Malassezia pachydermatis presence in canine external otitis and dermatopathies

1 Departamento de Parasitologia e Microbiologia, Instituto de Biologia, Universidade Federal de Pelotas, Pelotas, RS, Brasil. 2 Faculdade de Veterinária – Universidade Federal de Pelotas – Pelotas – RS – Brasil. 3 Departamento de Microbiologia Facultad de Medicina Veterinaria y Zootecnia Universidad Autonoma de Nuevo Leon – Monterrey México. 4 Programa de Pós Graduação em Ciências Veterinárias Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil.


INTRODUCTION
M. pachydermatis is both normal occurrence yeast and an opportunistic pathogen of the external ear canal of dogs and cats which can also be found on the skin, in the rectum, anal sacs and vagina (Bond et al., 1996;Dizotti and Coutinho, 2007).This yeast is one of the most frequent microorganisms associated to external otitis in dogs (Gentilini et al., 1991;Crespo et al., 2002;Nascente et al., 2004); recent studies have also referred to M. pachydermatis as being the cause of canine dermatitis (Nardoni et al., 2007;Leite et al., 2003).*Corresponding author.E-mail: josiara.mds@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Malasseziosis clinical manifestation installation indicates an alteration in the balance between the commensal and its host.The clinical disease occurs when there is an excessive multiplication of M. pachydermatis due to alterations in the host defense mechanism.The increase in M. pachydermatis cell number is facilitated when there are microenvironment changes, such as an excessive production of cerumen after therapy involving the use of antibiotics or corticoids (Ashbee, 2007;Nardoni et al., 2005), and also by concomitant primary conditions, such as hypersensitivity to food, flea bite hypersensitivity and/or atopy, pyodermitis, demodicosis and endocrine problems (hyperadrenocorticism, hyperthyreodism, diabetes mellitus) (Bond et al., 2002).
The aim of this paper was to research the diseases which predispose to the development of opportunistic M. pachydermatis in dogs with external otitis or dermatopathies.

MATERIALS AND METHODS
The study was performed in 70 dogs with dermatological clinical signs and/or external otitis, classified according to age, sex, and breed.All animals were submitted to a general clinical examination; the external ear canal and/or cutaneous tegument were also examined.Auxiliary examination methods were used to reach a final diagnosis.
Cerumen samples from animals with external otitis were collected with sterile swabs, rubbing the anterior part of the ear canal; samples from animals with dermatopathies were collected through the carpet technique (Mariat and Adan-Campos, 1967) by rubbing the carpet in the lesion area.
A direct examination was performed for the material collected from the external ear canal by using Gram coloration, which was analyzed under a microscope (1000X) in search for cells with morphology compatible to M. pachydermatis.Direct examination was done by counting the cells which were similar to the yeast in the microscopic field; six fields were observed.
The yeast cell/field absence was considered negative (-), and the following scores were considered positive: (+) up to five cells/field; (++), six to ten cells/field; (+++) more than ten cells/macroscopic field.All isolated samples of M. pachydermatis were cultured in Sabouraud dextrose agar medium (kindo et al., 2004;Prado et al., 2008) added with chloramphenicol and cycloheximide kept at 32ºC for ten days; their macroscopic characteristics were observed daily.Cell morphology was seen through colony smear, stained with Gram color and then biochemically characterized (Guého et al., 1996).
The results were coded according to animal characteristics parameters (sex, age and ear shape), direct examination and isolation; subsequently, data were analyzed by the EpiInfo software (Epi Info (TM) software), and distribution frequencies were carried out by the chi-square test for comparison.

