**2. Canine visceral Leishmaniasis and fisiopathology of VOC released from hair**

studies aim to develop new ways of diagnosing diseases based on identification of volatile organic compounds (VOC) as indicative of pathological states. Diseases such as diabetes, breast cancer, lung cancer and chronic obstructive lung disease, among others, have been target of metabolomics studies [2-4]. These diseases have in common the chronic condition in its pathogenesis, such as in leishmaniasis, and may develop over a long period without the manifestation of clinical indications but with the production of metabolites as biomarkers of

Different biological samples may be used for metabolomic studies and they commonly vary according to the pathogenesis of the disease and anatomical region with greatest pathological involvement. In the case of visceral leishmaniasis hair is noteworthy as a biological matrix for analysis of VOC resulting from final metabolism of *Leishmania* infection and due to the ease of sample collection and the nature of the hair follicle, which has the function to secrete and

Therefore, the identification of biomarkers in the dogs with visceral leishmaniasis can serve as a painless diagnostic tool with minimal invasivity, which represents a gain in the field of

We emphasize the use of different techniques for the extraction, separation and identification of these VOCs, as well as the use of univariate and multivariate planning in conducting the work. The use chemometric methods allow the analysis of a large number of variables, identifying similarities in complex samples, where several compounds are analyzed simulta‐

When metabolomics approach is performed on vector-borne diseases, another important aspect is the possibility of the identified VOC act as chemical attractants for vector insects. VOC produced by hosts which are detected by receivers and antennas of the vectors during the search the process of search and location of food are called kairomones [6]. The ability to detect these volatile compounds makes easier specific recognition of the host and increases the radius of food search by the vector. In the case of leishmaniasis VOC produced from final metabolism of *Leishmania* infection when released into the environment can attract phleboto‐ mine so that the insect feeds from the infected animals, thereby ensuring the dissemination of

The discovery of attractiveness of these compounds may aid studies about the presence and dispersion of phlebotomine providing subsidies for better understanding of the biology of these insects and in the future to substantiate effective programs to control the disease based

The present chapter contributes to a better understanding of the relationship parasite-dogsandfly in visceral leishmaniasis. Here we summarize the importance of studies involving metabolomics, identification of biomarkers for disease diagnosis, the key techniques and methods of extraction and analysis of VOCs, attract sandflies aspects of the VOC and its

the disease.

neously.

excrete endogenous produced substances [5].

316 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

the parasite and maintenance of disease cycle.

application in the control of visceral leishmaniasis.

on the manipulation of chemical communication of phlebotomine.

animal welfare and perspectives for disease control.

Assumed as a neglected disease by World Health Organization [7], visceral *Leishmania*sis (VL) are recorded in about 62 world counties and it mainly affects children, elderly and immuno‐ deficient people [8, 9]. The VL is a parasitic disease with zoonosis character caused in american continent by *Leishmania infantum,* protozoan transmited between hosts by phlebotomine vector of *Lutzomyia longipalpis* specie [10].

The VL is considered a serious public health problem due to its complex epidemiological characteristics, different reservoirs involved, besides fast geographic expansion as well as expensive and arduous control [11].

Domestic dog (*Canis familiaris*) is considered the main reservoir of urban area due to its high susceptibility to pathogen, the strong cutaneous parasitism and the fact that canine cases precedes humans [11-13]. Thus this species is considered protagonist in studies that aim the development of new forms of control and disease prevention.

In the wild, crab-eating fox *(Cerdocyon thous*), the hoary fox (*Lycalopex vetulus)*, the common opossum (*Didelphis marsupialis)* and the white-eared opossum (*Didelphis albiventris*) are used as natural reservoirs of infection and it is common to find positive animals in areas of human habitations which are related to the wild [9, 14].

The *Lutzomyia longipalpis* (Lutz & Neiva, 1912) is considered the main vector of VL in the American continent and it is widely studied because of its epidemiological importance for the transmission of this disease [15, 16].

*L. longipalpis* is one diptera of Psychodidae family and Phlebotominae subfamily. These phlebotomine insects measure approximately 2-3 mm, the body is quite hairy, light colored (light brown or straw color) and are easily recognized by their behavior of flying with small jumps and landing with half open wings [11].

