one situation in 25 cases treated with Alisine®) I needed to perform a third administration (again two doses 48h apart), in a female that was submitted to oral progestin treatment that started during estrus. Reduction of the mammary volume, in particular the mammary thickness, is the major parameter for assessment of the response to treatment. Mammary thickness can be assessed by ultrasonog‐ raphy. By using this schedule, it can be observed a slight reduction in the thickness of the disease mammary glands between days 1 and 3, which is predictive of the expected length of the treatment. For most cases, FEH recovery was obtained in 3 to 4 weeks, with only one situation (the one above mentioned) taking 6 weeks to obtain full regression of the mammary

Varying with the reports, the a mean of 4 to 5 treatments (Table 1) are needed to recover from FEH [23,42], and full recovery was obtained in varying periods that last for 3 to 11 weeks [23].

Occasionally, short-term skin irritation at the site of injection has been reported [23], but it

In some case descriptions, dopamine agonists such as Cabergoline and Bromocriptine were also used for FEH treatment [40]. Though these products were not licenced for cats in some countries, they are commonly used in the feline practice. Vomiting or anorexia are described as side effects in a small proportion of cases, as well as a slight depression of the blood pressure, although these symptoms tend to disappear with continued treatment. Nonetheless, its usefulness in the treatment of feline mammary fibroepithelial hyperplasia remain uncertain, as prolactin has not been described as one of the players in FEH pathogenesis and FEH lesions

as mastitis and ulceration, which will worsen the evolution of the primary condition.

progestin would take longer to regress if antiprogesterone drugs are not selected.

consequence, its application in cats is under the veterinarian responsibility.

condition.

226 Insights from Veterinary Medicine

seldom originates a problem.

Generally, the prognosis for uncomplicated feline mammary fibroepithelial hyperplasia is good. The co-existence of mastitis or ulceration may induce some concern, particularly when the situation was left untreated for a long period. In rare situations, abscess formation and systemic illness worsen the prognosis.

Spontaneous regression of the enlarged mammary glands after removal of the progesterone influences may occur, but it may take up to 11 months. Nevertheless, ovariohysterectomy or withdrawal of the progestin treatment does not always result in regression of the masses. With the available progesterone antagonist, medical treatment of the condition has improved, and regression of the mammary swelling is usually obtained within a 4-8 weeks interval. It is possible that the co-existing mammary abscesses may require the surgical drainage of the abscess content, in a way to hasten the FEH regression [10].

**Acknowledgements**

**Author details**

Rita Payan-Carreira

**References**

214-24.

474-85.

1699-703.

This work was supported by the project from CECAV/UTAD with the reference PEst-OE/AGR/

Feline Mammary Fibroepithelial Hyperplasia: A Clinical Approach

http://dx.doi.org/10.5772/55550

229

CECAV [Veterinary and Animal Research Centre] – University of Trás-os-Montes and Alto

[1] Murphy S. 2009. Mammary tumours in cats – causes and practical management. Conference proceedings of the European Society of Feline Medicine - ESFM Feline

[2] Giménez F, Hecht S, Craig LE, Legendre AM. 2010. Early detection, aggressive thera‐ py: optimizing the management of feline mammary masses. J Feline Med Surg. 12(3):

[3] Johnson C. 1994. Diseases of the mammary glands. In: Sherding R. (Ed). The Cat: Dis‐ eases and Clinical Management. 2nd Ed. Churchill Livingstone, New York: 1874-5.

[4] Johnston S, Root Kustritz M, Olson P. 2001. Disorders of the mammary gland of the Queen. In: Canine and Feline Theriogenology. W.B. Saunders Comp, Philadelphia:

[5] Hayden D, Johnston S, Kiang D, Johnson K, Barnes D. 1981. Feline mammary hyper‐ trophy/fibroadenoma complex: clinical and hormonal aspects. Am J Vet Res. 42(10):

[6] Loretti A, Ilha M, Breitsameter I, Faraco C. 2004. Clinical and pathological study of feline mammary fibroadenomatous changes associated with depot medroxyproges‐

[7] Loretti A, Ilha M, Ordás J, de las Mulas JM. 2005. Clinical, pathological and immuno‐ histochemical study of feline mammary fibroepithelial hyperplasia following a single injection of depot medroxyprogesterone acetate. J Feline Med Surg. 7(1): 43-52.

terone acetate therapy. Arq Bras Med Vet Zootec. 56(2): 270-4.

UI0772/2011, by the Portuguese Science and Technology Foundation.

Symposium, 1st April 2009, Birmingham, UK: 11-15.

Address all correspondence to: rtpayan@gmail.com

Douro, Dept. Zootechnics, Vila Real, Portugal

Recurrence of the disease is controversial. Some studies refer that it is rarely observed [11]. However, in the absence of neutering, several reports of FEH in females describe the recurrence of the condition at a variable timing after the initial treatment [6,23,31]. Consequently, recurrence of the mammary lesions is important concern particularly in females that can maintain their full reproductive activity.

Thus, when debating the prognosis with the cat owner, it is important to discuss also the measures need for avoiding the recurrence of FEH. If a progestin administration for contra‐ ception was the causative agent it should be advised the cat spaying. This should also be advised whenever the female is not intended for breeding. In cases where the surgery for neutering is decided it can be performed later, when mammary enlargement regressed, making the procedure easier for the surgeon and less traumatic for the cat, avoiding undesir‐ able trauma of the enlarged mammary glands during surgery. If progestin was used as treatment for skin disorders, alternative therapeutics should be found.

## **9. Concluding remarks**

FEH is a progesterone-associated disease that is characterized by a very rapid swelling of mammary gland, which onset is usually within 2 to 4 weeks from the occurrence of an estrus or the administration of a progestin treatment. Occasionally, this primary lesion can be complicated with mastitis, ulceration or abscessation of the diseased mammary glands. Diagnosis of feline mammary fibroepithelial hyperplasia is exclusively a clinical issue, though some complementary methods of diagnosis may be helpful aids to confirm the diagnosis.

Treatment of feline mammary fibroepithelial hyperplasia has undergone major changes in the past three decades, and considerable improvement of cat welfare was achieved with the introduction of successful medical treatment. Nowadays, antiprogesterone drugs are available that ease the therapeutics and hastens a favourable outcome. With these drugs, the treatment targets the major causal mechanism, interrupting the progesterone-mediated pathways of mammary development and growth. Antiprogesterone molecules are now in the first line treatments for FEH, allowing to avoid massive mastectomy, a very aggressive approach to the cat. However, relapses are possible, and most frequently ovariectomy or ovariohysterectomy are advised to avoid recurrence of the problem.

Nevertheless, new studies on molecular pathways involved in the disease might strengthen additional interplaying factors of interest to design additional therapeutic approaches, as well as to highlight the factors underlying the relapses described in the literature in order to improve the medical treatment and the animal welfare.

## **Acknowledgements**

withdrawal of the progestin treatment does not always result in regression of the masses. With the available progesterone antagonist, medical treatment of the condition has improved, and regression of the mammary swelling is usually obtained within a 4-8 weeks interval. It is possible that the co-existing mammary abscesses may require the surgical drainage of the

Recurrence of the disease is controversial. Some studies refer that it is rarely observed [11]. However, in the absence of neutering, several reports of FEH in females describe the recurrence of the condition at a variable timing after the initial treatment [6,23,31]. Consequently, recurrence of the mammary lesions is important concern particularly in females that can

Thus, when debating the prognosis with the cat owner, it is important to discuss also the measures need for avoiding the recurrence of FEH. If a progestin administration for contra‐ ception was the causative agent it should be advised the cat spaying. This should also be advised whenever the female is not intended for breeding. In cases where the surgery for neutering is decided it can be performed later, when mammary enlargement regressed, making the procedure easier for the surgeon and less traumatic for the cat, avoiding undesir‐ able trauma of the enlarged mammary glands during surgery. If progestin was used as

FEH is a progesterone-associated disease that is characterized by a very rapid swelling of mammary gland, which onset is usually within 2 to 4 weeks from the occurrence of an estrus or the administration of a progestin treatment. Occasionally, this primary lesion can be complicated with mastitis, ulceration or abscessation of the diseased mammary glands. Diagnosis of feline mammary fibroepithelial hyperplasia is exclusively a clinical issue, though some complementary methods of diagnosis may be helpful aids to confirm the diagnosis.

Treatment of feline mammary fibroepithelial hyperplasia has undergone major changes in the past three decades, and considerable improvement of cat welfare was achieved with the introduction of successful medical treatment. Nowadays, antiprogesterone drugs are available that ease the therapeutics and hastens a favourable outcome. With these drugs, the treatment targets the major causal mechanism, interrupting the progesterone-mediated pathways of mammary development and growth. Antiprogesterone molecules are now in the first line treatments for FEH, allowing to avoid massive mastectomy, a very aggressive approach to the cat. However, relapses are possible, and most frequently ovariectomy or ovariohysterectomy

Nevertheless, new studies on molecular pathways involved in the disease might strengthen additional interplaying factors of interest to design additional therapeutic approaches, as well as to highlight the factors underlying the relapses described in the literature in order to

abscess content, in a way to hasten the FEH regression [10].

treatment for skin disorders, alternative therapeutics should be found.

maintain their full reproductive activity.

228 Insights from Veterinary Medicine

**9. Concluding remarks**

are advised to avoid recurrence of the problem.

improve the medical treatment and the animal welfare.

This work was supported by the project from CECAV/UTAD with the reference PEst-OE/AGR/ UI0772/2011, by the Portuguese Science and Technology Foundation.

## **Author details**

Rita Payan-Carreira

Address all correspondence to: rtpayan@gmail.com

CECAV [Veterinary and Animal Research Centre] – University of Trás-os-Montes and Alto Douro, Dept. Zootechnics, Vila Real, Portugal

## **References**


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ry Congress, Jeju (Korea), 14 to 17 October. OC-I10: 764–767 (www.ivis.org/

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[20] Enginler SÖ, Şenünver A. 2011. The Effects of Progesterone Hormone Applications Used for Suppression of Estrus on Mammary Glands in Queens. Kafkas Üniversitesi

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**Chapter 9**

**Diseases of Thyroid in Animals and Their Management**

Disorders of the thyroid gland are well known in companion animals but less so in livestock. In livestock, nutritional iodine deficiencies have been of greater importance than thyroid-gland diseases, particularly in the iodine-deficient areas. Thyroid hormones have many functions in the body and, in general, regulate growth, differentiation, and the metabolism of lipids, proteins and carbohydrates. The thyroid gland of animals is a bilobed structure that overlays the trachea at a point just below the larynx. Anatomical varia‐ tions of the gland are quite marked between species and, to some extent, within a given species. The isthmus connecting the two lobes of the thyroid is the region that varies most markedly between species. Humans and the pig have a large discrete isthmus that forms a pyramidal lobe connecting the two lateral lobes. The cow has a fairly wide band of glandular tissue that forms the connecting isthmus. In the horse, sheep, goat, cat, and dog, the isthmus is a narrow remnant of tissue and may be nonexistent. The size of the gland relative to body weight is extremely small in all animals, approximating 0.20% of body weight. Accessory or extrathyroidal tissue is quite commonly seen in the dog, particular‐ ly near the thoracic inlet, though it may be found anywhere along the esophagus. This tissue is fully functional physiologically, synthesizes hormone, and can be located by its uptake of radionuclides. The thyroid gland is a highly vascularized tissue with a large blood flow. The functional unit of the thyroid gland is the thyroid follicle, a spherical structure composed of an outer monolayer of follicular cells surrounding an inner core of colloid, the thyroglobulin-hormone complex, which is the storage reservoir of thyroid hormone. The colloid stored in the lumen is a clear, viscous fluid. The individual follicu‐ lar cells vary from 5 to 10/zm in height and the entire follicle may vary from 25 to 250/zm in diameter. The size of the follicles and the height of their cells vary according to the functional state of the gland. The cells may vary from an inactive squamous cell to the highly active, tall columnar cell. Interspersed between the follicles are the thyroid C cells,

> © 2013 Singh and Beigh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Singh and Beigh; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

R. Singh and S. A. Beigh

http://dx.doi.org/10.5772/55377

**1. Introduction**

Additional information is available at the end of the chapter


## **Diseases of Thyroid in Animals and Their Management**

R. Singh and S. A. Beigh

[35] Dias Pereira P, Carvalheira J, Gärtner F. 2004. Cell proliferation in feline normal, hy‐ perplastic and neoplastic mammary tissue--an immunohistochemical study. Vet J.

[36] Schmidt PM, Chakraborty PK, Wildt DE. 1983. Ovarian activity, circulating hor‐ mones and sexual behavior in the cat. II. Relationships during pregnancy, parturi‐

[37] Moulton J. 1990. Mammary tumors of the cat. In: Moulton J. (Ed.) Tumours in Do‐

[38] Ginn P, Mansell J, Rakich P. 2007. Tumours of the mammary gland. In: Maxie M. (Ed). Jubb, Kennedy, and Palmer's Pathology of Domestic Animals. Saunders, Elsevi‐

[39] Buriticá EF, Echeverry DF, Lozada AF. 2010 Hiperplasia fibroepitelial mamaria feli‐

[40] Little S. 2011. Feline reproduction: common problems you will see in practice. Pro‐ ceedings of the 63rd Canadian Veterinary Medical Association Convention. Halifax, Nova Scotia, Canada, the 6 to 9 July 2011. Pp. 4. (http://canadianveterinarians.net/

SpeakerNotes2011/HTML/companion/companion\_little\_06-Feline\_Reproduc‐

[41] Chisholm H. 1993. Massive mammary enlargement in a cat. Can Vet J. 34(5): 315.

[42] Vitásek R, Dendisová H. 2006. Treatment of Feline Mammary Fibroepithelial Hyper‐ plasia Following a Single Injection of Proligestone. Acta Vet. Brno 75: 295-297.

[43] Trummel DK. 2007. Expression of Prolactin in Feline Mammary Adenomas and Ade‐

[44] Uçmak M, Enginler SÖ, Gündüz MG, Kirşan I, Sönmez K. 2011. Treatment of Feline Mammary Fibroepithelial Hyperplasia with the Combination of Aglepristone and

[45] Gonzalez-Lima F, Velez D, Blanco R. 1988. Antagonism of behavioral effects of bro‐

Cabergoline. İstanbul Üniversitesi Veteriner Fakültesi Dergisi 37 (1): 69-73.

mocriptine by prolactin in female cats. Behav Neural Biol. 49(1): 74-82.

nocarcinomas. BSc Thesis in Animal Sciences. Oregon State Univ.

tion, lactation and the postpartum estrus. Biol Reprod. 28(3): 657-71.

na: reporte de un caso. Rev Ces Med Vet Zootec. 5 (1):70-76.

tion.html). Accessed on the 18th September 2012.

mestic Animals. 3rd Ed. University of California Press, Berkeley. p. 547-8.

168(2): 180-5.

232 Insights from Veterinary Medicine

er, New York. p. 777-80.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55377

## **1. Introduction**

Disorders of the thyroid gland are well known in companion animals but less so in livestock. In livestock, nutritional iodine deficiencies have been of greater importance than thyroid-gland diseases, particularly in the iodine-deficient areas. Thyroid hormones have many functions in the body and, in general, regulate growth, differentiation, and the metabolism of lipids, proteins and carbohydrates. The thyroid gland of animals is a bilobed structure that overlays the trachea at a point just below the larynx. Anatomical varia‐ tions of the gland are quite marked between species and, to some extent, within a given species. The isthmus connecting the two lobes of the thyroid is the region that varies most markedly between species. Humans and the pig have a large discrete isthmus that forms a pyramidal lobe connecting the two lateral lobes. The cow has a fairly wide band of glandular tissue that forms the connecting isthmus. In the horse, sheep, goat, cat, and dog, the isthmus is a narrow remnant of tissue and may be nonexistent. The size of the gland relative to body weight is extremely small in all animals, approximating 0.20% of body weight. Accessory or extrathyroidal tissue is quite commonly seen in the dog, particular‐ ly near the thoracic inlet, though it may be found anywhere along the esophagus. This tissue is fully functional physiologically, synthesizes hormone, and can be located by its uptake of radionuclides. The thyroid gland is a highly vascularized tissue with a large blood flow. The functional unit of the thyroid gland is the thyroid follicle, a spherical structure composed of an outer monolayer of follicular cells surrounding an inner core of colloid, the thyroglobulin-hormone complex, which is the storage reservoir of thyroid hormone. The colloid stored in the lumen is a clear, viscous fluid. The individual follicu‐ lar cells vary from 5 to 10/zm in height and the entire follicle may vary from 25 to 250/zm in diameter. The size of the follicles and the height of their cells vary according to the functional state of the gland. The cells may vary from an inactive squamous cell to the highly active, tall columnar cell. Interspersed between the follicles are the thyroid C cells,

© 2013 Singh and Beigh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Singh and Beigh; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the source of calcitonin, the hypocalcemic hormone associated with calcium metabolism. A third type of hormonal tissue, the parathyroid, is embedded within the thyroid or located in close proximity. The parathyroids are the source of parathormone, the hypercalcemic hormone.

common. Dryness, excessive shedding, and retarded regrowth of hair are usually the earliest dermatologic changes. Nonpruritic hair thinning or alopecia (usually bilaterally symmetric) that may involve the ventral and lateral trunk, the caudal surfaces of the thighs, dorsum of the tail, ventral neck, and the dorsum of the nose occurs in about two-thirds of dogs with hypo‐ thyroidism. Alopecia, sometimes associated with hyperpigmentation, often starts over points of wear. Occasionally, secondary pyoderma (which may produce pruritus) is observed.

Diseases of Thyroid in Animals and Their Management

http://dx.doi.org/10.5772/55377

235

In moderate to severe cases, thickening of the skin occurs secondary to accumulation of glycosaminoglycans (mostly hyaluronic acid) in the dermis. In such cases, myxedema is most common on the forehead and face, resulting in a puffy appearance and thickened skin folds above the eyes. This puffiness, together with slight drooping of the upper eyelid, gives some dogs a "tragic" facial expression. These changes also have been described in the GI tract, heart,

In intact dogs, hypothyroidism may cause various reproductive disturbances: in females, failure to cycle (anestrus) or sporadic cycling, infertility, abortion, or poor litter survival; and in males, lack of libido, testicular atrophy, hypospermia, or infertility. During the fetal period and in the first few months of postnatal life, thyroid hormones are crucial for growth and development of the skeleton and CNS. Therefore, in addition to the well-recognized signs of adult-onset hypothyroidism, disproportionate dwarfism and impaired mental development (cretinism) are prominent signs of congenital and juvenile-onset hypothyroidism. In primary congenital hypothyroidism, enlargement of the thyroid gland (goiter) also may be detected, depending on the cause of the hypothyroidism. Radiographic signs of epiphyseal dysgenesis (underdeveloped epiphyses throughout the long bones), shortened vertebral bodies, and

In dogs with congenital hypopituitarism there may be variable degrees of thyroidal, adrenocortical, and gonadal deficiency, but clinical signs are primarily related to growth hormone deficiency. Signs include proportionate dwarfism, loss of primary guard hairs with retention of the puppy coat, hyperpigmentation of the skin, and bilaterally symmet‐

Clinical characteristics oh hypothyroidism in adult horses are poorly defined largely because of the difficulty of confirming the diagnosis and the pharmacological effect of exogenous thyroid hormone. Clinical abnormalities anecdotal attributed to hypothyroidism include exercise intolerance, infertility, weight gain, maldistribution of body fat, agalactia, anhidrosis, and laminitis among others. Congenital hypothyroid foals have a prolonged gestation but are born with a short silky hairs coat, soft pliable ears, difficulty in standing, lax joints and poorly ossified bones. The foals are referred to as dysmature. Characteristic musculoskeletal abnor‐ malities include inferior (mandubular) prognathism, flexural deformities, ruptured common

**Treatment:** Thyroxine (T4) is the thyroid hormone replacement compound of choice in dogs. With few exceptions, replacement therapy is necessary for the remainder of the dog's life; careful initial diagnosis and tailoring of treatment is essential. The reported replacement

and lateral extensor tendons, and poorly ossified cubiodal bones.

and skeletal muscles.

delayed epiphyseal closure are common.

ric alopecia of the trunk.

Thyroid hormones (T4, T3, and rT3) immediately on entering the circulation are bound to transport proteins, mainly to thyroxine binding globulin (TBG) and in lesser amounts to thyroxine binding prealbumin (TBPA) and to albumin. There is a wide spectrum of species variation in hormone binding by serum proteins. TBG is the major binding protein for hormone, but not all species have TBG however, TBPA is present in all species. In the cat, rabbit, rat, mouse, Guinea pig, pigeon, or chicken, TBG is absent and most of the hor‐ mone is transported by albumin. In these species without TBG, albumin transports 50-80% of the hormones. T3 (and likely rT3) appears to bind to these transport proteins in parallel with T4 binding.

## **2. Disorders of thyroid function**

#### **2.1. Hypothyroidism**

This disorder is most common in dogs but also develops rarely in other species, includ‐ ing cats, horses, and other large domestic animals. Hypothyroidism is most common in dogs 4-l0 yr old. It usually affects mid- to large-size breeds and is rare in toy and miniature breeds. Breeds reported to be predisposed include the Golden Retriever, Doberman Pinscher, Irish Setter, Miniature Schnauzer, Dachshund, Cocker Spaniel, and Airedale Terrier. There does not appear to be a sex predilection, but spayed females appear to have a higher risk of developing hypothyroidism than intact females. Clinical Hypothyroidism is usually the result of primary diseases of the thyroid gland, especially idiopathic follicular atrophy also termed "follicular collapse" and lymphocytic thyroiditis. In the adult dog, follicular atrophy is probably the most common cause of hypothyroidism. Hypothyroid‐ ism may be secondary to a pituitary insufficiency that prevents the release of either TSH or TRH. Other rare forms of hypothyroidism in dogs include neoplastic destruction of thyroid tissue and congenital (or juvenile-onset) hypothyroidism. Hypothyroidism is an extremely rare disorder in adult cats, iatrogenic hypothyroidism is the most common form congenital or juvenile-onset hypothyroidism does also occur. Hypothyroidism appears to be very rare in adult horses. In foals, congenital hypothyroidism may develop when pregnant mares graze plants that contain goitrogens, nitrate or are fed diets either deficient in or containing excessive amounts of iodine.