DISCUSSION
The high M. pachydermatis percentage (80%) in canine external otitis cases was similar to those previously found b y Gentilini et al. ( 1991) and Nascente et al. ( 2004), who found 91.8 and 76.5%, respectively.Feigl et al. (1981), isolated M. pachydermatis in 50.6% of external otitis cases.In other countries, the yeast frequency ranged from 23% to 76.3% (Nascente et al., 2004;Dworecka-Kaszak et al., 1994).Studies have demonstrated that M. pachydermatis frequency is variable and that the high percentage of isolations obtained was probably related to the great number of ceruminous otitis cases, which promotes an accumulation of secretion and cerumen (Nagata, 2013).The latter is a growth promoter to M. pachydermatis (Gabal, 1988); this frequency tends to be lower in bacterial otitis (Kiss et al., 1997) even though the yeast was isolated in the two cases presented in this study.It is also important to consider that the presence of the yeast in the external ear canal of dogs is more frequent than that in other parts of the body (Nascente et al., 2004;Guého et al., 1996;Bond et al., 2006;Machado et al., 2003).
In this study, there was a variation in cell number of the collected material from canine external otitis per microscopic field visualized by direct examination; in all cases where M. pachydermatis was isolated, however, direct examination presented yeast cells ranging from + to +++.Bond et al. (1996) considered the presence of many yeast cells per microscopic field to be a pathogenic activity sign.
Canine tegument infection by Malassezia is a chronic evolution dermatopathy because of the exacerbated multiplication of M. pachydermatis in certain cutaneous areas.W hen there is a rupture of the epidermal barrier, secondary infections caused by bacteria or yeasts may occur.There is a number of cutaneous or systemic conditions that can alter the protective skin barrier, such as food hypersensivity, atopy, flea bite hypersensitivity, pyodermitis, demodicosis and endocrine problems (hyperadrenocorticism, hypertireoidism, mellitus diabetes), directly contributing to the development of the disease (Gabal, 1988;Larsson et al., 1998;Nagata, 2013).
M. pachydermatis has also been isolated in animals with skin ceratinization disorders, hypersensibility, epidermal displasia, and immunosuppression, in addition to those showing external otitis.From the 25 dermatopathy samples, isolation was observed in 52% (13) of the cases, and the underlying diseases diagnosed in these cases were seborrhea (6), folding dermatitis (2), hot spot (2), atopy (1), callus pyodermatitis (1) and acanthosis nigricans (1).However, the fact the seborrhea was the most frequent dermatopathy found in the diagnosis (10) must be taken into consideration.The yeast pathogen has been discussed in several studies which have  described M. pachydermatis as a pathogenous opportunist which thrives in the medium where there is an increase in humidity and lipid cerumen composition, among other favorable factors (Amaral et al., 1998;Coutinho, 2005).Cocker Spaniel English, Daschund and Poodle breeds showing dermatopathies, as well as animals of no definite breed, were the ones which presented the most dermatological problems, followed by Sharpei, Labrador, German Shepherd, Boxer and Bulldog breeds, though their sampling was not significant due to the few individuals available for each breed.In previous studies, Basset Hound, Boxer, Sharpei, Lhasa Apso, Cocker Spaniel English, Poodle, German Shepherd, Daschund, German Shepherd and Labrador retriever breeds have been described as being more dermatitis-prone (Nascente et al., 2004;Bond et al., 2002;Larsson et al., 1998).The breeds most often stricken by external otitis were Poodle, Cocker Spaniel English, Labrador retriever, Sharpei, Rotweiller, Old English Sheep Dog, Siberian Husky, Boxer, as well as those animals of no definite breed.According to Feijó et al. (1998), the breed most affected with otic infection by Malassezia is the Poodle.In external otitis cases, a greater percentage of animals with malasseziosis infection presented pendulum-like ears, such being the case of the Poodle and the Cocker Spaniel English.These findings were justified by Mansifield et al. (1990) as being the result of an alteration in the external ear canal microclimate.German Shepherd dogs have prick ears but are external otitis prone due to the aprocrine glands (Guillot and Bond, 1999;Feijó et al., 1998).In our study, the Cocker Spaniel English presented dermatitis and external otitis, in agreement with the findings by Larsson et al. (1998) and Feijó et al (1998), who consider this breed as being malasseziosis prone due to idiopathic seborrhea.
Age predisposition was not observed, once there was a wide age range among the studied animals was; these results are in agreement with those by Reynoud and Chauve (Reynaud and Chauve, 1984), who found equivalent percentages for animals in every age range.Feijó et al. (1998), though, found a great percentage of affected animals ranging from two months to three years of age, considering that young animals are more affected than adults.Chengapa et al. (1983) also observed the disease caused by this yeast in every age range, but with a higher percentage in animals up to nine months.Other studies have reported a frequency in animals up to two years of age (Machado et al., 2003;Larsson et al., 1998).Choi et al. (2001) reported its occurrence in animals from one to three years of age and Dufait (1983) considered that most cases mostly occur in dogs older than two years.
In relation to sex, most of the animals referred to the Dermatological Clinic were male, in agreement with a study by Feijó et al. 1998.However, according to many authors (Coutinho et al., 2006;Girão et al., 2006;Larsson et al., 1998;Feijó et al., 1998;Mansifield et al., 1990), there is no sexual preference.

Conclusion
The results obtained confirms that the yeast may be a

Table 1 .
Features of the animals referred to veterinary clinic with skin disease or otitis external and its isolation Malassezia pachydermatis

Table 3 .
Distribution characteristics of 49 animals with Malassezia pachydermatis isolation associated with a skin disease or external otitis.
*No statistical difference between isolation of M. pachydermatis and gender, age or race.