The only proven way of transmission so far is through the bite of phlebotomine females that ingest amastigote forms with blood meal when it feeds from an infected mammal [9]. After undergoing processing in the digestive tract they are transmitted to other hosts already in the promastigote form while females feed for the second time [15, 17]. On the spot of inoculation an inflammatory process takes place and promastigostes are then phagocytosed by macro‐ phages, then they become amastigotes and multiply until macrophages break up and they are released to spread to the whole body [17].

The consequences, progression and severity of the disease depend on the immune response expressed by the animal, especially the cytokine production profile [1]. Cytokine production profile varies with environmental factors, nutritional and genetic aspects of the parasite like the species, strain and inoculum size [18].

In the early periods of post-infection there are changes in populations of T lymphocytes and it has been shown that there is a relationship between the pattern of produced cytokines and the manifestation of clinical signs [19]. This association is a result of the greater intensity of parasite load, when there is a lower production of Th1 cytokines (Lymphocyte T helper type I) in various organs such as lymph nodes [20], spleen [21], bone marrow [22] and skin [23].

Animals that produce Th1-type response develops immunity against the parasite cell type, resulting in resistance to infection [19]. In these animals there is a predominance of IFN-γ, TNFα and IL-12 release, which among other things promotes the production and activation of macrophages, nitric oxide and cell apoptosis [24].

Resistant dogs are characterized by low parasitic burden in several organs [25], no develop‐ ment of clinical signs [19], normal erythropoietic and leukopoietic activities [22] and low infectability to phlebotomine [26].

On other hand animals that produce Th2-type (Lymphocyte T helper type II) response are characterized by a humoral immune type response and consequent susceptibility to infection [19]. In these animals there is a predominance of IL4, IL10 and TGF-β that inhibit the production of IFN-γ and IL-2 then the cell type response [22, 27]. The antibodies produced by humoral response are unable to eradicate the infection resulting in high parasitic burden and expression of clinical signs of disease.

Thus in dogs with active VL the strength of parasite load is directly proportional to the intensity of clinical signs and disease severity (Figure 1). The most important clinical signs are ocular lesions, periocular and cutaneous, kidney diseases and their consequences; respiratory, digestive, cardiovascular and musculoskeletal disorders; hypertrophy of lymphoid organs and hematological disorders [28].

The transmission of *Leishmania* by dogs seems to be related to the extent of clinical manifestations of disease so how much more clinical signs present higher the ratio of infectivity, which is possibly related with high parasite load in different organs, especial‐ ly skin and lymph nodes [29].

Histological skin evaluations of animals which show the presence of perifollicular dermatitis with inflammatory infiltration generally extending by the sebaceous glands [30] and a predominance of macrophages, lymphocytes and polymorphonuclear neutrophils [31].

The hair is considered an appendix of the skin performing various functions in the body among which are included the secretion and excretion of endogenously produced substances by several metabolic processes [32].

The emission and accumulation of chemical compounds by hair are well-studied, in both humans and animals, for several purposes, either as indicative of the internal metabolism of drugs [33]; for investigation in toxicological forensic [34]; for evaluation of chemical contam‐ inants [35]; post-treatment accumulation of drugs [36] ; alcohol and drugs abuse [37]; evalua‐ tion of trace elements [38]; or for anti-doping investigations [39].

Hair is an interesting sample for standardization of biomarkers because of its role to excrete endogenous substances [5]. Furthermore, the exam of dogs hair allowed to find *Leishmania* DNA by standard PCR technique, reingorcing its importance for the diagnosis [40].

Perspectives for Diagnosis and Control of Leishmaniasis Based on Volatile Organic Compounds http://dx.doi.org/10.5772/57279 319

**Figure 1.** Some clinical signs found in dogs with leishmaniasis: A) Ulcerative erythematous lesions on the plantar sur‐ face of the paw; B) Dermatitis tip of ear; C) Onychogryphosis; D) Marked cachexia and generalized exfoliative alopecia; E) Ulcerative nasal mucocutaneous lesions and periocular alopecia.

Following the thought that hair is a biological sample that allows evaluation of internal metabolism of healthy and infected animals, Oliveira et al. [41] demonstrated that volatile organic compounds (VOC) emitted by the dogs clearly differ comparing healthy animals and infected by *Leishmania* sp., previously identified for ELISA and PCR. In this way, VOC profile also varies due to presence and manifestation of clinical signs for dogs infected by *Leishma‐ nia* sp. [42].