**Clinical Findings:** A deficiency of thyroid hormone affects the function of all organ systems; as a result, clinical signs are diffuse, variable, often nonspecific, and rarely pathognomonic. Slowing of cellular metabolism, results in development of mental dullness, lethargy, intoler‐ ance of exercise, and weight gain without a corresponding increase in appetite. Mild to marked obesity develops in some dogs. Difficulty in maintaining body temperature may lead to frank hypothermia; the classic hypothyroid dog is a heat-seeker. Alterations in the skin and coat are common. Dryness, excessive shedding, and retarded regrowth of hair are usually the earliest dermatologic changes. Nonpruritic hair thinning or alopecia (usually bilaterally symmetric) that may involve the ventral and lateral trunk, the caudal surfaces of the thighs, dorsum of the tail, ventral neck, and the dorsum of the nose occurs in about two-thirds of dogs with hypo‐ thyroidism. Alopecia, sometimes associated with hyperpigmentation, often starts over points of wear. Occasionally, secondary pyoderma (which may produce pruritus) is observed.

the source of calcitonin, the hypocalcemic hormone associated with calcium metabolism. A third type of hormonal tissue, the parathyroid, is embedded within the thyroid or located in close proximity. The parathyroids are the source of parathormone, the hypercalcemic

Thyroid hormones (T4, T3, and rT3) immediately on entering the circulation are bound to transport proteins, mainly to thyroxine binding globulin (TBG) and in lesser amounts to thyroxine binding prealbumin (TBPA) and to albumin. There is a wide spectrum of species variation in hormone binding by serum proteins. TBG is the major binding protein for hormone, but not all species have TBG however, TBPA is present in all species. In the cat, rabbit, rat, mouse, Guinea pig, pigeon, or chicken, TBG is absent and most of the hor‐ mone is transported by albumin. In these species without TBG, albumin transports 50-80% of the hormones. T3 (and likely rT3) appears to bind to these transport proteins in parallel

This disorder is most common in dogs but also develops rarely in other species, includ‐ ing cats, horses, and other large domestic animals. Hypothyroidism is most common in dogs 4-l0 yr old. It usually affects mid- to large-size breeds and is rare in toy and miniature breeds. Breeds reported to be predisposed include the Golden Retriever, Doberman Pinscher, Irish Setter, Miniature Schnauzer, Dachshund, Cocker Spaniel, and Airedale Terrier. There does not appear to be a sex predilection, but spayed females appear to have a higher risk of developing hypothyroidism than intact females. Clinical Hypothyroidism is usually the result of primary diseases of the thyroid gland, especially idiopathic follicular atrophy also termed "follicular collapse" and lymphocytic thyroiditis. In the adult dog, follicular atrophy is probably the most common cause of hypothyroidism. Hypothyroid‐ ism may be secondary to a pituitary insufficiency that prevents the release of either TSH or TRH. Other rare forms of hypothyroidism in dogs include neoplastic destruction of thyroid tissue and congenital (or juvenile-onset) hypothyroidism. Hypothyroidism is an extremely rare disorder in adult cats, iatrogenic hypothyroidism is the most common form congenital or juvenile-onset hypothyroidism does also occur. Hypothyroidism appears to be very rare in adult horses. In foals, congenital hypothyroidism may develop when pregnant mares graze plants that contain goitrogens, nitrate or are fed diets either deficient

**Clinical Findings:** A deficiency of thyroid hormone affects the function of all organ systems; as a result, clinical signs are diffuse, variable, often nonspecific, and rarely pathognomonic. Slowing of cellular metabolism, results in development of mental dullness, lethargy, intoler‐ ance of exercise, and weight gain without a corresponding increase in appetite. Mild to marked obesity develops in some dogs. Difficulty in maintaining body temperature may lead to frank hypothermia; the classic hypothyroid dog is a heat-seeker. Alterations in the skin and coat are

hormone.

234 Insights from Veterinary Medicine

with T4 binding.

**2.1. Hypothyroidism**

**2. Disorders of thyroid function**

in or containing excessive amounts of iodine.

In moderate to severe cases, thickening of the skin occurs secondary to accumulation of glycosaminoglycans (mostly hyaluronic acid) in the dermis. In such cases, myxedema is most common on the forehead and face, resulting in a puffy appearance and thickened skin folds above the eyes. This puffiness, together with slight drooping of the upper eyelid, gives some dogs a "tragic" facial expression. These changes also have been described in the GI tract, heart, and skeletal muscles.

In intact dogs, hypothyroidism may cause various reproductive disturbances: in females, failure to cycle (anestrus) or sporadic cycling, infertility, abortion, or poor litter survival; and in males, lack of libido, testicular atrophy, hypospermia, or infertility. During the fetal period and in the first few months of postnatal life, thyroid hormones are crucial for growth and development of the skeleton and CNS. Therefore, in addition to the well-recognized signs of adult-onset hypothyroidism, disproportionate dwarfism and impaired mental development (cretinism) are prominent signs of congenital and juvenile-onset hypothyroidism. In primary congenital hypothyroidism, enlargement of the thyroid gland (goiter) also may be detected, depending on the cause of the hypothyroidism. Radiographic signs of epiphyseal dysgenesis (underdeveloped epiphyses throughout the long bones), shortened vertebral bodies, and delayed epiphyseal closure are common.

In dogs with congenital hypopituitarism there may be variable degrees of thyroidal, adrenocortical, and gonadal deficiency, but clinical signs are primarily related to growth hormone deficiency. Signs include proportionate dwarfism, loss of primary guard hairs with retention of the puppy coat, hyperpigmentation of the skin, and bilaterally symmet‐ ric alopecia of the trunk.

Clinical characteristics oh hypothyroidism in adult horses are poorly defined largely because of the difficulty of confirming the diagnosis and the pharmacological effect of exogenous thyroid hormone. Clinical abnormalities anecdotal attributed to hypothyroidism include exercise intolerance, infertility, weight gain, maldistribution of body fat, agalactia, anhidrosis, and laminitis among others. Congenital hypothyroid foals have a prolonged gestation but are born with a short silky hairs coat, soft pliable ears, difficulty in standing, lax joints and poorly ossified bones. The foals are referred to as dysmature. Characteristic musculoskeletal abnor‐ malities include inferior (mandubular) prognathism, flexural deformities, ruptured common and lateral extensor tendons, and poorly ossified cubiodal bones.

**Treatment:** Thyroxine (T4) is the thyroid hormone replacement compound of choice in dogs. With few exceptions, replacement therapy is necessary for the remainder of the dog's life; careful initial diagnosis and tailoring of treatment is essential. The reported replacement dosages for T4 in dogs range from a total dose of 0.01-0.02 mg/lb (0.02-0.04 mg/kg), daily, given once or divided bid.

subtypes of goiters are recognized: diffuse hyperplastic, colloid iodide-excess, multifocal

Diseases of Thyroid in Animals and Their Management

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237

Diffuse thyroid hyperplasia due to iodine deficiency was common in many goitrogenic areas in India before the widespread supplementation of iodized salt to animal diets. Although outbreaks of iodine-deficient goiter are now sporadic and fewer animals are affected, iodine deficiency is still responsible for most goiters seen in large domestic animals. Marginally iodine-deficient diets containing certain goitrogenic substances may result in severe thyroid hyperplasia and clinical evidence of goiter. Goitrogenic substance include thiouracil, pro‐ pylthiouracil, sulfonamides, complex anions such as perchlorate (CLO4), perthecnetate(TcO4), perrhenate (ReO4) and tetrafluoroborate (TcO4). In addition, a number of plants from the genus *Brassica* contain thioglycosides which after digestion release thiocyanate and isothio‐ cyante. A particularly potent thioglycoside, goitrin (L-5-vinyl-2 thiooxazolidone) from plants is excreted in milk. Young animals born to females on iodine-deficient diets are more likely to develop severe thyroid hyperplasia and have clinical signs of hypothyroidism including palpable enlargement of the thyroid gland. Iodine deficiency may be conditioned by other anti-thyroid compounds present in animal feed and in particular situations, these can be responsible for higher incidence of goiter. Hyperplastic goiter in ruminants is associated with prolonged low-level exposure to thiocyanates produced by the ruminal degradation of cyanogenic glucosides of plants such as white clover (*Trifolium),* couch grass and linseed meal, and by degradation of glucosinolates of *Brassica* crops. *Leucaerne leucocephala* and other legumes

hyperplastic, and congenital dyshormonogenetic.

**Figure 1.** Goiter in a kid from Jammu region

## **3. Hyperthyroidism**

Among domestic animal species, disturbances of growth resulting from the production of excess thyroid hormones is most common in adult cats and often related to adenomas compared to hyperactive follicular cells. These neoplastic cells release both T4 and T3 at an uncontrolled rate resulting in the markedly elevated blood levels of both hormones. Cats with hyperthyroidism have elevated levels of total serum thyroxine and triiodothronine. Normal serum levels of T4 in cats, as measure by radioimmunoassay, are approximately 1.5 to 4.5 µg/ dl and serum T3 levels are 60 to 100 ng/dl. In hyperthyroid cats the total levels of T4 in the serum range from 5.0 to over 50 µg/dl and total levels of T3 in the serum range from 100 to 1,000 ng/ dl. Hyperthyroidism is associated with weight loss in spite of a normal or increased appetite and with restlessness and increased activity.

Dogs have a very efficient enteroheptic excretory mechanism for thyroid hormones that is difficult to overload, either from endogenous production by a tumor or by exogenous admin‐ istration of thyroid hormones. Hence, thyroid tumors in the dog only occasionally secrete sufficient amount of thyroid hormone to overload the highly efficient enterohepatic excretory pathways for the thyroid hormones and produce clinical signs of hyperthyroidism. The clinical signs of hyperthyroidism in dogs with functional thyroid tumors include polyuria and polydipsia and weight loss, despite increased appetite and polyphagia, leading to muscle atrophy and weakness. The levels of T3 and T4 in the serum of dogs with clinical hyperthyr‐ oidism are only mildly elevated: 300-400ng/dl and 5-7 µg/dl, respectively. As compared to dogs, cats are very sensitive to phenols and phenol derivatives. They have a poor ability to conjugate phenolic compounds such as T4 with glucuronic acid and to excrete the T4-glucur‐ onide into bile. In cats the capacity for conjugation of T3 with sulfate is also limited and can easily be overloaded.

## **4. Goiter**

Goiter is a clinical term for a non-neoplastic and non-inflamatory enlargement of the thyroid gland which develops in mammals, birds and submammalian vertebrate. The major pathogenic mechanism responsible for the development of thyroid hyperplasia include iodine-defeciant diets, goitrogenic compounds that interfere with hormone synthesis, dietary iodide-excess and genetic defects in the biosynthesis of thyroid hormone. All of these seemingly divergent factors result in deficient thyroxine and triiodothyronine synthesis and decreased blood levels of throid hormones. This is sensed by the hypothala‐ mus and pitituary gland and lead to an increased secretion of TSH, which results in hypertrophy and hyperplasia of the follicular cells in the thyroid gland. The following subtypes of goiters are recognized: diffuse hyperplastic, colloid iodide-excess, multifocal hyperplastic, and congenital dyshormonogenetic.

**Figure 1.** Goiter in a kid from Jammu region

dosages for T4 in dogs range from a total dose of 0.01-0.02 mg/lb (0.02-0.04 mg/kg), daily, given

Among domestic animal species, disturbances of growth resulting from the production of excess thyroid hormones is most common in adult cats and often related to adenomas compared to hyperactive follicular cells. These neoplastic cells release both T4 and T3 at an uncontrolled rate resulting in the markedly elevated blood levels of both hormones. Cats with hyperthyroidism have elevated levels of total serum thyroxine and triiodothronine. Normal serum levels of T4 in cats, as measure by radioimmunoassay, are approximately 1.5 to 4.5 µg/ dl and serum T3 levels are 60 to 100 ng/dl. In hyperthyroid cats the total levels of T4 in the serum range from 5.0 to over 50 µg/dl and total levels of T3 in the serum range from 100 to 1,000 ng/ dl. Hyperthyroidism is associated with weight loss in spite of a normal or increased appetite

Dogs have a very efficient enteroheptic excretory mechanism for thyroid hormones that is difficult to overload, either from endogenous production by a tumor or by exogenous admin‐ istration of thyroid hormones. Hence, thyroid tumors in the dog only occasionally secrete sufficient amount of thyroid hormone to overload the highly efficient enterohepatic excretory pathways for the thyroid hormones and produce clinical signs of hyperthyroidism. The clinical signs of hyperthyroidism in dogs with functional thyroid tumors include polyuria and polydipsia and weight loss, despite increased appetite and polyphagia, leading to muscle atrophy and weakness. The levels of T3 and T4 in the serum of dogs with clinical hyperthyr‐ oidism are only mildly elevated: 300-400ng/dl and 5-7 µg/dl, respectively. As compared to dogs, cats are very sensitive to phenols and phenol derivatives. They have a poor ability to conjugate phenolic compounds such as T4 with glucuronic acid and to excrete the T4-glucur‐ onide into bile. In cats the capacity for conjugation of T3 with sulfate is also limited and can

Goiter is a clinical term for a non-neoplastic and non-inflamatory enlargement of the thyroid gland which develops in mammals, birds and submammalian vertebrate. The major pathogenic mechanism responsible for the development of thyroid hyperplasia include iodine-defeciant diets, goitrogenic compounds that interfere with hormone synthesis, dietary iodide-excess and genetic defects in the biosynthesis of thyroid hormone. All of these seemingly divergent factors result in deficient thyroxine and triiodothyronine synthesis and decreased blood levels of throid hormones. This is sensed by the hypothala‐ mus and pitituary gland and lead to an increased secretion of TSH, which results in hypertrophy and hyperplasia of the follicular cells in the thyroid gland. The following

once or divided bid.

236 Insights from Veterinary Medicine

**3. Hyperthyroidism**

easily be overloaded.

**4. Goiter**

and with restlessness and increased activity.

Diffuse thyroid hyperplasia due to iodine deficiency was common in many goitrogenic areas in India before the widespread supplementation of iodized salt to animal diets. Although outbreaks of iodine-deficient goiter are now sporadic and fewer animals are affected, iodine deficiency is still responsible for most goiters seen in large domestic animals. Marginally iodine-deficient diets containing certain goitrogenic substances may result in severe thyroid hyperplasia and clinical evidence of goiter. Goitrogenic substance include thiouracil, pro‐ pylthiouracil, sulfonamides, complex anions such as perchlorate (CLO4), perthecnetate(TcO4), perrhenate (ReO4) and tetrafluoroborate (TcO4). In addition, a number of plants from the genus *Brassica* contain thioglycosides which after digestion release thiocyanate and isothio‐ cyante. A particularly potent thioglycoside, goitrin (L-5-vinyl-2 thiooxazolidone) from plants is excreted in milk. Young animals born to females on iodine-deficient diets are more likely to develop severe thyroid hyperplasia and have clinical signs of hypothyroidism including palpable enlargement of the thyroid gland. Iodine deficiency may be conditioned by other anti-thyroid compounds present in animal feed and in particular situations, these can be responsible for higher incidence of goiter. Hyperplastic goiter in ruminants is associated with prolonged low-level exposure to thiocyanates produced by the ruminal degradation of cyanogenic glucosides of plants such as white clover (*Trifolium),* couch grass and linseed meal, and by degradation of glucosinolates of *Brassica* crops. *Leucaerne leucocephala* and other legumes of this genus are native or cultivated in many subtropical areas and contain the toxic amino acid mimosine.

[6] Ferguson, D. C. (1994). Update on diagnosis of canine hypothyrodism. *Vet. Clin.*

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239

[7] Gerber, H, Peter, H, & Ferguson, D. C. (1994). Etiopathology of feline toxic nodular

[8] Gosselin, S. J, Capen, C. C, & Martin, S. L. (1981). Biochemical and immunological in‐

[9] Gosselin, S. J, Capen, C. C, & Martin, S. L. (1981). Histopathologic and ultrastructural evaluation of thyroid lesions associated with hypothyroidism in dogs. *Vet. Pathol*. 18:

[10] Gosselin, S. J, Capen, C. C, & Martin, S. L. (1981). Induced lymphocytic thyroditis in dogs: Effect of intrathyroidal injection of thyroid autoantibodies. *Am. J. Vet Res*. 42:

[11] Gosselin, S. J, Capen, C. C, & Krakowka, S. (1981). Lymphocytic Thyroiditis in dogs:

[12] Hoge, W. R, Lund, J. E, & Blackmore, J. C. (1974). Response to thyrotropin as a diag‐

[13] Holzworth, J, Theran, P, & Carpenter, J. L. (1980). Hyperthyroidism in the cat: Ten

[14] Kameda, Y. (1972). The accessory thyroid gland of the dogs around the intrapericar‐

[15] Kaptein, E. M, Hays, M. T, & Ferguson, D. C. (1994). Thryoid hormone metabolism:

[16] Mazzaferri, E. L. (1996). Radioiodine and other treatments and outcomes. *In*: Werner and Ingbar's the Thyroid: A Fundamental and Clinical Text, 7th edition, edited by

[17] Nachreiner, R. F, & Refsal, K. R. (1992). Radioimmunoassay monitoring of thyroid hormone concentration in dogs on thyroid replacement therapy: 2,674 cases

[18] Pammenter, M, Albrecht, C, & Leibenberg, W. (1978). Afrinkander cattle congenital goiter: characteristic of its morphological and iodoprotein pattern. *Endocrinology* 102:

[19] Peter, H. J, Gerber, H, & Studer, H. (1985). Pathogenesis of heterogeneity in human multinodular goiter: a study on growth and function of thyroid tissue transplanted

[20] Peterson, M. E. (1984). Feline hyperthyroidism. *Vet. Clin. North Am., Small Anim. Prac*.

A comparative evaluation. *Vet. Clin. North Am., Small Anim. Prac*. 24: 431.

L.E. Braverman and R.d.Utiger. Philadelphia, PA, J.b.Lipincott Co., , 922.

induction with local graft-versus-host reaction. *Am. J. Vet. Res.* 42: 1856.

nostic aid for canine hypothyroidism. *J.Am. Vet. Med. Assoc*. 10: 167.

*North Am.*, *Small Anim. Pract.* 24: 515.

cases. *J. Am. Vet. Med. Assoc.* 176: 345.

dial aorta. *Arch. Histol. Jpn.* 34: 375.

(1985-1987). *J. Am. Vet. Med. Assoc*. 201: 623.

onto nude mice. *J. Clin. Invest.* 76: 1992.

299.

1565

954.

14: 809.

goiter. *Vet. Clin. North Am.*, *Small Anim. Pract*. 24: 541.

vestigation of hypothyroidism in dogs. *Can.J.Comp.Med*. 44: 158.

Goiter in adult animals is usually of little significance and the general health of the animal is not impaired. However, goiter is of significance as a disease of the newborn, although the drastic losses of animals in endemic area are now controlled by the prophylactic use of iodized salt. Congenital hypothyroidism in domestic animals may be associated with iodine-deficient hyperplastic goiter, even though the dam shows no evidence of thyroid dysfunction. Gestation is often significantly prolonged, particularly for animals with large goiter, and there is increased incidence of dystocia with retention of fetal placenta. Foals affected with iodinedefecient goiter have moderately enlarged thyroids are waek at birth and frequently die within a few days after birth. Calves and kids with goiter are born partially or completely hairless and are either born dead or die soon after birth. Newborn goitrous pigs, goats and lambs frequently have myxedema and hair loss. The mortality rate is high in these species, with majority of offspring born dead or dying within a few hours of birth.

## **Author details**

R. Singh and S. A. Beigh

Division of Veterinary Clinical Medicine & Jurisprudence, Faculty of Veterinary Sciences & A. H., S.K.U.A.S.T.-Jammu, R. S. Pura, Jammu (J&K), India

#### **References**


[6] Ferguson, D. C. (1994). Update on diagnosis of canine hypothyrodism. *Vet. Clin. North Am.*, *Small Anim. Pract.* 24: 515.

of this genus are native or cultivated in many subtropical areas and contain the toxic amino

Goiter in adult animals is usually of little significance and the general health of the animal is not impaired. However, goiter is of significance as a disease of the newborn, although the drastic losses of animals in endemic area are now controlled by the prophylactic use of iodized salt. Congenital hypothyroidism in domestic animals may be associated with iodine-deficient hyperplastic goiter, even though the dam shows no evidence of thyroid dysfunction. Gestation is often significantly prolonged, particularly for animals with large goiter, and there is increased incidence of dystocia with retention of fetal placenta. Foals affected with iodinedefecient goiter have moderately enlarged thyroids are waek at birth and frequently die within a few days after birth. Calves and kids with goiter are born partially or completely hairless and are either born dead or die soon after birth. Newborn goitrous pigs, goats and lambs frequently have myxedema and hair loss. The mortality rate is high in these species, with

Division of Veterinary Clinical Medicine & Jurisprudence, Faculty of Veterinary Sciences &

[1] Belshaw, B. E, & Rijnberk, A. (1979). Radioimmunoassay of plasma T4 and T3 in the diagnosis of primary hypothyroidism in dogs. *J. Am. Anim. Hosp. Assoc*. 15:17.

