**4. Importance of animals' oral health**

Disorders of the oral cavity are of great importance in veterinary medicine due to their high prevalence in dogs and their serious consequences, which can even affect the systemic health of the animal [20]. Abnormalities, injuries or disorders of this organ can cause discomfort and pain, leading the animal to anorexia, due to lack of food, and adipsia, not water intake, predisposing it to conditions of decreased immunity and clinical complications [21].

In addition to this great discomfort and the involvement of other organs, the inflammatory response caused by diseases in the oral cavity can lead to the gingival tissue a progressive loss of tooth fixation to the alveolar bone and, consequently, the loosening and loss of this tooth [22].

The dentistry specialty in veterinary medicine has been evolving in recent years, gaining space in the curriculum of some colleges. Even though the food industry has undergone great advances in the production of diets aimed at improving oral health, the number of professionals who perform an adequate clinical examination is still not significant. In addition to this important factor, the lack of adequate provision of oral hygiene care is worrying [22, 23].

Among dogs over one year of age, 95% have some degree of the disease, and in the clinic, it is believed that 100% of adult animals have varying degrees of periodontal disease [23]. The most common signs associated with periodontal disease are halitosis, dental calculus, inflammation and gingival bleeding, anorexia and the consequent weight loss, ptyalism, difficulty in chewing and grinding food, mobility and migration of teeth, loss of alveolar bone, gingival retraction and behavioral changes [24].

Periodontal disease is, therefore, the most common disease affecting dogs of all breeds, formed from proliferative microorganisms, defense cells (leukocytes and macrophages), epithelial cells, bacterial polysaccharides and salivary glycoproteins, which over time become organize, occurring mineralization and formation of dental calculus [25]. It is believed that this clinical condition is usually caused by the formation of bacterial plaques, but the isolation of yeasts from the oral cavity of dogs with periodontal disease is frequent (**Figure 4**).

The greatest risk in periodontopathic is not only the loss of teeth or the development of local infections, but the possible systemic effects of the pathological agent in the bloodstream.

Thus, the oral health of dogs is extremely important and still needs a greater focus on microbiological research and awareness of those responsible, regarding food, the importance of oral hygiene, and the attention of the tutor and the veterinarian regarding the etiopathogenesis of diseases, such as yeasts.

**Figure 4.** *Connection: Presence of Periodontal Disease x Positive isolation of yeast [19].*

## **5. Main genera and species of yeasts isolated from the oral cavity of dogs and clinical signs**

Just like in humans, dogs have a known range of yeasts in their oral mucosa that still requires more studies regarding colonization and pathogenicity. Despite its remarkable importance in the health of dogs, studies involving the isolation and correct identification of yeasts began to be developed in the 20th century [26].

This microbiota is not yet fully described, due to its great complexity and diversity. Fungal colonization of the oral cavity of dogs is associated with yeasts of the genera *Candida*, *Malassezia*, *Trichosporon* and *Rhodotorula*. Less frequently, we can isolate yeasts of the genus *Cryptococcus* [27].

In a recent study conducted with 50 mixed breed dogs, a yeast profile was found, composed of *Candida albicans* (39.5%), *C. parapsilosis* (18.6%), *C. zeylanoides* (13.9%), *C. krusei* (7%), *C. tropicalis* (4.7%), *Trichosporon* spp. (4.7%), *T. asahii* (4.7%), *C. guilliermondii* (2.3%), *T. mucoides* (2.3%) and *Malassezia pachydermatis* (2.3%). The genus *Candida* showed a high prevalence, making up a total of 82.2% of the isolated yeast profile. It is worth mentioning here the isolation of *Candida zeylanoides*, a rare species, even in humans, and thus, the oral mucosa of dogs can harbor a new "ecological niche" of this fungus species, which can also act as an opportunistic pathogen [9].