[2] Broussard, J. D, Peterson, M. E, & Fox, P. R. (1995). Changes in clinical and laboratory findings in cats with hyperthyroidism from 1983 to 1993. *J. Am. Vet. Med. Assoc*. 206:

[3] Capen, C. C, Belshaw, B. E, & Martin, S. L. (1975). Endocrine diseases. *In*: Textbook of Veterinary Internal Medicine-Diseases of the dog and cat, section X, chapter 50, edit‐

[4] Carrasco, N. (1993). Iodide transport in the thyroid gland. Biochim. *Biophys. Acta*

[5] De Vihlder, J. J. M, Van Voorthuizen, W. F, & Van Dijk, J. E. (1978). Hereditary con‐ genital goiter with thyroglobulin deficiency in the breed of goats. *Endocrinology* 102:

ed by S.J. Ettinger. Philadelphia, PA, W.B. Saunders Co., , 1351.

majority of offspring born dead or dying within a few hours of birth.

A. H., S.K.U.A.S.T.-Jammu, R. S. Pura, Jammu (J&K), India

acid mimosine.

238 Insights from Veterinary Medicine

**Author details**

**References**

302.

1154: 65.

1214.

R. Singh and S. A. Beigh


[21] Rand, J. S, Levine, J, & Best, S. J. (1993). Spontaneous adult-onset hypothyroidism in a cat. *J.Vet.Intern.Med*.7: 272.

**Chapter 10**

**Crossregulation of the Thyroid Hormone and**

Thyroid hormones are involved in many physiological processes, during growth, develop‐ ment, behaviour, stress. Their actions are mediated by TH receptors (TR-alpha and TR-beta), which are members of the nuclear receptor (NR) superfamily and function as ligand-activat‐ ed transcription factors. In amphibians, TR-alpha is expressed shortly after hatching and is maintained at a relatively constant level throughout tadpole life and metamorphosis. Then amphibian metamorphosis is dependent on thyroid hormone (TH) changes, which induces the suite of molecular and cellular changes that cause a tadpole to transform into a frog.

Hormones other than TH play important roles in amphibian metamorphosis, in part by modifying the production and actions of TH. Corticosteroids (CS), hormones produced by adrenocortical cells (interrenal glands in frogs and in fish), synergize with TH at target tis‐ sues to promote morphogenesis [1,2]. The production of CS changes with development, ris‐ ing throughout metamorphosis and reaching a peak at metamorphic climax [2]. Like TH, CS actions are mediated by NRs encoded by two different genes: the glucocorticoid receptor

In the current study we examine molecular and physiological mechanisms involved in TH and CS axes regulation. We investigate the synergy between TH and CS not only during amphibian metamorphosis, but also in fish. Indeed TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species, including the Japanese flounder (*Paralichthys olivaceus*), the zebrafish (*Danio rerio*) and the seabream (*Sparus aurata*) [3]. Darras *et al*. [4] showed that exogenous T3 increased TH levels in zebrafish embryos and accelerated development and hatching, and it seems that

> © 2013 Terrien and Prunet; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Terrien and Prunet; licensee InTech. This is a paper distributed under the terms of the Creative Commons

**Corticosteroids in Amphibians and Fish:**

**The Effects of Endocrine Disruption**

Additional information is available at the end of the chapter

(GR) and the mineralocorticoid receptor (MR).

Xavier Terrien and Patrick Prunet

http://dx.doi.org/10.5772/54314

**1. Introduction**


## **Crossregulation of the Thyroid Hormone and Corticosteroids in Amphibians and Fish: The Effects of Endocrine Disruption**

Xavier Terrien and Patrick Prunet

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54314

## **1. Introduction**

[21] Rand, J. S, Levine, J, & Best, S. J. (1993). Spontaneous adult-onset hypothyroidism in

[22] Rijnberk, A. (1971). Iodine Metabolism and thyroid disease in the dog. University of

[23] Scott-moncrieff, J. C, Nelson, R. W, & Bruner, J. M. (1998). Comparison of serum con‐ centrations of thyroid-stimulating hormone in healthy dogs, hypothyroid dogs, and

euthyroid dogs with concurrent disease. *J. Am. Vet. Med. Assoc.* 212: 387.

a cat. *J.Vet.Intern.Med*.7: 272.

240 Insights from Veterinary Medicine

Utrecht, Utrecht, The Netherlands (thesis).

Thyroid hormones are involved in many physiological processes, during growth, develop‐ ment, behaviour, stress. Their actions are mediated by TH receptors (TR-alpha and TR-beta), which are members of the nuclear receptor (NR) superfamily and function as ligand-activat‐ ed transcription factors. In amphibians, TR-alpha is expressed shortly after hatching and is maintained at a relatively constant level throughout tadpole life and metamorphosis. Then amphibian metamorphosis is dependent on thyroid hormone (TH) changes, which induces the suite of molecular and cellular changes that cause a tadpole to transform into a frog.

Hormones other than TH play important roles in amphibian metamorphosis, in part by modifying the production and actions of TH. Corticosteroids (CS), hormones produced by adrenocortical cells (interrenal glands in frogs and in fish), synergize with TH at target tis‐ sues to promote morphogenesis [1,2]. The production of CS changes with development, ris‐ ing throughout metamorphosis and reaching a peak at metamorphic climax [2]. Like TH, CS actions are mediated by NRs encoded by two different genes: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR).

In the current study we examine molecular and physiological mechanisms involved in TH and CS axes regulation. We investigate the synergy between TH and CS not only during amphibian metamorphosis, but also in fish. Indeed TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species, including the Japanese flounder (*Paralichthys olivaceus*), the zebrafish (*Danio rerio*) and the seabream (*Sparus aurata*) [3]. Darras *et al*. [4] showed that exogenous T3 increased TH levels in zebrafish embryos and accelerated development and hatching, and it seems that

elevated T3 levels in culture medium regulates TR expression (for review see reference 4). Glucocorticoids are also key endocrine factors in teleost fishes, involved in metabo‐ lism, growth, reproduction [5], and GR can also regulate fish development. Hillegass *et al*. [6] showed that the embryonic zebrafish corticosteroids activate GR and modulate ex‐ pression of matrix metalloproteinases during development. It seems that TH and CS can regulate each other, in a positive or negative way depending on molecular, cellular and physiologic context. This cross-regulation is important to amplify hormone signals, regu‐ late hormone activity, and coordinate hormone action.

and 3, respectively, in humans). Like other nuclear receptors, TRs have modular struc‐

Crossregulation of the Thyroid Hormone and Corticosteroids in Amphibians and Fish: The Effects of…

http://dx.doi.org/10.5772/54314

243

TR is considered as a transcription factor: it regulates target genes expression directly through DNA response elements. The thyroid hormone response element (TRE) is com‐ posed of repeated DNA sequences [14]. Although TRs can bind to TREs as monomers or ho‐ modimers, the major form of TR bound to the TRE is the heterodimer with Retinoid X Receptor (RXR). An important property of TRs is their ability to bind TREs constitutively in‐ dependent of ligand occupancy [8,10,12,13]. Unliganded TR generally represses basal tran‐ scription. Ligand binding triggers a conformational change in the TR, resulting in activated transcription of its target gene. In the past few years, great progress in biochemical, func‐

A classical vertebrate model for thyroid hormone action in development is the amphibian tadpole. Thyroid hormone controls amphibian metamorphosis and thus plays an important role in the developmental changes in the nervous system that occur during metamorphosis. In anuran amphibians, thyroid function regulates the metamorphic process so these are one of the most commonly used *in vivo* systems for studying TH function [14]. TR mRNAs are present at very low levels in the oocyte and during embryogenesis [15-17]. High levels of TR-alpha mRNA are present after hatching and until the end of metamorphosis when levels decrease markedly, staying low in juveniles and adults [18]. TR-beta mRNA levels increase in parallel with endogenous TH levels. The promoter of TR-beta gene contains a thyroid re‐ sponse element, and its expression is induced by thyroid hormone itself in *X. laevis* [19]. As for TR-alpha mRNA, TR-beta mRNA levels decrease in juveniles and adults [18]. Similar profiles were observed when analyzing protein expression [20]. Furthermore, the proteins are functionally active as shown by T3 treatment inducing precocious metamorphosis [18].

Although TH effects have been mainly studied in mammals and amphibians for metamor‐ phosis process, more and more data show that TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species [3]. In zebrafish, the thyroid gland begins to develop during early embryogenesis and begins to be active around 55 hours post fertilization (hpf) [21]. Before this developmental stage, TH comes from the maternal stock in the egg [3]. The TH receptors (TR-alpha and TR-beta) are both present in prehatch fish embryos, and allow TH functions. Prehatch embryos possess all TH function components: TR-alpha and TR-beta and TH from the maternal stock [3]. TH are synthesized and secreted by the thyroid gland after TSH stimulation. TSH is produced by thyrotropes present in the fish adenopituitary. Terminal differentiation of thyrotropes in the zebrafish adenopituitary occurs around 48hpf. The thyroid gland is active later, about 55 h post fertili‐ zation. Concerning the thyroid gland tissue organization, the zebrafish thyroid gland de‐ rives from precursor cells located in the endoderm prior to pharynx formation. During two morphogenetic phases, the thyroid primordium first adopts a position close to the cardiac outflow tract, with the first differentiated thyroid follicle, that grows afterwards along the ventral pharyngeal midline. The thyroid gland in the adult zebrafish is a loose aggregation

tional, and structural studies has clarified the molecular mechanism of TR action.

tures with six regions (A–F) and three functional domains.

of follicles close to the ventral aorta.

Due to their aqueous exposure, fish and amphibians can be used as indicators for ecotoxico‐ logical studies and for detection of endocrine disruptors (ED) *in vivo*. Endocrine disruption has become one of the major topics in environmental research, but also in public health since the ED is known to affect reproductive biology, metabolism, growth and development. The aquatic environment exposes developing embryos to all compounds present in water. After hatching, penetration of ED into aquatic organism is even easier, resulting in high bioavaila‐ bility and bioaccumulation of chemicals. Amphibians (*Xenopus laevis*) and fish models (ze‐ brafish and medaka (*Oryzias latipes*)) are species widely used for the development of new tools for ED detection and screening of chemicals in the environment. They were firstly used to study endocrine disruption of estrogenic and androgenic systems. But now, more and more studies involve chemicals in thyroid and corticosteroid disruption. Indeed, the thyroid function and corticosteroid axis are both targets for ED. Being given the involvement of both TH and CS axis in amphibian and fish development, these two species were largely used to study TH and CS disruption. The abundant knowledge about endocrinology and develop‐ mental biology in amphibian and fish, and the general scientific interest about TH and CS disruption provide evidence for using these animal models for the study of ED of these two endocrine systems [7]. For that reason, we propose to summarize the impact of endocrine disruption on TH and CS axes.

## **2. Thyroid hormone and corticosteroid endocrine systems: current knowledge**

#### **2.1. Thyroid hormone**

Thyroid hormones (thyroxine T4 and tri-iodothyronine T3) play an important role in de‐ velopment, differentiation, and metabolism [8]. The lack of T3 in early human develop‐ ment results in growth disturbances and severe mental retardation, a disease called cretinism. TH action is also primary for developmental changes in the nervous system that occur during amphibian metamorphosis. Later in life, T3 plays an important role in metabolic balance [9]. T3 action is mediated by nuclear T3 receptors (TRs) that can bind T3 with high affinity [8]. TRs belong to the nuclear receptor superfamily that also in‐ cludes the receptors for retinoids, vitamin D, fatty acids, and prostaglandins, as well as "orphan receptors" with no identified ligands [10-13]. TR is encoded by two separate genes, which are designated TR-alpha and TR-beta, located in different chromosomes (17 and 3, respectively, in humans). Like other nuclear receptors, TRs have modular struc‐ tures with six regions (A–F) and three functional domains.

elevated T3 levels in culture medium regulates TR expression (for review see reference 4). Glucocorticoids are also key endocrine factors in teleost fishes, involved in metabo‐ lism, growth, reproduction [5], and GR can also regulate fish development. Hillegass *et al*. [6] showed that the embryonic zebrafish corticosteroids activate GR and modulate ex‐ pression of matrix metalloproteinases during development. It seems that TH and CS can regulate each other, in a positive or negative way depending on molecular, cellular and physiologic context. This cross-regulation is important to amplify hormone signals, regu‐

Due to their aqueous exposure, fish and amphibians can be used as indicators for ecotoxico‐ logical studies and for detection of endocrine disruptors (ED) *in vivo*. Endocrine disruption has become one of the major topics in environmental research, but also in public health since the ED is known to affect reproductive biology, metabolism, growth and development. The aquatic environment exposes developing embryos to all compounds present in water. After hatching, penetration of ED into aquatic organism is even easier, resulting in high bioavaila‐ bility and bioaccumulation of chemicals. Amphibians (*Xenopus laevis*) and fish models (ze‐ brafish and medaka (*Oryzias latipes*)) are species widely used for the development of new tools for ED detection and screening of chemicals in the environment. They were firstly used to study endocrine disruption of estrogenic and androgenic systems. But now, more and more studies involve chemicals in thyroid and corticosteroid disruption. Indeed, the thyroid function and corticosteroid axis are both targets for ED. Being given the involvement of both TH and CS axis in amphibian and fish development, these two species were largely used to study TH and CS disruption. The abundant knowledge about endocrinology and develop‐ mental biology in amphibian and fish, and the general scientific interest about TH and CS disruption provide evidence for using these animal models for the study of ED of these two endocrine systems [7]. For that reason, we propose to summarize the impact of endocrine

**2. Thyroid hormone and corticosteroid endocrine systems: current**

Thyroid hormones (thyroxine T4 and tri-iodothyronine T3) play an important role in de‐ velopment, differentiation, and metabolism [8]. The lack of T3 in early human develop‐ ment results in growth disturbances and severe mental retardation, a disease called cretinism. TH action is also primary for developmental changes in the nervous system that occur during amphibian metamorphosis. Later in life, T3 plays an important role in metabolic balance [9]. T3 action is mediated by nuclear T3 receptors (TRs) that can bind T3 with high affinity [8]. TRs belong to the nuclear receptor superfamily that also in‐ cludes the receptors for retinoids, vitamin D, fatty acids, and prostaglandins, as well as "orphan receptors" with no identified ligands [10-13]. TR is encoded by two separate genes, which are designated TR-alpha and TR-beta, located in different chromosomes (17

late hormone activity, and coordinate hormone action.

disruption on TH and CS axes.

**knowledge**

**2.1. Thyroid hormone**

242 Insights from Veterinary Medicine

TR is considered as a transcription factor: it regulates target genes expression directly through DNA response elements. The thyroid hormone response element (TRE) is com‐ posed of repeated DNA sequences [14]. Although TRs can bind to TREs as monomers or ho‐ modimers, the major form of TR bound to the TRE is the heterodimer with Retinoid X Receptor (RXR). An important property of TRs is their ability to bind TREs constitutively in‐ dependent of ligand occupancy [8,10,12,13]. Unliganded TR generally represses basal tran‐ scription. Ligand binding triggers a conformational change in the TR, resulting in activated transcription of its target gene. In the past few years, great progress in biochemical, func‐ tional, and structural studies has clarified the molecular mechanism of TR action.

A classical vertebrate model for thyroid hormone action in development is the amphibian tadpole. Thyroid hormone controls amphibian metamorphosis and thus plays an important role in the developmental changes in the nervous system that occur during metamorphosis. In anuran amphibians, thyroid function regulates the metamorphic process so these are one of the most commonly used *in vivo* systems for studying TH function [14]. TR mRNAs are present at very low levels in the oocyte and during embryogenesis [15-17]. High levels of TR-alpha mRNA are present after hatching and until the end of metamorphosis when levels decrease markedly, staying low in juveniles and adults [18]. TR-beta mRNA levels increase in parallel with endogenous TH levels. The promoter of TR-beta gene contains a thyroid re‐ sponse element, and its expression is induced by thyroid hormone itself in *X. laevis* [19]. As for TR-alpha mRNA, TR-beta mRNA levels decrease in juveniles and adults [18]. Similar profiles were observed when analyzing protein expression [20]. Furthermore, the proteins are functionally active as shown by T3 treatment inducing precocious metamorphosis [18].

Although TH effects have been mainly studied in mammals and amphibians for metamor‐ phosis process, more and more data show that TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species [3]. In zebrafish, the thyroid gland begins to develop during early embryogenesis and begins to be active around 55 hours post fertilization (hpf) [21]. Before this developmental stage, TH comes from the maternal stock in the egg [3]. The TH receptors (TR-alpha and TR-beta) are both present in prehatch fish embryos, and allow TH functions. Prehatch embryos possess all TH function components: TR-alpha and TR-beta and TH from the maternal stock [3]. TH are synthesized and secreted by the thyroid gland after TSH stimulation. TSH is produced by thyrotropes present in the fish adenopituitary. Terminal differentiation of thyrotropes in the zebrafish adenopituitary occurs around 48hpf. The thyroid gland is active later, about 55 h post fertili‐ zation. Concerning the thyroid gland tissue organization, the zebrafish thyroid gland de‐ rives from precursor cells located in the endoderm prior to pharynx formation. During two morphogenetic phases, the thyroid primordium first adopts a position close to the cardiac outflow tract, with the first differentiated thyroid follicle, that grows afterwards along the ventral pharyngeal midline. The thyroid gland in the adult zebrafish is a loose aggregation of follicles close to the ventral aorta.

Zebrafish genome encodes two TR-alpha genes and one TR-beta gene, which are expressed at different developmental stages, suggesting that they have different function during devel‐ opment (for review see reference 4). Essner *et al* [22] showed that TR-alpha functions mainly as a transcriptional repressor and may repress retinoic acid signalling in zebrafish early de‐ velopment, and its overexpression results in a loss of the midbrain-hindbrain border and a severe disruption of the rostral hindbrain, suggesting an important role for THs in brain de‐ velopment. Some studies showed that a T3 exposure of embryos up-regulates TR expression and accelerates developmental rate and hatching [4,23]. This suggests that TH can exert a positive auto-regulatory feedback control on the transcription of its receptors.

contributes to early developmental programming [26]. Further, Pikulkaew *et al*. [27] demon‐ strated that knocking down maternal GR leads to developmental defects in mesoderm for‐ mation in zebrafish. Recently, it has also been shown that GR signalling is essential for

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In mammals and non-mammalian vertebrates such as amphibians, the major mineralocorti‐ costeroid is aldosterone. However, aldosterone is not detected in fish, and deoxycorticoster‐ one (DOC) is considered as MR ligand in fish. Like aldosterone, DOC is a selective MR agonist, that does not activate trout GR. Cortisol is also a high-affinity ligand for MR. Fish MR is distributed beyond the tissue involved in salt and water balance, especially in gills and intestine, and MR mRNA is also high in rainbow trout brain. The role of MR and its ligand remains less clear than GR, especially concerning its implication during develop‐ ment. MR transcripts continuously increased between 1.5 and 97 hpf and remained at the same high level at 6 days. MR has been suggested as responsible for corticosteroid signalling

The corticosteroid and thyroid hormone receptors possess equivalent transactivation do‐ mains and have some structural functional similarity [10,29], suggesting that these nuclear receptors may enhance transcription of target genes by similar mechanisms as summarized above. The thyroid and corticosteroid systems interact at multiple levels to influence several

Thus, the hypothalamo-pituitary-interrenal axis modulates the thyroid axis in fishes and other vertebrates. Indeed heterologous CRH potently stimulated the release of TSH from cultured pituitary cells *in vitro* [30]. Cortisol exposure also downregulates T4 plasma level in

Conversely, T3 seems to be involved in corticosteroid receptors regulation. Recently, Terrien *et al*. showed that 48 hours of T3 exposure increased MR and GR genes expression in one day-old zebrafish embryos [23]. In another study in common carp (*Cyprinus carpio*), experi‐ mentally induced hyperthyroidism downregulates plasma cortisol level. Kelly *et al*. [33] demonstrated a synergistic effect of thyroid hormone and cortisol on cultured *O. mykiss*

Other studies have suggested a coincident expression and synergetic action of TH and corti‐ costeroids in other vertebrate models [34-37]. Indeed, corticosteroids and thyroid hormones act synergistically during some physiological processes such as amphibian metamorphosis, which is one of the most relevant biological models the most studied for TH and CR crossre‐ gulation [34]. Thus, it was shown that corticosteroids can synergize with thyroid hormone to accelerate tadpole metamorphosis, whether corticosteroids increase T3 binding capacity [2], or corticosteroids can increase the conversion of active T3 from T4, and decrease the degra‐ dation of T3 [38,39]. Moreover, corticosterone treatment upregulates TR-beta expression in the intestine of premetamorphic tadpoles and in tail explants cultures [37]. It is also known

just after hatching, since it could be activated by maternal cortisol [26].

physiological processes like development, growth or behaviour.

European eel (*Anguilla anguilla)*, but not in trout [31,32].

zebrafish muscle development [28].

**2.3. Relationship between TH and CR**

pavement cell epithelia *in vitro*.

that GR suppresses TSH expression [40].

#### **2.2. Corticosteroid**

Corticosteroids are implicated in many physiological process including osmoregulation, res‐ piration, immunity, reproduction, growth and metabolism. Like thyroid hormone, cortico‐ steroids production and action has been studied in amphibian models, for their implication in the positive control of metamorphosis [24]. In bony fishes, corticosteroids are secreted from the interrenal tissue located in the head kidney region. Cortisol is the major corticoste‐ roid in teleost fish and its release involves the coordinated activation of the hypothalamuspituitary-interrenal (HPI) axis. The key mediators include the release of corticotrophinreleasing factor (CRF) from the hypothalamus, and stimulating the release of adrenocorticotropic hormone (ACTH) from the pituitary. Circulating ACTH binds to mela‐ nocortin receptor 2 (MC2R) on the steroidogenic cells and activates the signalling pathway leading to cortisol biosynthesis [25].

In bony fishes, the corticosteroid receptor is a ligand-dependant transcription factor, with two major classes of receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Cortisol is the physiological ligand for GR. The molecular characterization of fish GR began with the cloning of rainbow trout (*Onchorynchus mykiss*) GR1 cDNA, followed by cloning of partial GR cDNA from tilapia (*Oreochromis mossambicus*) and full length GR cDNAs from Japanese flounder (*Paralichthys olivaceus*), rainbow trout GR2 and multiple GRs from cichlid fish (*Haplochromis burtoni*). The alignment of fish GR polypeptide sequences with mammalian GR showed very high homology with both C domain (DNA-binding) and E domain (hormone-binding), but the transcriptional transactivation domain has little ho‐ mology. The two rainbow trout GR (rtGR1 and rtGR2) showed high sequence homology, and it has been shown phylogenetically that these two GR are a result of gene duplication common to most of teleost fishes. Expression of trout GR transcript has been found in many tissues. Fish GR showed a wide distribution in the brain, gill, and liver.

Little is known about GR and MR function in fish development. Recently, zebrafish has been used to investigate the role of corticosteroid signalling in development. It was shown that both of these receptors are present during embryogenesis [26]. Indeed, during embryogene‐ sis, GR transcripts drops from 1.5 to 25 hpf, and then increases after hatching to the level at 1.5 hpf and was significantly higher at 25 hpf, and this level was maintained until 6 days. This study suggested a more important role for this receptor after hatch in zebrafish. It was also shown that cortisol synthesis occurred only after hatching and that maternal cortisol contributes to early developmental programming [26]. Further, Pikulkaew *et al*. [27] demon‐ strated that knocking down maternal GR leads to developmental defects in mesoderm for‐ mation in zebrafish. Recently, it has also been shown that GR signalling is essential for zebrafish muscle development [28].

In mammals and non-mammalian vertebrates such as amphibians, the major mineralocorti‐ costeroid is aldosterone. However, aldosterone is not detected in fish, and deoxycorticoster‐ one (DOC) is considered as MR ligand in fish. Like aldosterone, DOC is a selective MR agonist, that does not activate trout GR. Cortisol is also a high-affinity ligand for MR. Fish MR is distributed beyond the tissue involved in salt and water balance, especially in gills and intestine, and MR mRNA is also high in rainbow trout brain. The role of MR and its ligand remains less clear than GR, especially concerning its implication during develop‐ ment. MR transcripts continuously increased between 1.5 and 97 hpf and remained at the same high level at 6 days. MR has been suggested as responsible for corticosteroid signalling just after hatching, since it could be activated by maternal cortisol [26].

#### **2.3. Relationship between TH and CR**

Zebrafish genome encodes two TR-alpha genes and one TR-beta gene, which are expressed at different developmental stages, suggesting that they have different function during devel‐ opment (for review see reference 4). Essner *et al* [22] showed that TR-alpha functions mainly as a transcriptional repressor and may repress retinoic acid signalling in zebrafish early de‐ velopment, and its overexpression results in a loss of the midbrain-hindbrain border and a severe disruption of the rostral hindbrain, suggesting an important role for THs in brain de‐ velopment. Some studies showed that a T3 exposure of embryos up-regulates TR expression and accelerates developmental rate and hatching [4,23]. This suggests that TH can exert a

Corticosteroids are implicated in many physiological process including osmoregulation, res‐ piration, immunity, reproduction, growth and metabolism. Like thyroid hormone, cortico‐ steroids production and action has been studied in amphibian models, for their implication in the positive control of metamorphosis [24]. In bony fishes, corticosteroids are secreted from the interrenal tissue located in the head kidney region. Cortisol is the major corticoste‐ roid in teleost fish and its release involves the coordinated activation of the hypothalamuspituitary-interrenal (HPI) axis. The key mediators include the release of corticotrophinreleasing factor (CRF) from the hypothalamus, and stimulating the release of adrenocorticotropic hormone (ACTH) from the pituitary. Circulating ACTH binds to mela‐ nocortin receptor 2 (MC2R) on the steroidogenic cells and activates the signalling pathway

In bony fishes, the corticosteroid receptor is a ligand-dependant transcription factor, with two major classes of receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Cortisol is the physiological ligand for GR. The molecular characterization of fish GR began with the cloning of rainbow trout (*Onchorynchus mykiss*) GR1 cDNA, followed by cloning of partial GR cDNA from tilapia (*Oreochromis mossambicus*) and full length GR cDNAs from Japanese flounder (*Paralichthys olivaceus*), rainbow trout GR2 and multiple GRs from cichlid fish (*Haplochromis burtoni*). The alignment of fish GR polypeptide sequences with mammalian GR showed very high homology with both C domain (DNA-binding) and E domain (hormone-binding), but the transcriptional transactivation domain has little ho‐ mology. The two rainbow trout GR (rtGR1 and rtGR2) showed high sequence homology, and it has been shown phylogenetically that these two GR are a result of gene duplication common to most of teleost fishes. Expression of trout GR transcript has been found in many

Little is known about GR and MR function in fish development. Recently, zebrafish has been used to investigate the role of corticosteroid signalling in development. It was shown that both of these receptors are present during embryogenesis [26]. Indeed, during embryogene‐ sis, GR transcripts drops from 1.5 to 25 hpf, and then increases after hatching to the level at 1.5 hpf and was significantly higher at 25 hpf, and this level was maintained until 6 days. This study suggested a more important role for this receptor after hatch in zebrafish. It was also shown that cortisol synthesis occurred only after hatching and that maternal cortisol

tissues. Fish GR showed a wide distribution in the brain, gill, and liver.

positive auto-regulatory feedback control on the transcription of its receptors.

**2.2. Corticosteroid**

244 Insights from Veterinary Medicine

leading to cortisol biosynthesis [25].

The corticosteroid and thyroid hormone receptors possess equivalent transactivation do‐ mains and have some structural functional similarity [10,29], suggesting that these nuclear receptors may enhance transcription of target genes by similar mechanisms as summarized above. The thyroid and corticosteroid systems interact at multiple levels to influence several physiological processes like development, growth or behaviour.

Thus, the hypothalamo-pituitary-interrenal axis modulates the thyroid axis in fishes and other vertebrates. Indeed heterologous CRH potently stimulated the release of TSH from cultured pituitary cells *in vitro* [30]. Cortisol exposure also downregulates T4 plasma level in European eel (*Anguilla anguilla)*, but not in trout [31,32].

Conversely, T3 seems to be involved in corticosteroid receptors regulation. Recently, Terrien *et al*. showed that 48 hours of T3 exposure increased MR and GR genes expression in one day-old zebrafish embryos [23]. In another study in common carp (*Cyprinus carpio*), experi‐ mentally induced hyperthyroidism downregulates plasma cortisol level. Kelly *et al*. [33] demonstrated a synergistic effect of thyroid hormone and cortisol on cultured *O. mykiss* pavement cell epithelia *in vitro*.

Other studies have suggested a coincident expression and synergetic action of TH and corti‐ costeroids in other vertebrate models [34-37]. Indeed, corticosteroids and thyroid hormones act synergistically during some physiological processes such as amphibian metamorphosis, which is one of the most relevant biological models the most studied for TH and CR crossre‐ gulation [34]. Thus, it was shown that corticosteroids can synergize with thyroid hormone to accelerate tadpole metamorphosis, whether corticosteroids increase T3 binding capacity [2], or corticosteroids can increase the conversion of active T3 from T4, and decrease the degra‐ dation of T3 [38,39]. Moreover, corticosterone treatment upregulates TR-beta expression in the intestine of premetamorphic tadpoles and in tail explants cultures [37]. It is also known that GR suppresses TSH expression [40].

Like in fish models, TH seems to regulate CS in amphibians. Krain and Denver [37] showed that T3 upregulated the glucocorticoid receptor expression in tadpoles tail, and this regula‐ tion might be consistent with a physiological regulatory relationship, given the develop‐ mental pattern of thyroid hormone production and GR mRNA in the tail. In contrast, T3 has been shown to downregulate GR expression in the brain.

Moreover, transgenesis in zebrafish is fast and routinely used. For these reasons, the zebra‐ fish is now used to develop simple, rapid, cost-effective and innovative methods for screen‐ ing environmental pollutants [45,46]. Some stable transgenic zebrafish lines have recently been used for screening chemicals that can mimic the action of estrogens [47], and for devel‐ oping automated image acquisition and analysis in 96-well plates [46]. Recently, a fluores‐ cent transient fluorescent transgenic zebrafish model has been developed to easily and

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Another fish species, the medaka (*Oryzias latipes*) has evolved as an alternative model organ‐ ism for rapid screening of endocrine activities. Indeed, the medaka has the same advantages than zebrafish (transparent embryos, fishes easy to breed, a short life cycle, easy reproduc‐ tion and regular renewal of a large number of experiments). In addition to these technical advantages, the medaka has been widely used as a vertebrate model for the study of orga‐ nogenesis and embryogenesis, and is now increasingly used as a model for conducting tox‐ icity studies [48]. The growing interest in studies on the model of medaka was accompanied by a strong development of tools and methods available to study genetic aspects of such de‐ velopment. In this context, it is not surprising that medaka is now used to study the impact of endocrine disruptors, particularly for the characterization of the stress response and its genetic components [49]. Unlike other models of fish (zebrafish, stickelback), the medaka is more robust, easily exposable to environmental samples, while also the tools of transgenesis are more effective in this model. It is also one of the fish models referenced by the OECD for

Finally, amphibians are also used as indicators for ecotoxicological potencies of several envi‐ ronmental stressors. The aquatic larvae are continuously exposed to chemicals compounds present in water because the eggs are lacking a protective eggshell or membrane. After hatching, the skin of amphibians' larvae is still very permeable, allowing an easy penetra‐ tion of all compounds leading to high bioavailability and bioaccumulation of endocrine dis‐ ruptors. This development stage of amphibians is the most sensitive and the most used to study effects of environmental pollutants. Thus, Fini *et al*. used *in vivo X. laevis* tadpoles to monitor heavy metal pollution in water in continuous flow systems [50]. The same team al‐

**4. Impact of endocrine disruptors on thyroid hormone and corticosteroids**

Many natural and man-made chemicals (plasticizers, pesticides, detergents and pharma‐ ceuticals) interfere with the endocrine system and can result in adverse health effects in humans, mammals and fish. Wildlife living in or in closer association with the aquatic environment are especially impacted by these endocrine disruptors, because water act as sinks for chemical discharges. Thus, fish and amphibians are the main po‐ tential targets for endocrine disruption at multiple levels, either direct or indirect, through ingestion and accumulation of endocrine disruptor, the exposition or through

ready used *X. laevis* to detect the thyroid disrupting effect of BPA *in vivo* [51].

rapidly screen compounds capable of disrupting thyroid function [23].

the detection of estrogenic disruptors.

**systems in fish**

The coincident increase in cortisol and TH during flounder metamorphosis [35] and the regulation of GR mRNA expression after T3 treatment in *X. laevis* [37] support the idea of possible crosstalk between the thyroid hormones and the corticoid signalling system and are in agreement with previous studies highlighting the relationship between TH and the corticoid signalling system. More recently, corticosteroids have been shown to synergize with TH to promote morphogenesis [1], and that the synergistic actions of TH and corticosteroids occurs at the level of the TR-beta expression and deiodinase type 2, which converts T4 to T3 [34].

## **3. Use of aquatic organisms to investigate endocrine disruption**

Many natural or synthetic chemicals are now routinely observed in water. Evidence re‐ vealed that these compounds might interfere with the endogenous endocrine systems of wildlife and humans. Thus, it is now essential to monitor their presence in the environment. Aquatic organisms as amphibians and fish models (zebrafish and medaka) are species wide‐ ly used in ecotoxicology and for the development of transgenic techniques. These techniques allow development of new tools to detect and screen chemicals in the environment. The ze‐ brafish has numerous technical advantages, so that it can be considered as a model organ‐ ism: its complete embryogenesis occurs during the first 72 hours post-fertilization and most of the internal organs develop rapidly in the first 24-48 hours. They are easy to observe be‐ cause embryos are transparent, which allows to easily track their development and expres‐ sion of fluorescent proteins in transgenic fishes *in vivo*, until advanced stages [41]. In addition to rapid development (adult zebrafish can start to breed after 4 months), there are several other advantages of using zebrafish for assay development, including: small size, low cost to maintain, and easily bred in large numbers. Furthermore, a pair of zebrafish can produce over 100-200 eggs per day. Finally, single embryos can be maintained in small vol‐ umes during first days of development until hatching so that zebrafish can be used for auto‐ matic reading in 96-well plates. Chemicals can then be tested directly in the solution in which the embryos develop, facilitating high throughput screening [42]. These significant advantages over other species are making the zebrafish as a fully relevant biological model for detecting the effects of pollutants present in the water.

Zebrafish embryo bioassay has been extensively employed in drug and chemical screening [43,44] and the advantages promoting the use of the embryo assay for those purposes should also promote its use for endocrine disruptors phenotypic screening. Zebrafish em‐ bryos and larvae express hormones and receptors, and they possess all molecular actors to respond to exposure to endocrine disruptors.

Moreover, transgenesis in zebrafish is fast and routinely used. For these reasons, the zebra‐ fish is now used to develop simple, rapid, cost-effective and innovative methods for screen‐ ing environmental pollutants [45,46]. Some stable transgenic zebrafish lines have recently been used for screening chemicals that can mimic the action of estrogens [47], and for devel‐ oping automated image acquisition and analysis in 96-well plates [46]. Recently, a fluores‐ cent transient fluorescent transgenic zebrafish model has been developed to easily and rapidly screen compounds capable of disrupting thyroid function [23].

Like in fish models, TH seems to regulate CS in amphibians. Krain and Denver [37] showed that T3 upregulated the glucocorticoid receptor expression in tadpoles tail, and this regula‐ tion might be consistent with a physiological regulatory relationship, given the develop‐ mental pattern of thyroid hormone production and GR mRNA in the tail. In contrast, T3 has

The coincident increase in cortisol and TH during flounder metamorphosis [35] and the regulation of GR mRNA expression after T3 treatment in *X. laevis* [37] support the idea of possible crosstalk between the thyroid hormones and the corticoid signalling system and are in agreement with previous studies highlighting the relationship between TH and the corticoid signalling system. More recently, corticosteroids have been shown to synergize with TH to promote morphogenesis [1], and that the synergistic actions of TH and corticosteroids occurs at the level of the TR-beta expression and deiodinase type 2,

Many natural or synthetic chemicals are now routinely observed in water. Evidence re‐ vealed that these compounds might interfere with the endogenous endocrine systems of wildlife and humans. Thus, it is now essential to monitor their presence in the environment. Aquatic organisms as amphibians and fish models (zebrafish and medaka) are species wide‐ ly used in ecotoxicology and for the development of transgenic techniques. These techniques allow development of new tools to detect and screen chemicals in the environment. The ze‐ brafish has numerous technical advantages, so that it can be considered as a model organ‐ ism: its complete embryogenesis occurs during the first 72 hours post-fertilization and most of the internal organs develop rapidly in the first 24-48 hours. They are easy to observe be‐ cause embryos are transparent, which allows to easily track their development and expres‐ sion of fluorescent proteins in transgenic fishes *in vivo*, until advanced stages [41]. In addition to rapid development (adult zebrafish can start to breed after 4 months), there are several other advantages of using zebrafish for assay development, including: small size, low cost to maintain, and easily bred in large numbers. Furthermore, a pair of zebrafish can produce over 100-200 eggs per day. Finally, single embryos can be maintained in small vol‐ umes during first days of development until hatching so that zebrafish can be used for auto‐ matic reading in 96-well plates. Chemicals can then be tested directly in the solution in which the embryos develop, facilitating high throughput screening [42]. These significant advantages over other species are making the zebrafish as a fully relevant biological model

Zebrafish embryo bioassay has been extensively employed in drug and chemical screening [43,44] and the advantages promoting the use of the embryo assay for those purposes should also promote its use for endocrine disruptors phenotypic screening. Zebrafish em‐ bryos and larvae express hormones and receptors, and they possess all molecular actors to

**3. Use of aquatic organisms to investigate endocrine disruption**

been shown to downregulate GR expression in the brain.

for detecting the effects of pollutants present in the water.

respond to exposure to endocrine disruptors.

which converts T4 to T3 [34].

246 Insights from Veterinary Medicine

Another fish species, the medaka (*Oryzias latipes*) has evolved as an alternative model organ‐ ism for rapid screening of endocrine activities. Indeed, the medaka has the same advantages than zebrafish (transparent embryos, fishes easy to breed, a short life cycle, easy reproduc‐ tion and regular renewal of a large number of experiments). In addition to these technical advantages, the medaka has been widely used as a vertebrate model for the study of orga‐ nogenesis and embryogenesis, and is now increasingly used as a model for conducting tox‐ icity studies [48]. The growing interest in studies on the model of medaka was accompanied by a strong development of tools and methods available to study genetic aspects of such de‐ velopment. In this context, it is not surprising that medaka is now used to study the impact of endocrine disruptors, particularly for the characterization of the stress response and its genetic components [49]. Unlike other models of fish (zebrafish, stickelback), the medaka is more robust, easily exposable to environmental samples, while also the tools of transgenesis are more effective in this model. It is also one of the fish models referenced by the OECD for the detection of estrogenic disruptors.

Finally, amphibians are also used as indicators for ecotoxicological potencies of several envi‐ ronmental stressors. The aquatic larvae are continuously exposed to chemicals compounds present in water because the eggs are lacking a protective eggshell or membrane. After hatching, the skin of amphibians' larvae is still very permeable, allowing an easy penetra‐ tion of all compounds leading to high bioavailability and bioaccumulation of endocrine dis‐ ruptors. This development stage of amphibians is the most sensitive and the most used to study effects of environmental pollutants. Thus, Fini *et al*. used *in vivo X. laevis* tadpoles to monitor heavy metal pollution in water in continuous flow systems [50]. The same team al‐ ready used *X. laevis* to detect the thyroid disrupting effect of BPA *in vivo* [51].

## **4. Impact of endocrine disruptors on thyroid hormone and corticosteroids systems in fish**

Many natural and man-made chemicals (plasticizers, pesticides, detergents and pharma‐ ceuticals) interfere with the endocrine system and can result in adverse health effects in humans, mammals and fish. Wildlife living in or in closer association with the aquatic environment are especially impacted by these endocrine disruptors, because water act as sinks for chemical discharges. Thus, fish and amphibians are the main po‐ tential targets for endocrine disruption at multiple levels, either direct or indirect, through ingestion and accumulation of endocrine disruptor, the exposition or through the food chain. Chronic exposure to endocrine disruptors, such as the oestrogenic com‐ pounds used in birth control, can feminize male fish and decrease their capacity to re‐ produce. In the opposite, masculinised female specimens were found in effluent containing androgenic chemicals [52]. Endocrine disruption on thyroid hormone and corticosteroids in fish was also studied.

it significantly reduced T3-induced fluorescence suggesting disruption of the thyroid func‐ tion by BPA [23]. Many other natural (Rotenone) or synthetic (Malathion, Endosulfan) chemicals present in the environment produce thyroid disruption in fish, with varied re‐

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Because of the close relationship between thyroid and corticosteroid axis in fish and am‐ phibians, alteration of one of these axes could affect the other one. Indeed endocrine disrup‐ tion due to chemicals present in the environment affects these two endocrine systems, separately or concomitant. Further studies have to investigate the final effect of disruption

Nuclear receptor crossregulation are important mechanisms for amplifying hormone sig‐ nals, regulating hormone activity through negative feedback, and coordinating hormone ac‐ tion in a temporal and tissue-specific-manner. In this chapter, we were interested in crossregulation between thyroid hormone and corticosteroids. These two endocrine systems are keys actors of many physiological processes. Their coincident expression and synergetic action were studied in different models (amphibian metamorphosis, stress response in fish). Corticosteroids are known to synergize with thyroid hormone to promote metamorphosis, and links between the thyroid and corticosteroid axes are present at multiple levels. Under‐ standing the interactions between TH and CS will allow us to better understand the effects

The use of fish species as model organism for research on endocrine disruption is interesting the identification of potential new endocrine disruptors because of endocrine system and hormone signalling pathways are sufficiently similar to other vertebrates. In this context, we should observe more and more studies leading to a large development of screening tools based on these aquatic animals in a next future. However the consequences of the molecu‐ lar, physiological or organisms' effects for the population may be different between species. Confirmation in higher vertebrates or in humans of the effects observed in fish is necessary if we want to clearly identify new endocrine disruptors. For instance, the use of aquatic or‐ ganisms in endocrine disruption studies is relevant, because of their closeness with watersoluble chemicals. And the vast technical possibilities offered by the zebrafish and the

INRA, UR1037 Fish Physiology and Genomics, BIOSIT Rennes, F-35000 Rennes, France,

medaka models for functional genomics studies justify their use in ED research.

sponses (for review see reference 7).

**5. Conclusion**

of endocrine disruptors.

**Author details**

France

Xavier Terrien and Patrick Prunet

of one of these endocrine axes on the other one.

Endocrine disruptors may impact corticosteroid signalling system in a direct manner (competition with endogenous ligand) or indirect manner (alteration in accessory pro‐ teins, kinases, cytoskeleton...). Among corticosteroids disruptors, chemicals including Polychlorinated Biphenyls (PCB) and heavy metals were the most studied. For exam‐ ple, it has been shown that Arsenic affects GR signalling and one mechanism involves the downregulation of GR content in trout hepatocytes [53]. Another study showed that Copper exposure during 5 days *in vivo* reduced GR-immunoreactivity in rainbow trout gill cells [54].