#### **5.1 Genus** *Candida* **and Candidiasis**

Currently, 317 species of this genus are recognized. Several of these species, more precisely 20, have a pathogenic potential and can thus cause infections in several species of animals, such as dogs [17]. The relationship with the host can be commensal, parasitic or saprophytic. It can also be found in the usual form of a yeast, or in the form of pseudohyphae. *Candida albicans* is the most common colonizer in cases of infections, with a predilection for mucous surfaces and mucocutaneous areas. Other species, such as *C. kefyr, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. famata, C. parapsilosis*, can be isolated from animals (**Figure 5**) [15].

There were only few cases found in the literature in small animals, however, reports of candidiasis in various animal species are also increasingly common, described in photos of pyoderma of the lip folds, disseminated and localized mucocutaneous dermatitis, urinary tract infections, gastrointestinal and reproductive system, ear infections, systemic and oral infections [27]*.*

#### **Figure 5.**

*Presumptive result of* Candida *species isolated from the oral cavity of mixed breed dogs according to the CHROMAGAR Candida®.*

**97**

**Figure 7.**

*400x [15].*

**Figure 6.**

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

Candidiasis related to the digestive system of dogs, such as a clinical manifestation of glossitis, is characterized by the formation of pseudomembranous plaques, usually whitish in color, or yellowish beige. Once these plaques were removed, we

*Mixed breed dog with oral candidiasis (glossitis). Friable white and yellowish plates covering the tongue [28].*

The genus *Trichosporon* has 37 species that inhabit different ecological niches, such as water, soil and body and mucous surfaces of humans and animals. They can cause superficial and deep infections, such as *Trichosporon asahii*, *T. mucoides*,

No cases of *Trichosporon* infections have been reported in the oral mucosa of dogs, however, several species have already been isolated as colonizers. Clinical cases of nasal granuloma in other animals, cystitis in cats, mastitis in cows and dermatitis in horses and monkeys have already been described (**Figure 7**) [30].

The genus *Malassezia* has 15 species, mostly lipophilic yeasts, that can be part of the skin and mucous membranes of humans and dogs. They are opportunistic yeasts, and in certain circumstances, they can lead to clinical manifestations [31]. *Malassezia pachydermatis*, the most frequent in dogs, is not lipophilic and can grow

*A and B –Yeasts of Genus* Trichosporon *showing oval and rectangular artrospores - (A) 160x and (B)* 

noticed erythematous regions with the presence of ulcers (**Figure 6**) [28].

**5.2 Genus** *Trichosporon* **and Trichosporonose**

*T. ovoides*, *T. inkin*, *T. asteroides* and *T. cutaneum* [29].

in a culture medium common in mycology [19] (**Figure 8**).

**5.3 Genus** *Malassezia* **and Malasseziosis**

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

#### **Figure 6.**

*Canine Genetics, Health and Medicine*

can isolate yeasts of the genus *Cryptococcus* [27].

can also act as an opportunistic pathogen [9].

system, ear infections, systemic and oral infections [27]*.*

**5.1 Genus** *Candida* **and Candidiasis**

(**Figure 5**) [15].

**and clinical signs**

**5. Main genera and species of yeasts isolated from the oral cavity of dogs** 

Just like in humans, dogs have a known range of yeasts in their oral mucosa that still requires more studies regarding colonization and pathogenicity. Despite its remarkable importance in the health of dogs, studies involving the isolation and correct identification of yeasts began to be developed in the 20th century [26]. This microbiota is not yet fully described, due to its great complexity and diversity. Fungal colonization of the oral cavity of dogs is associated with yeasts of the genera *Candida*, *Malassezia*, *Trichosporon* and *Rhodotorula*. Less frequently, we

In a recent study conducted with 50 mixed breed dogs, a yeast profile was found, composed of *Candida albicans* (39.5%), *C. parapsilosis* (18.6%), *C. zeylanoides* (13.9%), *C. krusei* (7%), *C. tropicalis* (4.7%), *Trichosporon* spp. (4.7%), *T. asahii* (4.7%), *C. guilliermondii* (2.3%), *T. mucoides* (2.3%) and *Malassezia pachydermatis* (2.3%). The genus *Candida* showed a high prevalence, making up a total of 82.2% of the isolated yeast profile. It is worth mentioning here the isolation of *Candida zeylanoides*, a rare species, even in humans, and thus, the oral mucosa of dogs can harbor a new "ecological niche" of this fungus species, which