Cadmium is another metal that is widely distributed in the aquatic environment and is toxic to fish at sublethal concentrations [55]. Due to its long half-life and low excre‐ tion rate, Cadmium can also accumulate in various organs, primarily within the liver, kidney and reproductive and respiratory systems in fish [56]. This metal is known to disrupt head kidney corticosteroid production in fish. Vijayan *et al* [57] showed recent‐ ly that Cadmium exposure leads to suppression of the ACTH-stimulated cortisol pro‐ duction. This study suggested that MC2R signalling, the primary step in ACTHinduced cortocosteroidogenesis, is a key target for Cadmium-mediated disruption of cortisol production in trout.

Because Polybrominated Diphenyl Ethers (PBDE), used as flame retardants, are similar in structure to thyroxine T4 and tri-iodothyronine T3 [58], several teams have studied their ef‐ fect on thyroid function. Biologic effects of PBDEs in rodent are similar to those of PCB, in‐ creasing risks for reproductive and endocrine disruption. In 2011, Yu *et al*. [59] exposed zebrafish to low levels of PBDEs for most their lives. While the fish did accumulate different types of PBDE in their tissues, there was no toxicity. But the PBDEs were present in the eggs, and the chemicals and their associated toxic effects passed along the progeny and reduced hatch rates and altered thyroid hormone system of the next generation. This study is impor‐ tant because it shows that PBDEs trigger thyroid hormone disruption not only in the ex‐ posed population but also in the subsequent generation.

Due to its structural homology with thyroid hormone, Bisphenol A (BPA) is also frequently studied as endocrine disruptor for thyroid function. Is has been shown that BPA can interact with TR and it can be considered as a TR antagonist [60,61]. First, transgenic *X. laevis* were used to test *in vivo* whether BPA interferes with TH and to create an *in vivo* detection system for BPA endocrine disruptive properties [62]. More recently, Terrien *et al*. [23] used zebrafish receiving transient transgenesis of TR-sensitive reporter systems to study BPA disrupting ef‐ fects. In this fish model, the green fluorescent protein (GFP) is expressed under the control of the TH/bZip promoter from *X. laevis* known to contain two thyroid hormone responsive elements (TRE). Exposure to T3 increased the GFP fluorescence in these transient transgenic fish. When tested alone, Bisphenol A did not modify fluorescence, but when tested with T3, it significantly reduced T3-induced fluorescence suggesting disruption of the thyroid func‐ tion by BPA [23]. Many other natural (Rotenone) or synthetic (Malathion, Endosulfan) chemicals present in the environment produce thyroid disruption in fish, with varied re‐ sponses (for review see reference 7).

Because of the close relationship between thyroid and corticosteroid axis in fish and am‐ phibians, alteration of one of these axes could affect the other one. Indeed endocrine disrup‐ tion due to chemicals present in the environment affects these two endocrine systems, separately or concomitant. Further studies have to investigate the final effect of disruption of one of these endocrine axes on the other one.

## **5. Conclusion**

the food chain. Chronic exposure to endocrine disruptors, such as the oestrogenic com‐ pounds used in birth control, can feminize male fish and decrease their capacity to re‐ produce. In the opposite, masculinised female specimens were found in effluent containing androgenic chemicals [52]. Endocrine disruption on thyroid hormone and

Endocrine disruptors may impact corticosteroid signalling system in a direct manner (competition with endogenous ligand) or indirect manner (alteration in accessory pro‐ teins, kinases, cytoskeleton...). Among corticosteroids disruptors, chemicals including Polychlorinated Biphenyls (PCB) and heavy metals were the most studied. For exam‐ ple, it has been shown that Arsenic affects GR signalling and one mechanism involves the downregulation of GR content in trout hepatocytes [53]. Another study showed that Copper exposure during 5 days *in vivo* reduced GR-immunoreactivity in rainbow

Cadmium is another metal that is widely distributed in the aquatic environment and is toxic to fish at sublethal concentrations [55]. Due to its long half-life and low excre‐ tion rate, Cadmium can also accumulate in various organs, primarily within the liver, kidney and reproductive and respiratory systems in fish [56]. This metal is known to disrupt head kidney corticosteroid production in fish. Vijayan *et al* [57] showed recent‐ ly that Cadmium exposure leads to suppression of the ACTH-stimulated cortisol pro‐ duction. This study suggested that MC2R signalling, the primary step in ACTHinduced cortocosteroidogenesis, is a key target for Cadmium-mediated disruption of

Because Polybrominated Diphenyl Ethers (PBDE), used as flame retardants, are similar in structure to thyroxine T4 and tri-iodothyronine T3 [58], several teams have studied their ef‐ fect on thyroid function. Biologic effects of PBDEs in rodent are similar to those of PCB, in‐ creasing risks for reproductive and endocrine disruption. In 2011, Yu *et al*. [59] exposed zebrafish to low levels of PBDEs for most their lives. While the fish did accumulate different types of PBDE in their tissues, there was no toxicity. But the PBDEs were present in the eggs, and the chemicals and their associated toxic effects passed along the progeny and reduced hatch rates and altered thyroid hormone system of the next generation. This study is impor‐ tant because it shows that PBDEs trigger thyroid hormone disruption not only in the ex‐

Due to its structural homology with thyroid hormone, Bisphenol A (BPA) is also frequently studied as endocrine disruptor for thyroid function. Is has been shown that BPA can interact with TR and it can be considered as a TR antagonist [60,61]. First, transgenic *X. laevis* were used to test *in vivo* whether BPA interferes with TH and to create an *in vivo* detection system for BPA endocrine disruptive properties [62]. More recently, Terrien *et al*. [23] used zebrafish receiving transient transgenesis of TR-sensitive reporter systems to study BPA disrupting ef‐ fects. In this fish model, the green fluorescent protein (GFP) is expressed under the control of the TH/bZip promoter from *X. laevis* known to contain two thyroid hormone responsive elements (TRE). Exposure to T3 increased the GFP fluorescence in these transient transgenic fish. When tested alone, Bisphenol A did not modify fluorescence, but when tested with T3,

corticosteroids in fish was also studied.

trout gill cells [54].

248 Insights from Veterinary Medicine

cortisol production in trout.

posed population but also in the subsequent generation.

Nuclear receptor crossregulation are important mechanisms for amplifying hormone sig‐ nals, regulating hormone activity through negative feedback, and coordinating hormone ac‐ tion in a temporal and tissue-specific-manner. In this chapter, we were interested in crossregulation between thyroid hormone and corticosteroids. These two endocrine systems are keys actors of many physiological processes. Their coincident expression and synergetic action were studied in different models (amphibian metamorphosis, stress response in fish). Corticosteroids are known to synergize with thyroid hormone to promote metamorphosis, and links between the thyroid and corticosteroid axes are present at multiple levels. Under‐ standing the interactions between TH and CS will allow us to better understand the effects of endocrine disruptors.

The use of fish species as model organism for research on endocrine disruption is interesting the identification of potential new endocrine disruptors because of endocrine system and hormone signalling pathways are sufficiently similar to other vertebrates. In this context, we should observe more and more studies leading to a large development of screening tools based on these aquatic animals in a next future. However the consequences of the molecu‐ lar, physiological or organisms' effects for the population may be different between species. Confirmation in higher vertebrates or in humans of the effects observed in fish is necessary if we want to clearly identify new endocrine disruptors. For instance, the use of aquatic or‐ ganisms in endocrine disruption studies is relevant, because of their closeness with watersoluble chemicals. And the vast technical possibilities offered by the zebrafish and the medaka models for functional genomics studies justify their use in ED research.

## **Author details**

Xavier Terrien and Patrick Prunet

INRA, UR1037 Fish Physiology and Genomics, BIOSIT Rennes, F-35000 Rennes, France, France

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**Section 4**

**Highlights for Homeopathic Therapeuthicals**


**Highlights for Homeopathic Therapeuthicals**

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**Chapter 11**

**Acute Toxicity Profiles of Aqueous and Ethanolic**

**Uganda**

Charles Lagu and Frederick I. B. Kayanja

http://dx.doi.org/10.5772/53599

**1. Introduction**

the seed oil.

thocyanosides

Additional information is available at the end of the chapter

**Extracts of** *Capsicum annum* **Seeds from South Western**

*Capsicum annum* belongs to the Kingdom Plantae plants, subkingdom of Tracheobiota (vas‐ cular plants), super-division of Spermatophyta (seed plants), division of Magnoliophyta (flowering plants), class Magnoliopsida (dicotyledons), subclass Asteridae, order Solanels, family Solanaceae (potato family), genus Capsicum L. (pepper), species *Capsicum annum* L.

*Capsicuma annum* is a perennial shrub growing up to 2 m (6') in height, having a woody trunk. It has green fruits that ripen to red. The active ingredient in the plant is *capsaicin* that is used for the management of various medical conditions. The varieties of this "fruit" vary greatly in size, colour and pungency. The plant extract that provides therapeutic action is

Globally, *Capsicum annum* has many uses. It is used as a feed additive and as spices. The cor‐ rosive nature of capsaicin, its gross irritating effects and its toxicities are well documented. A qualitative assessment of bioactive chemical compounds in *Capsicum annum* established the presence of reducing compounds, saponins, alkaloid salts, alkaloids, quarternary bases, anthracenosides, flavanosides, flavonds, coumourin derivatives, steroid glycosides and an‐

It is indeed true that *Capsicum annum* possesses many medicinal plant bioactive substances that are active against certain bacteria, viruses and protozoa [1-3]. In-vitro studies indicated that *Capsicum annum* fruits are efficacious against common bacteria e.g. Staphylococcus, Streptococcus species etc. The widespread use of *Capsicum annum* as an herbal remedy by smallholder poultry farmers is based on a wide range of doses [3]. A wide range of medici‐

> © 2013 Lagu and Kayanja; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Lagu and Kayanja; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(Cayenne pepper) and variety *Capsicum annum* L. var annum (Cayenne pepper).

## **Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of** *Capsicum annum* **Seeds from South Western Uganda**

Charles Lagu and Frederick I. B. Kayanja

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53599

## **1. Introduction**

*Capsicum annum* belongs to the Kingdom Plantae plants, subkingdom of Tracheobiota (vas‐ cular plants), super-division of Spermatophyta (seed plants), division of Magnoliophyta (flowering plants), class Magnoliopsida (dicotyledons), subclass Asteridae, order Solanels, family Solanaceae (potato family), genus Capsicum L. (pepper), species *Capsicum annum* L. (Cayenne pepper) and variety *Capsicum annum* L. var annum (Cayenne pepper).

*Capsicuma annum* is a perennial shrub growing up to 2 m (6') in height, having a woody trunk. It has green fruits that ripen to red. The active ingredient in the plant is *capsaicin* that is used for the management of various medical conditions. The varieties of this "fruit" vary greatly in size, colour and pungency. The plant extract that provides therapeutic action is the seed oil.

Globally, *Capsicum annum* has many uses. It is used as a feed additive and as spices. The cor‐ rosive nature of capsaicin, its gross irritating effects and its toxicities are well documented. A qualitative assessment of bioactive chemical compounds in *Capsicum annum* established the presence of reducing compounds, saponins, alkaloid salts, alkaloids, quarternary bases, anthracenosides, flavanosides, flavonds, coumourin derivatives, steroid glycosides and an‐ thocyanosides

It is indeed true that *Capsicum annum* possesses many medicinal plant bioactive substances that are active against certain bacteria, viruses and protozoa [1-3]. In-vitro studies indicated that *Capsicum annum* fruits are efficacious against common bacteria e.g. Staphylococcus, Streptococcus species etc. The widespread use of *Capsicum annum* as an herbal remedy by smallholder poultry farmers is based on a wide range of doses [3]. A wide range of medici‐

nal plant users do not believe that plant extracts can be toxic, yet standard laboratory tests [1, 3-5] indicate profound toxicity from some medicinal plants.

**2.2. Acute toxicity study protocol**

**Groups**

the use of experimental animals for the experiments.

mice each and kept in different cages for easy observation.

ried out earlier. The doses rates were as shown in table 1.

**Table 1.** Preliminary acute toxicity levels of crude extracts of *Capsicum annum*

(mg/ml). The extracts were administered by gavage orally.

and brain were observed and recorded.

aqueous extracts were 10,000, 12,000, 14,000 and 16,000 mg/ml.

The ethical committee of Mbarara University of Science and Technology (MUST) approved

Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda

Swiss albino mice of 16-25 gr bodyweight from both sexes were selected and labelled using tail markers of different colours. The animals (n=30) were divided into five groups of six

The dose levels were determined after a preliminary acute toxicity trial, which had been car‐

**Dose levels (mg/kg)** *n*

Group 1 10,000 3500 6 0 0 Group 2 12,000 5000 6 2 2 Group 3 14,000 6500 6 4 4 Group 4 16,000 8000 6 6 6 Control 1ml dH20 6 0 0

The volumes of the drug doses administered to the mice were calculated using the following formula [4]: Volume (ml) = body weight (kg) x dose levels (mg/kg)/stock drug concentration

The derived dose levels for ethanolic extracts were 3,500, 5000, 6500 and 8,000 mg/kg and for

**Postmortem procedure:** The numbers of mice which died after being subjected to the medic‐ inal plant crude extracts were noted. Randomly selected animals (n=2) from each group were submitted to necropsy for organ collection. The surviving mice were sacrificed at the end of the experiment after observation for 7 days and subjected to post mortem analysis. Within 24hrs, organs from mice that died or were sacrificed were collected and fixed in jars containing formalin (10% formaldehyde) and subsequently submitted to routine paraffin embedding. The liver, kidney, lungs, intestines and brain were collected for histopathology analysis. Four (4) micrometer sections of the collected organs were routinely stained with haematoxylin and eosin for histological examination, performed by a Pathologist. Putative histopathological changes in the structural organization of the liver, kidney, lungs, intestines

**Data collection and analysis**: Mortality (number of dead mice) were counted in each group and recorded. The median lethal dose that killed 50% of the test animals was determined

**Aqueous (a)** *Ethanolic (b)* **Aqueous (a)** *Ethanolic (b)*

**Nº dead**

http://dx.doi.org/10.5772/53599

259

**Extracts**

The smallholder farmers in the South Western Agro-ecological Zone of Uganda (SWAEZ) use *Capsicum annum* [6] as a medicinal plant for controlling various poultry diseases like Newcastle disease. They would pick 3-4 seeds of Capsicum annum seeds from their gardens or neighbours gardens and crush the harvested seeds in a cup and then mixed it in various proportions of water, from half a litre to up to two litres. The mixed extract is then given to the suspected sick birds infected with Newcastle disease. The birds may be assisted to drink the extract or the birds may drink it freely at will. The shelf life of the prepared extracts is usually one to at least two days then fresh mixtures are constituted depending on the severi‐ ty and improvements in the health conditions of the bird(s).

Despite the plant and its compounds being used by the farmers, the acute toxicities of *Capsi‐ cum annum* extracted under aqueous and ethanolic solvent systems are not yet known. The study hypothesized that medicinal plant with unknown acute toxicity levels exist in the SWAEZ of Uganda. The study assessed the acute toxicity levels of *Capsicum annum* in the SWAEZ under aqueous and ethanolic solvent systems.

## **2. Materials and methods**

#### **2.1. Plant seed collection and preparation**

The *Capsicum annum* seeds were collected from Rubindi Sub County of Mbarara district in the South Western Agro-Ecological Zone (SWAEZ). A total of 2 kilograms of the sample was harvested and labelled with the laboratory number (CA 001 Lag), date and kept in black a polythene bag in a cupboard. The seeds were packed in a paper box wrapped in polythene and taken to the Division of Pharmacology and Physiological Sciences, College of Veterinary Medicine, Animal Resources and Bio-security (COVAB), Makerere University (Uganda). The seeds were then dried at room temperature of 250 C for 7 days. Afterwards, 200 gr were weighed for water extraction and another 200 gr for ethanolic extraction. The seeds were manually ground to fine powder using a mortar and pestle.

**Weighing and extraction process:** A total of 160 gr of the powdered sample was weighed and soaked in a dark bottle containing 1 litre of 70% ethanol with intermittent shaking for 72 hours. Similarly, 160 gr were weighed for extraction in 1 litre of water. After three days of extraction the extract was filtered using cotton wool fixed within a funnel. The filtrate was collected in a glass conical flask and labelled, and the residue discarded. The filtrate was then subjected to a rotary evaporator at reduced pressure and temperature for concentration to a constant volume. After concentration the extract was put in an oven at 450 c until a semisolid form of extract was obtained. This was put in bijous bottle, well labelled, and kept in a refrigerator at 40 c for future use. The percentage yield (%yield) =10g×100/160=6.25%. The percentage yield (%yield) was 6.25% of the fruit extract.

#### **2.2. Acute toxicity study protocol**

nal plant users do not believe that plant extracts can be toxic, yet standard laboratory tests

The smallholder farmers in the South Western Agro-ecological Zone of Uganda (SWAEZ) use *Capsicum annum* [6] as a medicinal plant for controlling various poultry diseases like Newcastle disease. They would pick 3-4 seeds of Capsicum annum seeds from their gardens or neighbours gardens and crush the harvested seeds in a cup and then mixed it in various proportions of water, from half a litre to up to two litres. The mixed extract is then given to the suspected sick birds infected with Newcastle disease. The birds may be assisted to drink the extract or the birds may drink it freely at will. The shelf life of the prepared extracts is usually one to at least two days then fresh mixtures are constituted depending on the severi‐

Despite the plant and its compounds being used by the farmers, the acute toxicities of *Capsi‐ cum annum* extracted under aqueous and ethanolic solvent systems are not yet known. The study hypothesized that medicinal plant with unknown acute toxicity levels exist in the SWAEZ of Uganda. The study assessed the acute toxicity levels of *Capsicum annum* in the

The *Capsicum annum* seeds were collected from Rubindi Sub County of Mbarara district in the South Western Agro-Ecological Zone (SWAEZ). A total of 2 kilograms of the sample was harvested and labelled with the laboratory number (CA 001 Lag), date and kept in black a polythene bag in a cupboard. The seeds were packed in a paper box wrapped in polythene and taken to the Division of Pharmacology and Physiological Sciences, College of Veterinary Medicine, Animal Resources and Bio-security (COVAB), Makerere University (Uganda).

weighed for water extraction and another 200 gr for ethanolic extraction. The seeds were

**Weighing and extraction process:** A total of 160 gr of the powdered sample was weighed and soaked in a dark bottle containing 1 litre of 70% ethanol with intermittent shaking for 72 hours. Similarly, 160 gr were weighed for extraction in 1 litre of water. After three days of extraction the extract was filtered using cotton wool fixed within a funnel. The filtrate was collected in a glass conical flask and labelled, and the residue discarded. The filtrate was then subjected to a rotary evaporator at reduced pressure and temperature for concentration

solid form of extract was obtained. This was put in bijous bottle, well labelled, and kept in a

c for future use. The percentage yield (%yield) =10g×100/160=6.25%. The

to a constant volume. After concentration the extract was put in an oven at 450

C for 7 days. Afterwards, 200 gr were

c until a semi-

[1, 3-5] indicate profound toxicity from some medicinal plants.

ty and improvements in the health conditions of the bird(s).

SWAEZ under aqueous and ethanolic solvent systems.

The seeds were then dried at room temperature of 250

percentage yield (%yield) was 6.25% of the fruit extract.

manually ground to fine powder using a mortar and pestle.

**2. Materials and methods**

258 Insights from Veterinary Medicine

refrigerator at 40

**2.1. Plant seed collection and preparation**

The ethical committee of Mbarara University of Science and Technology (MUST) approved the use of experimental animals for the experiments.

Swiss albino mice of 16-25 gr bodyweight from both sexes were selected and labelled using tail markers of different colours. The animals (n=30) were divided into five groups of six mice each and kept in different cages for easy observation.

The dose levels were determined after a preliminary acute toxicity trial, which had been car‐ ried out earlier. The doses rates were as shown in table 1.


**Table 1.** Preliminary acute toxicity levels of crude extracts of *Capsicum annum*

The volumes of the drug doses administered to the mice were calculated using the following formula [4]: Volume (ml) = body weight (kg) x dose levels (mg/kg)/stock drug concentration (mg/ml). The extracts were administered by gavage orally.

The derived dose levels for ethanolic extracts were 3,500, 5000, 6500 and 8,000 mg/kg and for aqueous extracts were 10,000, 12,000, 14,000 and 16,000 mg/ml.

**Postmortem procedure:** The numbers of mice which died after being subjected to the medic‐ inal plant crude extracts were noted. Randomly selected animals (n=2) from each group were submitted to necropsy for organ collection. The surviving mice were sacrificed at the end of the experiment after observation for 7 days and subjected to post mortem analysis. Within 24hrs, organs from mice that died or were sacrificed were collected and fixed in jars containing formalin (10% formaldehyde) and subsequently submitted to routine paraffin embedding. The liver, kidney, lungs, intestines and brain were collected for histopathology analysis. Four (4) micrometer sections of the collected organs were routinely stained with haematoxylin and eosin for histological examination, performed by a Pathologist. Putative histopathological changes in the structural organization of the liver, kidney, lungs, intestines and brain were observed and recorded.

**Data collection and analysis**: Mortality (number of dead mice) were counted in each group and recorded. The median lethal dose that killed 50% of the test animals was determined using the graphical and Probit analysis. Any signs of toxicity observed in the collected or‐ gans were also recorded.

**Quality assurance:** The calyces were shade dried to prevent loss of essential chemical com‐ ponents and the voucher specimen were taken to the herbarium (Biology Department, Mbarara University of Science and Technology) for identification and also to ensure the quality of the techniques adopted from world Health Organization (WHO) guidelines on herbal quality control.

Distilled water was used for extraction to prevent contamination of the extract. Both the aqueous and ethanolic extracts of *Capsicum annum* seeds were kept in sterilized bottles and placed in a refrigerator at 4o C to prevent mould formation. All the reagents used were ana‐ lytical grade.

The Swiss albino mice of the same age were used for the study to minimize variation in the test results. The control group given 1 ml of distilled water was used for comparison with the groups given the plant extract.

## **3. Results**

### **3.1. Aqueous acute toxicity profile of** *Capsicum annum* **seeds**

The aqueous acute toxicity profile of *Capsicum annum* seeds under various dose levels is shown in table 2.

**Graph 1.** Probit for aqueous *Capsicum annum* extracts. From the equation 1**Y=mx-c**,where: **Y**=0.0737(4.15)+3.7749 =

Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda

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261

There were notable side effects associated to higher doses of *Capsicum* extracts, such as limb movements, gut irritation, loss of balance, GIT muscle twitching. Hyperactivity was noted within 2 minutes after administration. Vocalization, urination, drowsiness, reddening of lips, dyspnoea and circling movement were noted and convulsions occurred before death.

The ethanolic acute toxicity profile of *Capsicum annum* seeds under various dose levels is shown in table 2. The LD50 was calculated according to Fisher's and Yates as in Table 2. The

For the ethanolic extracts at a dosage of 3500mg/kg and 5000mg/kg, the following signs were observed: irritation shown by the use of forelimbs to scratch the areas of the mouth, gasping for air and dyspnoea. For aqueous extracts at a dosage of 6500mg/kg and 8000mg/kg there were observed twitching of GIT muscles, urination, dry mouth, reddening

**3.5. Comparison of key histo-pathological findings due to aqueous and ethanolic extracts**

The key histopathological findings due to the effects of aqueous and 70% ethanolic extracts

0.305855+ 3.7749 = 4.080755; **Ld50** = antilog of 4.080755 =12,043.563 mg/kg.

**3.2. Toxicity signs observed with dose increase within the groups**

**3.3. Acute toxicity of 70% ethanolic extract of** *Capsicum annum*

lethal dose was established to be 5,492mg/kg (Graph 2).

**3.4. Signs of acute toxicity observed**

of *Capsicum annum* are detailed in table 3.

of lips and convulsions.


**Table 2.** Acute toxicity profile of aqueous and ethanolic *Capsicum annum extracts*

It was observed that it would take 12043 mg/kg to kill 50% of the test animals (Graph 1). This gives a wide safety margin for the lethal dose of *Capsicum annum* fruits extracts in test animals. This is the LD 50 for the aqueous extract; which is the most commonly method used by the local farmers.

**Graph 1.** Probit for aqueous *Capsicum annum* extracts. From the equation 1**Y=mx-c**,where: **Y**=0.0737(4.15)+3.7749 = 0.305855+ 3.7749 = 4.080755; **Ld50** = antilog of 4.080755 =12,043.563 mg/kg.

#### **3.2. Toxicity signs observed with dose increase within the groups**

There were notable side effects associated to higher doses of *Capsicum* extracts, such as limb movements, gut irritation, loss of balance, GIT muscle twitching. Hyperactivity was noted within 2 minutes after administration. Vocalization, urination, drowsiness, reddening of lips, dyspnoea and circling movement were noted and convulsions occurred before death.

#### **3.3. Acute toxicity of 70% ethanolic extract of** *Capsicum annum*

The ethanolic acute toxicity profile of *Capsicum annum* seeds under various dose levels is shown in table 2. The LD50 was calculated according to Fisher's and Yates as in Table 2. The lethal dose was established to be 5,492mg/kg (Graph 2).

#### **3.4. Signs of acute toxicity observed**

using the graphical and Probit analysis. Any signs of toxicity observed in the collected or‐

**Quality assurance:** The calyces were shade dried to prevent loss of essential chemical com‐ ponents and the voucher specimen were taken to the herbarium (Biology Department, Mbarara University of Science and Technology) for identification and also to ensure the quality of the techniques adopted from world Health Organization (WHO) guidelines on

Distilled water was used for extraction to prevent contamination of the extract. Both the aqueous and ethanolic extracts of *Capsicum annum* seeds were kept in sterilized bottles and

The Swiss albino mice of the same age were used for the study to minimize variation in the test results. The control group given 1 ml of distilled water was used for comparison with

The aqueous acute toxicity profile of *Capsicum annum* seeds under various dose levels is

**Extracts Group Dose (mg/kg) log dose dead/total dead% Probit**

It was observed that it would take 12043 mg/kg to kill 50% of the test animals (Graph 1). This gives a wide safety margin for the lethal dose of *Capsicum annum* fruits extracts in test animals. This is the LD 50 for the aqueous extract; which is the most commonly method

 10,000 4.00 0/6 0 3.04 12,000 4.08 2/6 33.33 4.53 14,000 4.15 3/6 50.00 4.95 16,000 4.20 4/6 66.66 5.36 18,000 4.26 6/6 100.00 6.75

 3500 3.54 0/6 0 3.04 5000 3.70 2/6 33.33 4.53 6500 3.81 4/6 66.67 5.36 8000 3.90 6/6 100 6.75

C to prevent mould formation. All the reagents used were ana‐

gans were also recorded.

260 Insights from Veterinary Medicine

herbal quality control.

lytical grade.

**3. Results**

shown in table 2.

Aqueous

Ethanolic

used by the local farmers.

placed in a refrigerator at 4o

the groups given the plant extract.

**3.1. Aqueous acute toxicity profile of** *Capsicum annum* **seeds**

**Table 2.** Acute toxicity profile of aqueous and ethanolic *Capsicum annum extracts*

For the ethanolic extracts at a dosage of 3500mg/kg and 5000mg/kg, the following signs were observed: irritation shown by the use of forelimbs to scratch the areas of the mouth, gasping for air and dyspnoea. For aqueous extracts at a dosage of 6500mg/kg and 8000mg/kg there were observed twitching of GIT muscles, urination, dry mouth, reddening of lips and convulsions.

#### **3.5. Comparison of key histo-pathological findings due to aqueous and ethanolic extracts**

The key histopathological findings due to the effects of aqueous and 70% ethanolic extracts of *Capsicum annum* are detailed in table 3.

**Organs examined**

Lungs

Small intestine

Liver

**Control group Toxic effects associated to**

D E

D −Kidney (Control) Medulla. The scale bar is 100µm

G −Lungs (Control). The scale bar is 100µm

J −Small intestine (Control). The scale bar is 100µm

**Capsicum annum aqueous extracts**

Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda

E −Kidney renal haemorrhages, congestion and tissue degeneration at 12,000mg/kg. The scale bar is 100µm

G H I

H −Mild Lung emphysema noted at 10,000mg/kg. The scale bar is 100µm

<sup>J</sup> <sup>K</sup> <sup>L</sup>

K −Erosions of the small intestine at 14,000mg/kg. The scale bar is 100µm

<sup>M</sup> <sup>N</sup> <sup>O</sup>

**Toxic effects associated to Capsicum annum 70% alcohol extracts**

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263

F −Haemorrhages and congestion at 5,00mg/kg. The scale bar is 100µm

I −Lungs haemorrhages and congestion at 5,000mg/kg. The scale bar is 100µm

L −Intestines villus sloughing off and mucosal erosion at 6,500mg/kg. The scale bar is 100µm

**Graph 2.** Probit for 70% ethanolic *Capsicum annum* extracts. From the equation 2**y=bx-c**, where **Y**=0.0989 (4.945) + 3.2507 = 0.4890605 + 3.2507 = 3.7397605..**Ld50** = antilog (3.7397605) = 5492.3790

#### **3.6. Histopathology findings**


Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda http://dx.doi.org/10.5772/53599 263


**Graph 2.** Probit for 70% ethanolic *Capsicum annum* extracts. From the equation 2**y=bx-c**, where **Y**=0.0989 (4.945) +

**Capsicum annum aqueous extracts**

A B C

B −No significant effects on the brain at 10,000mg/kg. The scale bar is 100µm

**Toxic effects associated to Capsicum annum 70% alcohol extracts**

C −Slight congestion of the meninges at 3500mg/kg. The scale bar is 100µm

F

**Control group Toxic effects associated to**

3.2507 = 0.4890605 + 3.2507 = 3.7397605..**Ld50** = antilog (3.7397605) = 5492.3790

**3.6. Histopathology findings**

262 Insights from Veterinary Medicine

A −Brain (Control). The scale bar is 100µm

**Organs examined**

Brain

Kidney (Medulla)


The extracts derived from ethanolic solvents have lower LD50 of 5492mg/kg because ethanol is less polar. The ethanolic extracts have yet another advantage: the longer shelf life for use because of the preservative effects of alcohol on the extracts when compared to that of the water in aqueous extracts [5]. It can be noted here that extracts, which seem to show high toxicity in many circumstances, have no traditional uses, which is in agreement with previ‐

Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda

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265

After administration of the higher extract dosages there were often observed notable clinical signs in test animals, due to the side effects of the extracts. Key clinical signs noted were irri‐ tation, evidenced by the use of fore limbs to scratch the areas of the mouth, gasping for air, dyspnoea, twitching of GIT muscle, urination, dry mouth, reddening of lips and convul‐ sions. The effects of bioavailability of the drugs on major organs like the intestines, liver, lungs, kidneys and brain showed clear manifestations of the presence of the active substan‐ ces. Bioavailability is the rate and extent to which the active drug ingredient is absorbed from a drug product and becomes available at the site of drug action. The effects of the ac‐ tive substances in the body of the mice could affect by the rate of disintegration of the drug product, dissolution of the drug in the fluids at the absorption site or the transfer of drug molecule across the membrane lining the gastrointestinal tract into the systemic circulation.

The key physiologic factors that could affect the availability of the active substances in the body include: variations in absorption power along GI tract, variations in pH of GI fluids, gastric emptying rate, intestinal motility, perfusion of GI tract, presystemic and first-pass metabolism, age, sex, weight and disease states. The interactions with other substances like

There exist some reports of seeds side effects by *Capsicum annum* on chickens reared under the traditional use system. This finding concurs with the experimental trials. Aware of this fact, is to note that mice, chickens compared to other mammals hence exhibit a large surface

The histopathological findings showed significant effects on brain, kidney, lungs, small in‐ testines, liver and heart. Studies in India indicated that necropsy examination is paramount in linking the general and target organ specific toxic effects of phytomedicine [9]. Many oth‐ ers are in agreement OECD [10-12]. Absence of any significant gross pathological lesions in treated rats and mice at the terminal sacrifice indicates the justifiable harmless nature of the

Results of study indicated that the lethal dose (LD50) of aqueous extracts of *Capsicum annum* was12043 mg/kg compared with 5,492mg/kg for 70% ethanolic extracts. The clinical signs noted depended on the level of concentration of the plant extracts.The study concludes that 70% ethanol extracts of *Capsicum annum and aqueous extracts* of capsicum annum are safe to use and are classified as practically non-toxic (5-15g/kg body weight). Both extracts are suit‐

food, fluid volume and other drugs or chemicals could also play an important role.

ous findings [1,8].

area compared to other mammals.

phytomedicine [9].

**5. Conclusion**

**Table 3.** Comparison of key histopathological findings of aqueous and 70% ethanolic extracts for the collected organs

## **4. Discussion**

### **4.1. Lethal dose (LD50) levels**

The lethal dose (LD50) of aqueous extracts of *Capsicum annum* was found to be12043 mg/kg. The 70% ethanolic extracts of *Capsicum annum* had a lethal dose (LD50) of 5492 mg/kg. Con‐ sequently the *Capsicum annum* is classified as practically non-toxic because the LD50 results fall in the dose range of 5-15g/kg of body weight [4].

Local smallholder farmers are using aqueous extracts of *Capsicum annum* seeds to manage various disorders. This study has confirmed that the use of the aqueous extract of *Capsicum annum* by the local poultry farmers is safe. Save to note here that the smallholder farmer's dose levels greatly vary from one farmer to another and in varying levels of concoctions [6]. The estimate dose for local farmers range between 4014-6022mg/kg for aqueous extracts. The farmers' dose levels are two-three times below the determined LD50 of 12043mg/Kg body weight for aqueous extracts.

The Organization for Economic Cooperation Development (OECD) Guidelines for the Test‐ ing of Chemicals [7] recommended that the maximum dose levels for any chemical com‐ pounds should not exceed 5000 mg/kg of the animal body weight. The ethanolic extraction of Capsicum annum has advantages compared to the aqueous because ethanol is better sol‐ vent for extraction of bioactive compounds.

The extracts derived from ethanolic solvents have lower LD50 of 5492mg/kg because ethanol is less polar. The ethanolic extracts have yet another advantage: the longer shelf life for use because of the preservative effects of alcohol on the extracts when compared to that of the water in aqueous extracts [5]. It can be noted here that extracts, which seem to show high toxicity in many circumstances, have no traditional uses, which is in agreement with previ‐ ous findings [1,8].

After administration of the higher extract dosages there were often observed notable clinical signs in test animals, due to the side effects of the extracts. Key clinical signs noted were irri‐ tation, evidenced by the use of fore limbs to scratch the areas of the mouth, gasping for air, dyspnoea, twitching of GIT muscle, urination, dry mouth, reddening of lips and convul‐ sions. The effects of bioavailability of the drugs on major organs like the intestines, liver, lungs, kidneys and brain showed clear manifestations of the presence of the active substan‐ ces. Bioavailability is the rate and extent to which the active drug ingredient is absorbed from a drug product and becomes available at the site of drug action. The effects of the ac‐ tive substances in the body of the mice could affect by the rate of disintegration of the drug product, dissolution of the drug in the fluids at the absorption site or the transfer of drug molecule across the membrane lining the gastrointestinal tract into the systemic circulation.

The key physiologic factors that could affect the availability of the active substances in the body include: variations in absorption power along GI tract, variations in pH of GI fluids, gastric emptying rate, intestinal motility, perfusion of GI tract, presystemic and first-pass metabolism, age, sex, weight and disease states. The interactions with other substances like food, fluid volume and other drugs or chemicals could also play an important role.

There exist some reports of seeds side effects by *Capsicum annum* on chickens reared under the traditional use system. This finding concurs with the experimental trials. Aware of this fact, is to note that mice, chickens compared to other mammals hence exhibit a large surface area compared to other mammals.

The histopathological findings showed significant effects on brain, kidney, lungs, small in‐ testines, liver and heart. Studies in India indicated that necropsy examination is paramount in linking the general and target organ specific toxic effects of phytomedicine [9]. Many oth‐ ers are in agreement OECD [10-12]. Absence of any significant gross pathological lesions in treated rats and mice at the terminal sacrifice indicates the justifiable harmless nature of the phytomedicine [9].

## **5. Conclusion**

**Organs examined**

264 Insights from Veterinary Medicine

Heart

**4. Discussion**

**4.1. Lethal dose (LD50) levels**

body weight for aqueous extracts.

vent for extraction of bioactive compounds.

**Control group Toxic effects associated to**

M −Liver (Control). The scale bar is 100µm

P

P −Heart (Control). The scale bar is 100µm

fall in the dose range of 5-15g/kg of body weight [4].

**Capsicum annum aqueous extracts**

N −Hepatic degeneration and congestion of the liver at 14,000mg/kg. The scale bar is 100µm

Q

Q −Myocardial haemorrhages at 12,000mg/kg. The scale bar is 100µm

**Table 3.** Comparison of key histopathological findings of aqueous and 70% ethanolic extracts for the collected organs

The lethal dose (LD50) of aqueous extracts of *Capsicum annum* was found to be12043 mg/kg. The 70% ethanolic extracts of *Capsicum annum* had a lethal dose (LD50) of 5492 mg/kg. Con‐ sequently the *Capsicum annum* is classified as practically non-toxic because the LD50 results

Local smallholder farmers are using aqueous extracts of *Capsicum annum* seeds to manage various disorders. This study has confirmed that the use of the aqueous extract of *Capsicum annum* by the local poultry farmers is safe. Save to note here that the smallholder farmer's dose levels greatly vary from one farmer to another and in varying levels of concoctions [6]. The estimate dose for local farmers range between 4014-6022mg/kg for aqueous extracts. The farmers' dose levels are two-three times below the determined LD50 of 12043mg/Kg

The Organization for Economic Cooperation Development (OECD) Guidelines for the Test‐ ing of Chemicals [7] recommended that the maximum dose levels for any chemical com‐ pounds should not exceed 5000 mg/kg of the animal body weight. The ethanolic extraction of Capsicum annum has advantages compared to the aqueous because ethanol is better sol‐

**Toxic effects associated to Capsicum annum 70% alcohol extracts**

O −Liver wide necrosis and tissue degeneration at 6,500mg/kg. The scale bar is 100µm

R

R −Wide spread haemorrhages on the myocardium at 5,000mg/kg. The scale bar is 100µm

> Results of study indicated that the lethal dose (LD50) of aqueous extracts of *Capsicum annum* was12043 mg/kg compared with 5,492mg/kg for 70% ethanolic extracts. The clinical signs noted depended on the level of concentration of the plant extracts.The study concludes that 70% ethanol extracts of *Capsicum annum and aqueous extracts* of capsicum annum are safe to use and are classified as practically non-toxic (5-15g/kg body weight). Both extracts are suit‐

able for traditional poultry disease management by farmers. Further toxicity studies using chicken as animal species are necessary. Sub-acute and chronic toxicity tests are recom‐ mended in order to determine the long-term effects of the extract.

**Author details**

Mbarara, Uganda

**References**

ogy, Mbarara, Uganda

and Frederick I. B. Kayanja2

1 Department of Biology, Faculty of Science, Mbarara University of Science and Technology,

Acute Toxicity Profiles of Aqueous and Ethanolic Extracts of *Capsicum annum* Seeds from South Western Uganda

http://dx.doi.org/10.5772/53599

267

2 Department of Anatomy, Faculty of Medicine, Mbarara University of Science and Technol‐

[1] Chandra P, Sachan N, Ghosh A K and Kishore K (2010).Acute and Sub-chronic Oral Toxicity Studies of a Mineralo-herbal Drug Amlena on Experimental Rats.Interna‐

[2] Atsamo, A. D., Nguelefack, T. B., Datte, J.Y and Kamanyi, A (2011).Acute and sub‐ chronic oral toxicity assessment of the aqueous extract from stem bark of *Erythrinase‐*

[3] Wang Cuina, Zhang Tiehua, Liu Jun, Lu Shuang, Zhang Cheng, Wang Erlei, Wang Zuozhao, Zhang Yan, Liu Jingbo (2011).Subchronic toxicity study of corn silk with

[4] Ghosh, M.N.1984.Fundamentals of Experimental Pharmacology.2nd Edition.Culcut‐

[5] BussmannR.W.., Malca G., Glenn A., Sharon D., Nilsen B., Parris B. , Dubose D., Ruiz D., Saleda J., Martinez M., Carillo L. , Walker K., Kuhlman A., Townesmith (2011). Toxicity of medicinal plants used in traditional medicine in Northern Peru. J. Ethno‐

[6] Lagu C and Kayanja F I B 2010: Medicinal plant extracts widely used in the control of Newcastle disease (NCD) and helminthosis among village chickens of South Western Uganda. *Livestock Research for Rural Development.Volume 22, Article #200.*http://

[7] Organization for Economic Co-operation and Development (OECD), 1995 Guidelines for the Testing of Chemicals (No. 407, Section 4: Health Effects) "Repeated Dose 28- Day Oral Toxicity in Rodents" (Adopted on 12 May 198 1 and Updated on 27 July

www.lrrd.org/lrrd22/11/lagu22200.htm (accessed 2 November, 2011).

tional Journal of Pharmaceutical Research and Innovation, Vol. I: 15-18, 2010

*negalensis* DC (Fabaceae) in rodents. J. Ethnopharmacology. (2011).

\*Address all correspondence to: chlaguu@gmail.com

rats.J.Ethnopharmacol. (2011).

pharmacol. (2011).

1995.)

ta: Scientific Book Agency; 1984. pp. 153–158.

Charles Lagu1

#### **Conflict of interest**

The authors declare here that there is no conflict of interest.

### **Acknowledgement**

We profoundly thank the Belgian Technical Cooperation (BTC) for funding this research work. The vital roles of Dr.Vudriko Patrick, Mr. James Ndukui in laboratory services and coordination are appreciated. Dr.Nanyingi Mark and Dr. Sarah Nalule are recognized for their cordial advice and peer review.


#### **Appendix**

The percentage dead for 0 and 100 will be corrected before determination of LD50 using the following formula [4]: *For 0% dead: 100(0.25/n); for 100% dead: 100(n-0.25/n).*

**Table 4.** Probit conversion tables. Source: [4]

## **Author details**

able for traditional poultry disease management by farmers. Further toxicity studies using chicken as animal species are necessary. Sub-acute and chronic toxicity tests are recom‐

We profoundly thank the Belgian Technical Cooperation (BTC) for funding this research work. The vital roles of Dr.Vudriko Patrick, Mr. James Ndukui in laboratory services and coordination are appreciated. Dr.Nanyingi Mark and Dr. Sarah Nalule are recognized for

**% 0 1 2 3 4 5 6 7 8 9** 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45 4.48 4.50 4.53 4.56 4.49 4.61 4.64 4.67 4.69 4.72 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33

The percentage dead for 0 and 100 will be corrected before determination of LD50 using the following formula [4]:

mended in order to determine the long-term effects of the extract.

The authors declare here that there is no conflict of interest.

**Conflict of interest**

266 Insights from Veterinary Medicine

**Acknowledgement**

**Appendix**

their cordial advice and peer review.

*For 0% dead: 100(0.25/n); for 100% dead: 100(n-0.25/n).*

**Table 4.** Probit conversion tables. Source: [4]

Charles Lagu1 and Frederick I. B. Kayanja2

\*Address all correspondence to: chlaguu@gmail.com

1 Department of Biology, Faculty of Science, Mbarara University of Science and Technology, Mbarara, Uganda

2 Department of Anatomy, Faculty of Medicine, Mbarara University of Science and Technol‐ ogy, Mbarara, Uganda

## **References**


[8] Bizimenyera E.S., Swam G.E., Samdumu F. B., McGaw L.J and Eloff J. N (2007). Safe‐ ty profiles of peltophorumafricanumsond. (Fabaceae) extracts. PhD thesis, University of Pretoria; 2007.