Currently, 317 species of this genus are recognized. Several of these species, more precisely 20, have a pathogenic potential and can thus cause infections in several species of animals, such as dogs [17]. The relationship with the host can be commensal, parasitic or saprophytic. It can also be found in the usual form of a yeast, or in the form of pseudohyphae. *Candida albicans* is the most common colonizer in cases of infections, with a predilection for mucous surfaces and mucocutaneous areas. Other species, such as *C. kefyr, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. famata, C. parapsilosis*, can be isolated from animals

There were only few cases found in the literature in small animals, however, reports of candidiasis in various animal species are also increasingly common, described in photos of pyoderma of the lip folds, disseminated and localized mucocutaneous dermatitis, urinary tract infections, gastrointestinal and reproductive

*Presumptive result of* Candida *species isolated from the oral cavity of mixed breed dogs according to the* 

**96**

**Figure 5.**

*CHROMAGAR Candida®.*

*Mixed breed dog with oral candidiasis (glossitis). Friable white and yellowish plates covering the tongue [28].*

Candidiasis related to the digestive system of dogs, such as a clinical manifestation of glossitis, is characterized by the formation of pseudomembranous plaques, usually whitish in color, or yellowish beige. Once these plaques were removed, we noticed erythematous regions with the presence of ulcers (**Figure 6**) [28].

#### **5.2 Genus** *Trichosporon* **and Trichosporonose**

The genus *Trichosporon* has 37 species that inhabit different ecological niches, such as water, soil and body and mucous surfaces of humans and animals. They can cause superficial and deep infections, such as *Trichosporon asahii*, *T. mucoides*, *T. ovoides*, *T. inkin*, *T. asteroides* and *T. cutaneum* [29].

No cases of *Trichosporon* infections have been reported in the oral mucosa of dogs, however, several species have already been isolated as colonizers. Clinical cases of nasal granuloma in other animals, cystitis in cats, mastitis in cows and dermatitis in horses and monkeys have already been described (**Figure 7**) [30].

#### **5.3 Genus** *Malassezia* **and Malasseziosis**

The genus *Malassezia* has 15 species, mostly lipophilic yeasts, that can be part of the skin and mucous membranes of humans and dogs. They are opportunistic yeasts, and in certain circumstances, they can lead to clinical manifestations [31]. *Malassezia pachydermatis*, the most frequent in dogs, is not lipophilic and can grow in a culture medium common in mycology [19] (**Figure 8**).

**Figure 7.**

*A and B –Yeasts of Genus* Trichosporon *showing oval and rectangular artrospores - (A) 160x and (B) 400x [15].*

**Figure 8.**

*Yeasts of the Genus* Malassezia *spp. - single budding on wide base - Panotic, 1000x.*

**Figure 9.** Cryptococcus *spp. Encapsulated yeasts - Nigrosina, 1000x.*

Several clinical symptoms can be associated with *Malassezia* spp. and, particularly, in cases of otitis and dermatitis in dogs. Cases of otitis by *Malassezia pachydermatis* are frequency, but oral infection caused by this agent have not been described or has not yet been well studied [19].

#### **5.4 Genus** *Cryptococcus* **and cryptococcosis**

In the *Cryptococcus* genus, we found 38 species, with *Cryptococcus neoformans* and *C. gattii* being the most prominent in medical mycology in man and animals (**Figure 9**). The species can be found in different places in the environment, primarily in association with birds' droppings, mainly pigeons, but have an ecological association with trees too, such as eucalyptus [32].

In dogs, can enter the body through the lung causing pulmonary disease, and several clinical signs can be presented, such as skin lesions, nasal mucosa ("clown nose"), and can hit the central nervous system, for its neurotropic nature. These lesions in the nasal mucosa can extend into the oral cavity of the animals [28].

### **6. Collection of clinical material and identification of yeasts**

#### **6.1 Material collection**

The collection of the oral cavity of dogs is performed with the aid of a sterile, alginate swab, moistened with sterile saline solution. The swab is introduced,

**99**

mucosa [19] (**Figure 10**).