**Chapter 12**

**The** *in vitro* **Antihelminthic Efficacy of** *Erythrina*

Helminth Infestation can lead to reduced growth and egg production in poultry. Coupled with high costs in 'wasted feeding' and demand for de-worming, this result in considerable economic losses in poultry enterprises and directly affects livelihood of small holder farmers [1]. In the birds, there is slow growth rate hence reduced body weight, delayed market weight attainment because of competition for nutrients by the bird and parasites. For the farmer, there is loss of income, reduced employment, and compromised household welfare,

Though an important veterinary practice, helminths control is largely neglected in villagelevel chicken production. The situation demands for alternative and inexpensive helminths control measures. Locally available medicinal plants have traditionally been used by small holder farmers to manage various livestock and human ailments [1-4], However, scientific data on the efficacy of these plants in helminthic control is lacking [5-8], It has been reported that local communities in the south-western agro-ecological zone (SWAEZ), Uganda, use *Er‐*

Ascaridiasis is a common disease of poultry (especially chicken and turkeys) in Uganda; it's caused by a nematode, *Ascaridia galli*. *A. galli* is a highly pathogenic worm residing in small

Ascaridiasis lead to weight depression and in severe cases causes intestinal blockage, loss of blood, reduced sugar content, retarded growth and mortality. It was noted that the age of the host and severity of exposure play a role in *A. galli* infections. Chickens older than 3-months are largely resistant to *A. galli* infection. *A galli* larvae undergo little to no development in older chicken.Larvaldevelopmentisarrestedinthethirdstageathighdoseratesasaresultofresistance

> © 2013 Lagu and Kayanja; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Lagu and Kayanja; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

difficulty to raise educational fees, health fess, and social security activities.

*ythrina abyssinica* (Leguminocae) extracts to deworm village chicken [9].

intestines and is transmitted through eggs.

*Abyssinica* **Extracts on** *Ascaridia galli*

Additional information is available at the end of the chapter

Charles Lagu and FIB Kayanja

http://dx.doi.org/10.5772/53708

**1. Introduction**


## **Chapter 12**

## **The** *in vitro* **Antihelminthic Efficacy of** *Erythrina Abyssinica* **Extracts on** *Ascaridia galli*

Charles Lagu and FIB Kayanja

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53708

## **1. Introduction**

[8] Bizimenyera E.S., Swam G.E., Samdumu F. B., McGaw L.J and Eloff J. N (2007). Safe‐ ty profiles of peltophorumafricanumsond. (Fabaceae) extracts. PhD thesis, University

[9] Joshua A.J., Goudar K.S., Sameera N., Pavan Kumar G., Murali B., Dinakar N. and Amit A (2010).Safety Assessment of Herbal Formulations, Rumbion™ and Tyrel™ in Albino Wistar Rats.American Journal of Pharmacology and Toxicology 5 (1): 42-47,

[10] Organization for Economic Co-operation and Development (OECD), 2000. Guidance document on the recognition, assessment and use of clinical signs as humane end‐ points for experimental animals used in safety evaluation.Environmental Health and

[11] Gad, S.C., 2007.The Rat. In: Animal Models in Toxicology, Gad, S.C. (Ed.). CRC

[12] Hayes, A.W., 2007. Principles of Pathology for Toxicological Studies In: Principles and Methods of Toxicology, Hayes, A.W. (Ed.), 5th Edn., CRC Press, New York,

Safety Monograph Series on Testing and Assessment No.

Press, Boca Raton, FL, ISBN: 10: 0824754077, pp: 193-195.

of Pretoria; 2007.

268 Insights from Veterinary Medicine

2010 ISSN 1557-4962.

ISBN: 084933778X pp: 592.

Helminth Infestation can lead to reduced growth and egg production in poultry. Coupled with high costs in 'wasted feeding' and demand for de-worming, this result in considerable economic losses in poultry enterprises and directly affects livelihood of small holder farmers [1]. In the birds, there is slow growth rate hence reduced body weight, delayed market weight attainment because of competition for nutrients by the bird and parasites. For the farmer, there is loss of income, reduced employment, and compromised household welfare, difficulty to raise educational fees, health fess, and social security activities.

Though an important veterinary practice, helminths control is largely neglected in villagelevel chicken production. The situation demands for alternative and inexpensive helminths control measures. Locally available medicinal plants have traditionally been used by small holder farmers to manage various livestock and human ailments [1-4], However, scientific data on the efficacy of these plants in helminthic control is lacking [5-8], It has been reported that local communities in the south-western agro-ecological zone (SWAEZ), Uganda, use *Er‐ ythrina abyssinica* (Leguminocae) extracts to deworm village chicken [9].

Ascaridiasis is a common disease of poultry (especially chicken and turkeys) in Uganda; it's caused by a nematode, *Ascaridia galli*. *A. galli* is a highly pathogenic worm residing in small intestines and is transmitted through eggs.

Ascaridiasis lead to weight depression and in severe cases causes intestinal blockage, loss of blood, reduced sugar content, retarded growth and mortality. It was noted that the age of the host and severity of exposure play a role in *A. galli* infections. Chickens older than 3-months are largely resistant to *A. galli* infection. *A galli* larvae undergo little to no development in older chicken.Larvaldevelopmentisarrestedinthethirdstageathighdoseratesasaresultofresistance

© 2013 Lagu and Kayanja; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lagu and Kayanja; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

rather than a density-dependent phenomenon. Also, heavier broiler breeds are known to be more resistant to Ascarid infections than lighter white leghorn chicken [1,4],

week. The plant materials were then pounded into powder (in a mortar) for chemical extrac‐

The *in vitro* Antihelminthic Efficacy of *Erythrina Abyssinica* Extracts on *Ascaridia galli*

http://dx.doi.org/10.5772/53708

271

Two hundred fifty grams (250gr) of freshly dried powdered root, stem barks and leaves were macerated in 2000 ml of 70% ethanol for 72 hours with intermittent shaking. Filtration through cotton wool was done to remove coarse particles (residues) and filter paper 12.5mm (Whitman®, No.1). The filtrate was concentrated on rota-vapour under reduced pressure at

C. The concentrated extracts were later dried on weighed kidney dishes to a constant

A 100% *Piperazine citrate* powder was bought from a known Veterinary pharmacy in Kam‐ pala. Of this 30gr were weighed and dissolved in 600mls of Goodwin's solution to make a stock concentration of 50mg/ml of the drug as the highest concentrated dose level. The stock concentration was then serially diluted to make final concentrations of 25.00mg/ml,

C. The above procedures were repeated with water as solvent. The dried ex‐

C until needed for bioassays.

tion at the Uganda Natural Chemotherapeutics Research laboratories (UNCRL).

**Figure 1.** Picture of *Erythrina abyssinica*

tracts were packed into universal bottles and kept at 40

12.50mg/ml and 6.25mg/ml for the experiment.

**c.** Preparation of piperazine citrate stock concentration

400

weight at 500

**d.** Experimental design

The study hypothesized that plants with known but undocumented anthelmintic activity exist inthe SWAEZofUganda.The efficacyofmedicinalplantvarieswiththe locationinthe SWAEZ. This study aimed to investigate the efficacy of *Erythrina abyssinica* under in vitro conditions and to compare its efficacy with that of a conventional drug, the piperazine citrate.

## **2. Materials and methods**

**a.** Collection and maintenance of the worms

Ascarid worms have a large, thick, yellowish white head with 3 large lips. The male is 50-76 mm long, 490-1.21 mm wide. It has a preanal sucker oval or circular, with strong chitinous wall with a papilliform interruption on its posterior rim; tail with narrow caudal alae or membranes and 10 pairs of papillae. The female is 60-116 mm long, 900-1.8mm wide; the vulva is the anterior part of the body, and eggs are elliptical, thick Shelled and not embryo‐ nated at time of deposition [10].

The worms used in this study were collected from fresh intestines taken from slaughtered indigenous chicken from the Bwaise market. The intestines were grossly evaluated and the worms removed and preserved in Goodwin's solution at a temp of 37˚c in an incubator.

The Goodwin's physiological solution was prepared as following [11,12,13,14, 15]. The con‐ stituents of Goodwin's physiological solution were calcium chloride (0.20g), glucose (5g), magnesium chloride (0.10g), potassium chloride (0.2g), sodium bicarbonate (0.15g), sodium chloride (8g) and sodium hydrogen phosphate (0.5g), all quantities dissolved in one (1) litre of distilled water. Calcium chloride was added later after dissolving other salts to discour‐ age its precipitation. The solution was pre warmed to 370 C before placing in the worms.

#### **b.** *Erythrina abysinnica* selection and extraction procedures

*Erythrina abyssinica* (local and Luganda names: *Muyigiti;* Runyakole name*:Ekiko* ) (Figure 1), is a deciduous savannah species. It grows in open woodland and grassland. It has character‐ istic red overflowing flowers. It can be propagated through seedlings, cuttings and trun‐ cheons. In the south-western rangelands of Uganda, it is sometimes planted along fences of paddocks to support barbed wires. It has various traditional medicinal applications in live‐ stock. It is also used in traditional human medicine [2, 14].

The leaves, stem and root barks of the plant *Erythrina abyssinica* were collected in the four districts of southwestern agro-ecological zone of Uganda. The Rubare subcounty in Ntunga‐ mo, the Rubindi subcounty in Mbarara, the Bugongi subcounty in Bushenyi and the Lugu‐ sulu subcounty in Rakai districts.

The collected *Erythrina abyssinica* materials were pressed and voucher specimen deposited with the Botany Department at Mbarara University of Science and Technology. The remain‐ ing plant materials were taken to Mbarara Zonal Agricultural Research and Development Institute (Mbarara ZARDI) for drying. The plants were dried in the shade at 250 C for one week. The plant materials were then pounded into powder (in a mortar) for chemical extrac‐ tion at the Uganda Natural Chemotherapeutics Research laboratories (UNCRL).

**Figure 1.** Picture of *Erythrina abyssinica*

rather than a density-dependent phenomenon. Also, heavier broiler breeds are known to be

The study hypothesized that plants with known but undocumented anthelmintic activity exist inthe SWAEZofUganda.The efficacyofmedicinalplantvarieswiththe locationinthe SWAEZ. This study aimed to investigate the efficacy of *Erythrina abyssinica* under in vitro conditions and

Ascarid worms have a large, thick, yellowish white head with 3 large lips. The male is 50-76 mm long, 490-1.21 mm wide. It has a preanal sucker oval or circular, with strong chitinous wall with a papilliform interruption on its posterior rim; tail with narrow caudal alae or membranes and 10 pairs of papillae. The female is 60-116 mm long, 900-1.8mm wide; the vulva is the anterior part of the body, and eggs are elliptical, thick Shelled and not embryo‐

The worms used in this study were collected from fresh intestines taken from slaughtered indigenous chicken from the Bwaise market. The intestines were grossly evaluated and the worms removed and preserved in Goodwin's solution at a temp of 37˚c in an incubator.

The Goodwin's physiological solution was prepared as following [11,12,13,14, 15]. The con‐ stituents of Goodwin's physiological solution were calcium chloride (0.20g), glucose (5g), magnesium chloride (0.10g), potassium chloride (0.2g), sodium bicarbonate (0.15g), sodium chloride (8g) and sodium hydrogen phosphate (0.5g), all quantities dissolved in one (1) litre of distilled water. Calcium chloride was added later after dissolving other salts to discour‐

*Erythrina abyssinica* (local and Luganda names: *Muyigiti;* Runyakole name*:Ekiko* ) (Figure 1), is a deciduous savannah species. It grows in open woodland and grassland. It has character‐ istic red overflowing flowers. It can be propagated through seedlings, cuttings and trun‐ cheons. In the south-western rangelands of Uganda, it is sometimes planted along fences of paddocks to support barbed wires. It has various traditional medicinal applications in live‐

The leaves, stem and root barks of the plant *Erythrina abyssinica* were collected in the four districts of southwestern agro-ecological zone of Uganda. The Rubare subcounty in Ntunga‐ mo, the Rubindi subcounty in Mbarara, the Bugongi subcounty in Bushenyi and the Lugu‐

The collected *Erythrina abyssinica* materials were pressed and voucher specimen deposited with the Botany Department at Mbarara University of Science and Technology. The remain‐ ing plant materials were taken to Mbarara Zonal Agricultural Research and Development

Institute (Mbarara ZARDI) for drying. The plants were dried in the shade at 250

C before placing in the worms.

C for one

more resistant to Ascarid infections than lighter white leghorn chicken [1,4],

to compare its efficacy with that of a conventional drug, the piperazine citrate.

**2. Materials and methods**

270 Insights from Veterinary Medicine

nated at time of deposition [10].

sulu subcounty in Rakai districts.

**a.** Collection and maintenance of the worms

age its precipitation. The solution was pre warmed to 370

**b.** *Erythrina abysinnica* selection and extraction procedures

stock. It is also used in traditional human medicine [2, 14].

Two hundred fifty grams (250gr) of freshly dried powdered root, stem barks and leaves were macerated in 2000 ml of 70% ethanol for 72 hours with intermittent shaking. Filtration through cotton wool was done to remove coarse particles (residues) and filter paper 12.5mm (Whitman®, No.1). The filtrate was concentrated on rota-vapour under reduced pressure at 400 C. The concentrated extracts were later dried on weighed kidney dishes to a constant weight at 500 C. The above procedures were repeated with water as solvent. The dried ex‐ tracts were packed into universal bottles and kept at 40 C until needed for bioassays.

**c.** Preparation of piperazine citrate stock concentration

A 100% *Piperazine citrate* powder was bought from a known Veterinary pharmacy in Kam‐ pala. Of this 30gr were weighed and dissolved in 600mls of Goodwin's solution to make a stock concentration of 50mg/ml of the drug as the highest concentrated dose level. The stock concentration was then serially diluted to make final concentrations of 25.00mg/ml, 12.50mg/ml and 6.25mg/ml for the experiment.

**d.** Experimental design

The conical flasks of 250ml capacity were labelled according to the different extracts and pi‐ perazine doses namely 0, 6.25, 12.5, 25 and 50 mg/ml. In each of the dose rate concentrations 200, 187.5, 175, 150 and 100 mls respectively of Goodwin solutions were added. The extracts added were 0, 12.5, 25, 50 and 100 mls respectively. The final volume per conical flask is 200mls of a solution containing Godwin solution and extracts. Ten worms were placed in each flask and these were incubated at 37˚C in water bath. The worms were monitored ev‐ ery 12hrs for a period of 48hrs (table 1).

means where ANOVA showed significant difference. Graphs were drawn to illustrate the

The *in vitro* Antihelminthic Efficacy of *Erythrina Abyssinica* Extracts on *Ascaridia galli*

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273

The Comparison among specific plant extracts (Root-R, Stem-S and Leaves-L) and Districts (B-Bushenyi, M-Mbarara, N-Ntungamo, R-Rakai) was carried out using one way Anova then Post tested using Tukey test followed by Bonferroni post hoc *t*-test. P-value = 0.05 was used for significance level. The comparison in variations within concentrations of different extracts were conducted using Newman-Keuls Multiple Comparison Test P- value = 0.05 was used for significance level. The following parameters were tested: districts (Bushenyi, Mbarara, Ntungamo and Rakai), log transformation of the dose levels 0, 6.25, 12.5, 25 and 50

The mean of action irrespective of the districts for Piperazine (P), Rootbark (R), Stem bark (S) and Leaves (L) at different concentrations 0, 6.25, 12.5, 25 and 50 mg/ml of the extracts. The *A. galli* were subjected to extracts for 48hours, monitored at each 12 hours interval, and

0 10 0 0 0 0

6.25 10 2.67±0.14 1.42±0.48 1.83±0.27 3.58±0.96

12.5 10 6.25±0.33 3.42±0.39 3.58±0.45 7.75±0.60

25 10 8.75±0.28 6.50±0.75 5.00±0.55 8.08±0.38

50 10 10.00 7.92±0.98 7.17±0.91 9.46±0.53

**Table 1.** Mean of action irrespective of the districts (Generally for Piperazine (P), Rootbark (R), Stem bark (S) and

=0.924 and Leaves R2=0.721

5. The *Piperazine citrate* has a rank correlation coefficient of R2

The rank correlation coefficient for the different extracts Piperazine (P), Root bark (R), Stem bark (S) and Leaves (L) at concentrations 0, 6.25,12.5,25,50 mg/ml are detailed in Figures 2 to

**worms used Total No. of worms immobilized (paralysed +dead) after 48 hours**

Piperazine Root barks Stem barks Leaves

=0.7701; Root bark (R)

trends in activity by the *Erythrina* extracts and *Piperazine citrate* against *Ascaridia galli*.

mg/ml, corresponding to 0.000, 0,796, 1.097, 1.398, 1.699.

**Total No. of**

Leaves (L) for different concentrations of the extract

=0.8966, Stem bark (S) R2

**5. Results presentation**

results are detailed in Table 1.

**Concentrations (mg/ml)**

R2

The experiment had the following treatment groups: Negative control (N), Positive control involving three replicates of Piperazine citrate (P1,P2,P3), and the testing extracts, involving three replicates of Root bark (RB1, RB2, RB3), of Stem barks (SB1, SB2, SB3) and of Leaves (L1, L2,L3).

## **3. Worm motility assessment**

In preliminary experiments, a criteria used for assessing the effects of crude plant extracts on the motility of adult *Ascaridia galli* was developed, combining the procedures previously described in literature [14], [28]. Worm motility was assessed at 12hours, 24hours, 36 hours and 48hours post treatment.

After 12, 24, 36 and 48 hours of incubation at 370 C, the worms were gently removed from the testing treatment and re-suspended in Goodwin's physiological solution at the same tem‐ perature for 30 seconds for possible recovery of parasite motility. The worms were assessed for death or paralysis. A worm was considered to be motile if it moved in a sinusoidal mo‐ tion when stimulated by water at 40-50°C. Similarly it was considered paralyzed if on stimu‐ lating it by water at 500 C only part of the body responded either by raising the head and whether some parts showed autolysis and change of colour to pale white. Motility was also assessed by pressing the worm with an index finger, or using water at 50 - 600 C to differenti‐ ate dead from paralyzed worms.

The percentage of immotile or dead worm was calculated as the number of dead worms div‐ ided by the total number of worms per flask multiplied by 100 to represent percentage para‐ lyzed or dead.

## **4. Data analysis**

The data collected were first entered in a laboratory counter book and then entered in a computer database (Microsoft Excel). The bioassay data was analyzed by the General Linear Model Procedures with multiple comparisons (Bonferroni method) and regression, using the Graph Pad Prism Version 5.01 software, Inc San Diego, CA USA. P value <0.05 was tak‐ en for significance level. The differences between the controls and treated means were ana‐ lysed using one-way analysis of variance (ANOVA). Student t-test was used to separate means where ANOVA showed significant difference. Graphs were drawn to illustrate the trends in activity by the *Erythrina* extracts and *Piperazine citrate* against *Ascaridia galli*.

The Comparison among specific plant extracts (Root-R, Stem-S and Leaves-L) and Districts (B-Bushenyi, M-Mbarara, N-Ntungamo, R-Rakai) was carried out using one way Anova then Post tested using Tukey test followed by Bonferroni post hoc *t*-test. P-value = 0.05 was used for significance level. The comparison in variations within concentrations of different extracts were conducted using Newman-Keuls Multiple Comparison Test P- value = 0.05 was used for significance level. The following parameters were tested: districts (Bushenyi, Mbarara, Ntungamo and Rakai), log transformation of the dose levels 0, 6.25, 12.5, 25 and 50 mg/ml, corresponding to 0.000, 0,796, 1.097, 1.398, 1.699.

## **5. Results presentation**

The conical flasks of 250ml capacity were labelled according to the different extracts and pi‐ perazine doses namely 0, 6.25, 12.5, 25 and 50 mg/ml. In each of the dose rate concentrations 200, 187.5, 175, 150 and 100 mls respectively of Goodwin solutions were added. The extracts added were 0, 12.5, 25, 50 and 100 mls respectively. The final volume per conical flask is 200mls of a solution containing Godwin solution and extracts. Ten worms were placed in each flask and these were incubated at 37˚C in water bath. The worms were monitored ev‐

The experiment had the following treatment groups: Negative control (N), Positive control involving three replicates of Piperazine citrate (P1,P2,P3), and the testing extracts, involving three replicates of Root bark (RB1, RB2, RB3), of Stem barks (SB1, SB2, SB3) and of Leaves

In preliminary experiments, a criteria used for assessing the effects of crude plant extracts on the motility of adult *Ascaridia galli* was developed, combining the procedures previously described in literature [14], [28]. Worm motility was assessed at 12hours, 24hours, 36 hours

testing treatment and re-suspended in Goodwin's physiological solution at the same tem‐ perature for 30 seconds for possible recovery of parasite motility. The worms were assessed for death or paralysis. A worm was considered to be motile if it moved in a sinusoidal mo‐ tion when stimulated by water at 40-50°C. Similarly it was considered paralyzed if on stimu‐

whether some parts showed autolysis and change of colour to pale white. Motility was also

The percentage of immotile or dead worm was calculated as the number of dead worms div‐ ided by the total number of worms per flask multiplied by 100 to represent percentage para‐

The data collected were first entered in a laboratory counter book and then entered in a computer database (Microsoft Excel). The bioassay data was analyzed by the General Linear Model Procedures with multiple comparisons (Bonferroni method) and regression, using the Graph Pad Prism Version 5.01 software, Inc San Diego, CA USA. P value <0.05 was tak‐ en for significance level. The differences between the controls and treated means were ana‐ lysed using one-way analysis of variance (ANOVA). Student t-test was used to separate

assessed by pressing the worm with an index finger, or using water at 50 - 600

C only part of the body responded either by raising the head and

C, the worms were gently removed from the

C to differenti‐

ery 12hrs for a period of 48hrs (table 1).