**Figure 10.**

**Figure 11.**

up to two weeks [19].

**6.2 Yeast identification**

(**Figure 12**).

(**Figure 13**).

(**Figure 14**).

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

carefully, in the oral cavity, in circular movements, passing through the entire oral

*Collection with sterile swab of the oral mucosa of a mixed breed dog - City of Campinas, São Paulo - Brazil.*

After this procedure, the collected samples must be sent to the laboratory and sown in Petri dishescontaining basic mycology medium (Sabouraud dextrose agar), plus antibiotics (chloramphenicol - 0.05 g/L concentration). Incubation at 25°C for

There are several procedures that can be used to identify yeasts. Direct examina-

The identification of yeasts can be performed by means of macro and micro-

In the more specific micro morphological identification, we must observe the characteristics of the cells (oval, round, unipolar bud, or multiple buds), pseudohyphae, hyphae and structures characteristic of *C. albicans*, such as chlamydoconidia

The formation of a germ tube, another important characteristic of *C. albicans*,

morphological, biochemical, proteome (MALDI-TOF) and molecular tests. In macromorphological characterization, we studied color, texture and edges

originates from blastoconidium when the yeast is sown in fetal bovine serum

tion (fresh), or with Gram stain is also highlighted (**Figure 11**).

*Yeasts of Genus* Candida *in an abdominal dog fluid sample - Fuchsin, 1000x.*

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

#### **Figure 10.**

*Canine Genetics, Health and Medicine*

or has not yet been well studied [19].

**5.4 Genus** *Cryptococcus* **and cryptococcosis**

Cryptococcus *spp. Encapsulated yeasts - Nigrosina, 1000x.*

*Yeasts of the Genus* Malassezia *spp. - single budding on wide base - Panotic, 1000x.*

association with trees too, such as eucalyptus [32].

Several clinical symptoms can be associated with *Malassezia* spp. and, particularly, in cases of otitis and dermatitis in dogs. Cases of otitis by *Malassezia pachydermatis* are frequency, but oral infection caused by this agent have not been described

In the *Cryptococcus* genus, we found 38 species, with *Cryptococcus neoformans* and *C. gattii* being the most prominent in medical mycology in man and animals (**Figure 9**). The species can be found in different places in the environment, primarily in association with birds' droppings, mainly pigeons, but have an ecological

In dogs, can enter the body through the lung causing pulmonary disease, and several clinical signs can be presented, such as skin lesions, nasal mucosa ("clown nose"), and can hit the central nervous system, for its neurotropic nature. These lesions in the nasal mucosa can extend into the oral cavity of the

The collection of the oral cavity of dogs is performed with the aid of a sterile, alginate swab, moistened with sterile saline solution. The swab is introduced,

**6. Collection of clinical material and identification of yeasts**

**98**

animals [28].

**Figure 9.**

**Figure 8.**

**6.1 Material collection**

*Collection with sterile swab of the oral mucosa of a mixed breed dog - City of Campinas, São Paulo - Brazil.*

**Figure 11.** *Yeasts of Genus* Candida *in an abdominal dog fluid sample - Fuchsin, 1000x.*

carefully, in the oral cavity, in circular movements, passing through the entire oral mucosa [19] (**Figure 10**).

After this procedure, the collected samples must be sent to the laboratory and sown in Petri dishescontaining basic mycology medium (Sabouraud dextrose agar), plus antibiotics (chloramphenicol - 0.05 g/L concentration). Incubation at 25°C for up to two weeks [19].

There are several procedures that can be used to identify yeasts. Direct examination (fresh), or with Gram stain is also highlighted (**Figure 11**).

#### **6.2 Yeast identification**

The identification of yeasts can be performed by means of macro and micromorphological, biochemical, proteome (MALDI-TOF) and molecular tests. In macromorphological characterization, we studied color, texture and edges (**Figure 12**).

In the more specific micro morphological identification, we must observe the characteristics of the cells (oval, round, unipolar bud, or multiple buds), pseudohyphae, hyphae and structures characteristic of *C. albicans*, such as chlamydoconidia (**Figure 13**).