**3. Worm motility assessment**

After 12, 24, 36 and 48 hours of incubation at 370

and 48hours post treatment.

lating it by water at 500

lyzed or dead.

**4. Data analysis**

ate dead from paralyzed worms.

(L1, L2,L3).

272 Insights from Veterinary Medicine

The mean of action irrespective of the districts for Piperazine (P), Rootbark (R), Stem bark (S) and Leaves (L) at different concentrations 0, 6.25, 12.5, 25 and 50 mg/ml of the extracts. The *A. galli* were subjected to extracts for 48hours, monitored at each 12 hours interval, and results are detailed in Table 1.


**Table 1.** Mean of action irrespective of the districts (Generally for Piperazine (P), Rootbark (R), Stem bark (S) and Leaves (L) for different concentrations of the extract

The rank correlation coefficient for the different extracts Piperazine (P), Root bark (R), Stem bark (S) and Leaves (L) at concentrations 0, 6.25,12.5,25,50 mg/ml are detailed in Figures 2 to 5. The *Piperazine citrate* has a rank correlation coefficient of R2 =0.7701; Root bark (R) R2 =0.8966, Stem bark (S) R2 =0.924 and Leaves R2=0.721

**Figure 4.** The effects of Stem barks on the number of worms immobilized (paralysed +dead)

The *in vitro* Antihelminthic Efficacy of *Erythrina Abyssinica* Extracts on *Ascaridia galli*

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275

**Figure 5.** The effects of Leaves on the number of worms immobilized (paralysed +dead)

ferent parts (root, stem and leaves) of the plant irrespective of the plant origin.

The study established the existence of a statistical significant relationship (P<0.05) between the Positive control (*Piperazine citrate*) at different concentrations and the extracts from dif‐

**Figure 2.** The effects of *Piperazine citrate* on the number of worms immobilized (paralysed +dead)

**Figure 3.** The effects of Root barks on the number of worms immobilized (paralysed +dead)

**Figure 4.** The effects of Stem barks on the number of worms immobilized (paralysed +dead)

**Figure 2.** The effects of *Piperazine citrate* on the number of worms immobilized (paralysed +dead)

274 Insights from Veterinary Medicine

**Figure 3.** The effects of Root barks on the number of worms immobilized (paralysed +dead)

**Figure 5.** The effects of Leaves on the number of worms immobilized (paralysed +dead)

The study established the existence of a statistical significant relationship (P<0.05) between the Positive control (*Piperazine citrate*) at different concentrations and the extracts from dif‐ ferent parts (root, stem and leaves) of the plant irrespective of the plant origin.

Further, the results found statistically insignificant differences (p>0.05) in activity against *A.galli* among specific plant extracts (Root-R, Stem-S and Leaves-L) and their location (Bush‐ enyi, Mbarara, Ntungamo and Rakai)

erties in order to remove the dead worm load, or it may induce the death of the host as a

The *in vitro* Antihelminthic Efficacy of *Erythrina Abyssinica* Extracts on *Ascaridia galli*

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277

It has been reported that some plant metabolites like tannins bind to glycoprotein on the cu‐

The crude extracts yielded significant positive activity on *A.galli* as detailed in Table 1 with‐ in 48 hours. The study established that there were insignificant differences in plant parts in the various localities viz; Ntungamo, Mbarara, Bushenyi and Rakai. The differences between the therapeutic potential for root, stem and leaf vary between regions in a similar way/ proportion. The South Western Agro-ecological Zone of Uganda in which Ntungamo, Mbar‐ ara, Bushenyi and Rakai districts lies in the same agro ecological conditions, similar rainfall,

However, it has been found that ecological, genetic and environmental differences of plants harvested from the wild may vary in quality, consistency of active bio-compounds [21]. The variation in medicinal plants may also be linked to age of the plant, seasonal variation and

The study also indicated both plant extracts, in particular the leaves, and *Piperazine citrate* response did not differ significantly (Figures 2 to 5). The use the plant leaves crude extract as alternative de-wormer but dosages need to be standardized. This would make farmers save on cost of livestock production. These findings agree with previous farmer's claims that the plant is useful in the treatment of helminthosis [4,9,14].Repeated exposure to insufficient crude extract concentration could lead to worm resistance. This would explain the contin‐ ued reports of helminthosis and low livestock productivity despite farmers use of medicinal

Ethanolic extraction was selected in this work to extract active substances from the root bark, stem bark and leaves of *Erythrina abyssinica*. Farmers use water to extract the active substances. There may be variation in extraction potential using aqueous and ethanol sol‐ vent systems due to the kind of bioactive substances extracted by the two solvents since different solvents extract different compounds depending on type of substances and po‐

Further, alcohol is a "good for all-purpose" solvent for preliminary extraction [12,22]. In this study the use of ethanol has an added advantage of extract preservation and increasing the shelf life of the medicinal plant extracts. This would not only reduce labour of repeated

The anthelmintic activities observed might be due the *Erythrina* condensed tannins, though synergy by other compounds could have enhanced the activity. The role of condensed tan‐

Chemically, tannins are polyphenolic compounds [30] and some synthetic phenolic anthel‐ mintics, like niclosamide and oxyclozanide are said to interfere with energy generation in helminth parasites by uncoupling oxidative phosphorylation. It is possible that tannins con‐ tained in the extracts of *Erythrina abyssinica* produced similar effects. It was also suggested

ticle of the parasite and disturb the physiological functions like motility [20].

temperature, humidity, soil ingredients and other biotic factors.

preparation but also promote the plant species conservation.

nins in helminth control has been demonstrated [23-24].

consequence of toxic syndromes.

geographical deviation at harvest site.

plants to contain the parasites.

larity [22].

The activity of the leaf extracts from Bushenyi, Mbarara, Ntungamo and Rakai districts were comparable to the conventional drug in the management of *Ascaridia galli as detailed* in Table 2.


**Table 2.** Details the concentration of the extracts and the levels of worm immobilization (paralysed +dead) after 48 hours (R – Root barks; S – Stem barks; L – Leaves)

## **6. Discussion**

The research showed that the leaves, stem barks and root barks of *Erythrina abyssinica* may be useful in poultry helminthosis control. This information supports its used as antihel‐ minthic in ethno-veterinary medicine as previously defended [2-3]. The percentage of motil‐ ity inhibition is an estimate of anthelmintic efficacy by comparing worm motility before and after incubation with plant extracts and *Piperazine citrate*. In this study the *Ascaridia galli* mo‐ tility was assessed at 12, 24, 36 and 48 hours post treatment. The motility decreased with in‐ creasing extract concentration and increase in the incubation period. The anthelmintic property of plants is dependent on secondary plant metabolites [16-17] which in turn may depend on solvent of extraction [18] Paralyses of *Ascaridia galli* were very evident in treated groups that progressed till death of the parasite.

Piperazine is a GABA receptor agonist. Piperazine binds directly and selectively to muscle membrane GABA receptors, presumably causing hyperpolarization of nerve endings, result‐ ing in flaccid paralysis of the worm. Similar observations have been reported showing that anthelmintic drugs kill worms either by starving them to death or by causing paralysis, which impairs the worm to store energy to meet their metabolic energy requirements [19]. The worms probably died from energy deficiencies (starvation) since they became paralyzed and fail to feed. It was further explained that interfering with feeding for up to 24 hours is sufficient to kill most adult parasites [12].

It is very crucial to note here that immobilization of the worms is ideal but it may lead to stenosis of the intestinal lumen. Thus it is important that a substance possess laxative prop‐ erties in order to remove the dead worm load, or it may induce the death of the host as a consequence of toxic syndromes.

Further, the results found statistically insignificant differences (p>0.05) in activity against *A.galli* among specific plant extracts (Root-R, Stem-S and Leaves-L) and their location (Bush‐

The activity of the leaf extracts from Bushenyi, Mbarara, Ntungamo and Rakai districts were comparable to the conventional drug in the management of *Ascaridia galli as detailed*

**Plant parts** R S L R S L R S L R S L R S L **Bushenyi** 0 0 0 0 2 3 2 4 4 5 5 6 7 8 10 **Mbarara** 0 0 0 1 2 2 2 5 4 5 7 6 7 7 8 **Ntungamo** 0 0 0 1 2 3 2 4 5 5 5 7 7 8 9 **Rakai** 0 0 0 2 2 2 3 3 6 6 6 8 9 7 9

**Table 2.** Details the concentration of the extracts and the levels of worm immobilization (paralysed +dead) after 48

The research showed that the leaves, stem barks and root barks of *Erythrina abyssinica* may be useful in poultry helminthosis control. This information supports its used as antihel‐ minthic in ethno-veterinary medicine as previously defended [2-3]. The percentage of motil‐ ity inhibition is an estimate of anthelmintic efficacy by comparing worm motility before and after incubation with plant extracts and *Piperazine citrate*. In this study the *Ascaridia galli* mo‐ tility was assessed at 12, 24, 36 and 48 hours post treatment. The motility decreased with in‐ creasing extract concentration and increase in the incubation period. The anthelmintic property of plants is dependent on secondary plant metabolites [16-17] which in turn may depend on solvent of extraction [18] Paralyses of *Ascaridia galli* were very evident in treated

Piperazine is a GABA receptor agonist. Piperazine binds directly and selectively to muscle membrane GABA receptors, presumably causing hyperpolarization of nerve endings, result‐ ing in flaccid paralysis of the worm. Similar observations have been reported showing that anthelmintic drugs kill worms either by starving them to death or by causing paralysis, which impairs the worm to store energy to meet their metabolic energy requirements [19]. The worms probably died from energy deficiencies (starvation) since they became paralyzed and fail to feed. It was further explained that interfering with feeding for up to 24 hours is

It is very crucial to note here that immobilization of the worms is ideal but it may lead to stenosis of the intestinal lumen. Thus it is important that a substance possess laxative prop‐

0 0.79588 1.09691 1.39794 1.69897

enyi, Mbarara, Ntungamo and Rakai)

hours (R – Root barks; S – Stem barks; L – Leaves)

groups that progressed till death of the parasite.

sufficient to kill most adult parasites [12].

in Table 2.

276 Insights from Veterinary Medicine

**Concentrati on mg/ml**

**6. Discussion**

It has been reported that some plant metabolites like tannins bind to glycoprotein on the cu‐ ticle of the parasite and disturb the physiological functions like motility [20].

The crude extracts yielded significant positive activity on *A.galli* as detailed in Table 1 with‐ in 48 hours. The study established that there were insignificant differences in plant parts in the various localities viz; Ntungamo, Mbarara, Bushenyi and Rakai. The differences between the therapeutic potential for root, stem and leaf vary between regions in a similar way/ proportion. The South Western Agro-ecological Zone of Uganda in which Ntungamo, Mbar‐ ara, Bushenyi and Rakai districts lies in the same agro ecological conditions, similar rainfall, temperature, humidity, soil ingredients and other biotic factors.

However, it has been found that ecological, genetic and environmental differences of plants harvested from the wild may vary in quality, consistency of active bio-compounds [21]. The variation in medicinal plants may also be linked to age of the plant, seasonal variation and geographical deviation at harvest site.

The study also indicated both plant extracts, in particular the leaves, and *Piperazine citrate* response did not differ significantly (Figures 2 to 5). The use the plant leaves crude extract as alternative de-wormer but dosages need to be standardized. This would make farmers save on cost of livestock production. These findings agree with previous farmer's claims that the plant is useful in the treatment of helminthosis [4,9,14].Repeated exposure to insufficient crude extract concentration could lead to worm resistance. This would explain the contin‐ ued reports of helminthosis and low livestock productivity despite farmers use of medicinal plants to contain the parasites.

Ethanolic extraction was selected in this work to extract active substances from the root bark, stem bark and leaves of *Erythrina abyssinica*. Farmers use water to extract the active substances. There may be variation in extraction potential using aqueous and ethanol sol‐ vent systems due to the kind of bioactive substances extracted by the two solvents since different solvents extract different compounds depending on type of substances and po‐ larity [22].

Further, alcohol is a "good for all-purpose" solvent for preliminary extraction [12,22]. In this study the use of ethanol has an added advantage of extract preservation and increasing the shelf life of the medicinal plant extracts. This would not only reduce labour of repeated preparation but also promote the plant species conservation.

The anthelmintic activities observed might be due the *Erythrina* condensed tannins, though synergy by other compounds could have enhanced the activity. The role of condensed tan‐ nins in helminth control has been demonstrated [23-24].

Chemically, tannins are polyphenolic compounds [30] and some synthetic phenolic anthel‐ mintics, like niclosamide and oxyclozanide are said to interfere with energy generation in helminth parasites by uncoupling oxidative phosphorylation. It is possible that tannins con‐ tained in the extracts of *Erythrina abyssinica* produced similar effects. It was also suggested that tannins bind to free proteins in the gastrointestinal tract of host animal [2,25] or glyco‐ protein on the cuticle of the parasite disturbing the physiological functions like motility, feed absorption and reproduction [20,26-27,] or by interference with morphology and pro‐ teolytic activity of microbes [13,28] and cause death.

**Author details**

ara, Uganda

Mbarara, Uganda

**References**

Charles Lagu1\* and FIB Kayanja2

giene, 71, 5, 608-616.

Sciences Department; 2001.

naria 173.

opment Research Foundation, Pune, India.

\*Address all correspondence to: chlaguu@gmail.com

1 Department of Biology, Faculty of Science, University of Science and Technology, Mbar‐

The *in vitro* Antihelminthic Efficacy of *Erythrina Abyssinica* Extracts on *Ascaridia galli*

http://dx.doi.org/10.5772/53708

279

2 Department of Anatomy, Faculty of Medicine, University of Science and Technology,

[1] Masimba E S, Mbiriri D T, Kashangura M T and Mutibvu T 2011: Indigenous practi‐ ces for the control and treatment of ailments in Zimbabwe's village poultry. Live‐ stock Research for Rural Development. Volume 23, Article #257. Retrieved

[2] Kotze, A. C., Clifford, S., O'Grady, J., Behnke, J. M. and McCarthy, J. S., 2004. An in vitro larval motility assay to determine anthelmintic sensitivity for human hook‐ worm and strongyloides species. American Journal of Tropical Medicine and Hy‐

[3] Mathias, E., D.V. Rangnekar, and C.M. McCorkle, 1999. Ethnoveterinary Medicine: Alternatives for Livestock Development. Proceedings of an International Conference held in Pune, India, on November 4-6, 1997. Volume 1: Selected Papers. BAIF Devel‐

[4] Nalule A S, Mbaria J M, Olila D and Kimenju J W , 2011: Ethnopharmacological prac‐ tices in management of livestock helminthes by pastoral communities in the dry‐ lands of Uganda. Livestock Research for Rural Development. Volume 23, Article

#36.Retrieved May 18, 2011, from http://www.lrrd.org/lrrd23/2/nalu23036.htm

asite control: facts and fictions. Animal (2007), 1:9, pp1397-1400.

[5] Athanasiadou S, Githiori J and Kyriazakis I, 2007 Medicinal plants for Helminth par‐

[6] Gakuya D .W, 2001. Pharmacological and clinical evaluation of the anthelmintic ac‐ tivity of Albizia anthelmintica Brogn, Maerua edulis De wolf and Maerua subcordata De wolf plant extracts in sheep and mice. PhD thesis. University of Nairobi, Clinical

[7] Githiori J B, 2004. Evaluation of anthelmintic properties of ethno veterinary plant preparations used as livestock dewormers by pastoralists and small holder farmers in Kenya. PhD thesis, Uppsala 2004. Acta Universitatis Agriculturae Sueciae Veteri‐

September 4, 2012, from http://www.lrrd.org/lrrd23/12/masi23257.htm

Alternatively, the presence of alkaloids salts which are physiologically active with sedative and analgesic properties could have contributed to the paralysis and consequent death of the worms. Alkaloids are toxic due to their stimulatory effects, leading to excitation of cells and neurological dysfunction.

## **7. Conclusion and recommendations**

The study validates the farmers efforts, who have been using for long these medicinal plants and stem barks for management of diverse ailments in poultry and other livestock diseases. The findings of the current study provide evidence that *Erythrina abyssinica* can be used by local farmers to control poultry helminthosis. Our study found that leaves had very good activity on *Ascaridia galli* comparable with the conventional *piperazine citrate*. This finding provides a new innovation in the utilization of plants parts to solve helminthes problems in local chicken. The use of leaves is important for sustainable conservation of plants. Plants where the community use the root barks are more endangered than plants whereby the community use plant leaves. The use of leaves is an opportunity to conservation of *Erythrina abyssinica* without tampering with the root barks.

Nevertheless, there is the need to conduct acute toxicity test to establish safety of the plant extracts. The use *Erythrina abyssinica* leaves other than root or stem is sustainable way of conserving the medicinal plants.

## **Acknowledgements**

We thank Belgian Technical Cooperation (BTC) for funding the research. The contributions of Mr. Francis Omujal, Amai Corn, Henry Tumusiime, and Moses Agwaya of Uganda Natu‐ ral chemotherapeutics of Wandegeya laboratories are applauded in guiding the extraction and concentration of plants extracts process. The roles of Dr. Patrick Vudriko, Mr. James Ndukui and Ms. Kibui Pauline of the Toxicology and Division of Pharmacological sciences laboratories, College of Veterinary Medicine,Animal Resources and Biosecurity, Makerere University in the in vitro studies are highly appreciated. Dr. Nsubuga Mutaka and Ms. Betty Laura Ayoo of Mbarara Zonal Agriculture Research and Development Institute played vital technical advisory roles during the in vitro studies. The vital role of Kuria Anthony of Tropi‐ cal Biology Association, Nairobi is highly appreciated.

## **Author details**

that tannins bind to free proteins in the gastrointestinal tract of host animal [2,25] or glyco‐ protein on the cuticle of the parasite disturbing the physiological functions like motility, feed absorption and reproduction [20,26-27,] or by interference with morphology and pro‐

Alternatively, the presence of alkaloids salts which are physiologically active with sedative and analgesic properties could have contributed to the paralysis and consequent death of the worms. Alkaloids are toxic due to their stimulatory effects, leading to excitation of cells

The study validates the farmers efforts, who have been using for long these medicinal plants and stem barks for management of diverse ailments in poultry and other livestock diseases. The findings of the current study provide evidence that *Erythrina abyssinica* can be used by local farmers to control poultry helminthosis. Our study found that leaves had very good activity on *Ascaridia galli* comparable with the conventional *piperazine citrate*. This finding provides a new innovation in the utilization of plants parts to solve helminthes problems in local chicken. The use of leaves is important for sustainable conservation of plants. Plants where the community use the root barks are more endangered than plants whereby the community use plant leaves. The use of leaves is an opportunity to conservation of *Erythrina*

Nevertheless, there is the need to conduct acute toxicity test to establish safety of the plant extracts. The use *Erythrina abyssinica* leaves other than root or stem is sustainable way of

We thank Belgian Technical Cooperation (BTC) for funding the research. The contributions of Mr. Francis Omujal, Amai Corn, Henry Tumusiime, and Moses Agwaya of Uganda Natu‐ ral chemotherapeutics of Wandegeya laboratories are applauded in guiding the extraction and concentration of plants extracts process. The roles of Dr. Patrick Vudriko, Mr. James Ndukui and Ms. Kibui Pauline of the Toxicology and Division of Pharmacological sciences laboratories, College of Veterinary Medicine,Animal Resources and Biosecurity, Makerere University in the in vitro studies are highly appreciated. Dr. Nsubuga Mutaka and Ms. Betty Laura Ayoo of Mbarara Zonal Agriculture Research and Development Institute played vital technical advisory roles during the in vitro studies. The vital role of Kuria Anthony of Tropi‐

teolytic activity of microbes [13,28] and cause death.

**7. Conclusion and recommendations**

*abyssinica* without tampering with the root barks.

cal Biology Association, Nairobi is highly appreciated.

conserving the medicinal plants.

**Acknowledgements**

and neurological dysfunction.

278 Insights from Veterinary Medicine

Charles Lagu1\* and FIB Kayanja2

\*Address all correspondence to: chlaguu@gmail.com

1 Department of Biology, Faculty of Science, University of Science and Technology, Mbar‐ ara, Uganda

2 Department of Anatomy, Faculty of Medicine, University of Science and Technology, Mbarara, Uganda

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operative Extension.


## *Edited by Rita Payan-Carreira*

Under the title "Insights from Veterinary Medicine", this book presents original research results and reviews flashing several distinctive aspects of the Veterinary Medicine Sciences, in which the knowledge has continuously increased over the past decades. Veterinary and Human Medicine have been developed in close association, in part as a reflection of the intertwined relationship found between animals and humans since the establishment of first civilizations. Humans and animals share common mechanisms of disease, thus serving as spontaneous models for the study of particular disorders, such as tumors and cardiac diseases. Furthermore, concerns on the deleterious side-effects of contaminants, in particular over the endocrine axis regulating different body functions and fertility, are on the table, as animals may serve as sentinels for environmental quality. In addition, the quality of the animal life with regards to both health and welfare also contribute to the quality of human life, and food-animal health and safety are safeguards against the disruption of the food chain. The Veterinary Medicine field has broadened its scope of action, and nowadays it encompasses much more than the veterinary medical practice. Similarly, a wide field of knowledge was covered in this book. Even so, it was our goal to provide you with current advanced resources in different veterinary science disciplines.

Insights from Veterinary Medicine

Insights from

Veterinary Medicine

*Edited by Rita Payan-Carreira*

Photo by Oranit6666 / iStock