The formation of a germ tube, another important characteristic of *C. albicans*, originates from blastoconidium when the yeast is sown in fetal bovine serum (**Figure 14**).

**Figure 12.** *Culture of yeasts on Sabouraud dextrose agar.*

#### **Figure 13.**

Candida albicans *in culture broth. Globose, or elongated cells, pseudohyphae, hyphae, blastoconidia and characteristic chlamydoconidia, 1000x.*

Tests of assimilation of sources of nitrogen and carbohydrates can be performed (auxanogram), as well as fermentation tests (zymogram). The protocol followed for these methods is from the manual "The Yeasts: a taxonomic study" (volumes 1, 2 and 3). The MALDI-TOF technique is a mass spectrometry, which determines the protein profile (proteome) of the yeast under study. It is a fast technique (15–20), simple, excellent cost–benefit, however, there are limitations to the use of the laboratory routine, as the device is expensive and requires specialists to use it, as well as a robust base of standard strains.

**101**

**Figure 16.**

**Figure 15.**

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

*Germ tube on bovine serum –* Candida albicans*, 400x.*

specialized laboratories [33].

**Figure 14.**

For the identification of yeasts, we also count on molecular biology techniques, which are sensitive and specific. For the differentiation, for example, of *C. albicans* and *C. dubliniensis* is the most accurate technique. There are several methods such as PCR (Polymerase chain reaction), RFLP (Restriction fragment length polymorphism) and RAPD (Random amplified polymorphic DNA). One of the most used is PCR, which detects minimal amounts of DNA, or RNA. But not all laboratories can use these methods, due to the higher costs and the needs of

*Yeasts of genus Candida on CHROMAGAR Candida® -* Candida albicans *with green color.*

*API20CAUX method (bioMérieux®) –profile of carbohydrates assimilation.*

*Importance of Yeasts in Oral Canine Mucosa DOI: http://dx.doi.org/10.5772/intechopen.95905*

*Canine Genetics, Health and Medicine*

*Culture of yeasts on Sabouraud dextrose agar.*

**100**

**Figure 13.**

**Figure 12.**

a robust base of standard strains.

*characteristic chlamydoconidia, 1000x.*

Tests of assimilation of sources of nitrogen and carbohydrates can be performed (auxanogram), as well as fermentation tests (zymogram). The protocol followed for these methods is from the manual "The Yeasts: a taxonomic study" (volumes 1, 2 and 3). The MALDI-TOF technique is a mass spectrometry, which determines the protein profile (proteome) of the yeast under study. It is a fast technique (15–20), simple, excellent cost–benefit, however, there are limitations to the use of the laboratory routine, as the device is expensive and requires specialists to use it, as well as

Candida albicans *in culture broth. Globose, or elongated cells, pseudohyphae, hyphae, blastoconidia and* 

**Figure 14.** *Germ tube on bovine serum –* Candida albicans*, 400x.*

For the identification of yeasts, we also count on molecular biology techniques, which are sensitive and specific. For the differentiation, for example, of *C. albicans* and *C. dubliniensis* is the most accurate technique. There are several methods such as PCR (Polymerase chain reaction), RFLP (Restriction fragment length polymorphism) and RAPD (Random amplified polymorphic DNA). One of the most used is PCR, which detects minimal amounts of DNA, or RNA. But not all laboratories can use these methods, due to the higher costs and the needs of specialized laboratories [33].

**Figure 15.** *Yeasts of genus Candida on CHROMAGAR Candida® -* Candida albicans *with green color.*

#### **Figure 16.** *API20CAUX method (bioMérieux®) –profile of carbohydrates assimilation.*

#### **6.3 Chromogenic medium: CHROMagarCandida®**

Sowing in chromogenic media, such as CHROMagarCandida®, can provide presumptive identification according to the color developed by the yeast. In this medium, the specie *Candida albicans* develops a light green color; *C. tropicalis* it is blue/green and *C. krusei* light pink, for example (**Figure 15**).

In addition to these identification methods, there are several automated and manual systems that facilitate the laboratory routine, such as Vitek and API20C, as examples (**Figure 16**).
