Comparison of two sampling techniques for the detection of Malassezia pachydermatis on the skin of dogs with chronic dermatitis

Adhesive tape strip and dry swab sampling techniques were compared for the detection of Malassezia pachydermatis on the skin of dogs with chronic dermatitis. One hundred and four dogs were sampled by each of the techniques. Two methods, a culture method and a stain method, were used to assess the sampling techniques. By the adhesive tape strip sampling technique, M. pachydermatis was detected on 83 (80%) dogs using the culture method and on 45 (43%) dogs using the stain method. By the dry swab sampling technique, M. pachydermatis was detected on 55 (53%) dogs using the culture method and on 33 (32%) dogs using the stain method. The study showed that the adhesive tape strip sampling technique, using the culture method, detected Malassezia on the skin of significantly more dogs (P<0.001) than the same technique using the stain method and also significantly more than the dry swab sampling technique, using either the culture or stain methods. It was also shown that an adhesive tape sample could be used to transfer cells to a slide for staining and microscopy prior to being used for culturing Malassezia.     

Humoral measurement of type-1 hypersensitivity reactions to a commercial Malassezia allergen

Malassezia pachydermatis is considered to be a contributing factor to canine atopic dermatitis (AD). The purpose of this study was to investigate the humoral response to a commercially produced M. pachydermatis extract. Fifteen atopic dogs with Malassezia overgrowth on the skin (MD), 16 atopic dogs without MD, three atopic dogs with overgrowth of Malassezia in the ears only (MO), and 12 normal dogs were intradermally tested with M. pachydermatis extract at 50, 100, 250, 500, 1000, 2000 and 4000 PNU mL(-1). All dogs were evaluated cytologically by cutaneous tape strip and bilateral ear exudate sampling to determine presence of MD or MO. Each had serum evaluated for anti-Malassezia IgE using three Malassezia extracts with an ELISA assay. The irritant threshold concentration at which healthy nonatopic dogs ceased to react was 1000 PNU mL(-1). There was a significant difference in intradermal test reactivity between the atopic groups. At this dilution, 93% (14/15) of the atopic MD group, 31% (5/16) of the atopic group without MD or MO, and 100% (3/3) of the atopic MO only group reacted. There were no significant differences in the serum IgE levels as measured by the Greer ELISA assay, between any groups using any of the three extracts. These results support that Greer's M. pachydermatis extract is useful for intradermal testing of dogs with an allergic phenotype, and that atopics with MD are more likely to have a type-1 Malassezia hypersensitivity than those without. The ELISA assay may require further development in order to be useful for the diagnosis of Malassezia hypersensitivity.     

Macroscopic,cytological and bacteriological evaluation of anal sac content in normal dogs and in dogs with selected dermatological diseases

Abstract Macroscopic and cytological aspects of anal sac content were evaluated in 40 normal dogs and 10 dogs each with pyoderma, Malassezia dermatitis associated with atopic dermatitis and uncomplicated atopic dermatitis. Bacteria isolated from anal sacs were compared with those from abdominal skin and hair in 20 normal dogs and 10 dogs with pyoderma. There was no difference between the groups in anal sac dimension, or in the colour, consistency or presence of granules in their content. Extracellular bacteria were found in higher numbers in diseased animals, whereas intracellular bacteria were observed in 40% of dogs with pyoderma and in only 2.5% of normal dogs. Malassezia spp. were present in 15.7% of dogs, with no difference between groups. Neutrophils were observed in 12.5% of normal dogs, 30% of dogs with Malassezia dermatitis with underlying atopic dermatitis and in 70 and 80% of dogs with pyoderma and uncomplicated atopic dermatitis, respectively. Seven bacterial species were isolated from anal sacs, with no difference between normal dogs and dogs with pyoderma. In five normal animals and in four dogs with pyoderma the same bacterial strains were isolated from anal sacs and from abdominal skin and hair.     

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.     

Serum antibodies to Malassezia yeasts in canine atopic dermatitis

Significant numbers of humans with atopic dermatitis develop Malassezia-specific IgE. Immediate skin-test reactivity to Malassezia has been demonstrated in atopic dogs. The aim of this study was to compare the serum IgG and IgE response to Malassezia in atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis, nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis and healthy dogs. Cytology was used to diagnose clinically significant Malassezia dermatitis and otitis. Contact plate cultures confirmed the validity of this technique. Reproducible enzyme-linked immunosorbent assays for Malassezia-specific IgG and IgE in canine serum were established. Atopic dogs had significantly higher serum IgG and IgE levels than either healthy dogs or nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis. There was no significant difference in IgG and IgE levels between atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis. The implications of these findings in the pathogenesis and management of canine atopic dermatitis are discussed.     

Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions.

Malassezia species are commensal organisms of human and animal skin that occasionally act as opportunistic pathogens. The lipid-dependent species are associated with human skin disorders, whereas the non-lipid-dependent species (Malassezia pachydermatis) is considered as an opportunistic secondary pathogen affecting the canine skin surface and ear canal. This study evaluated the relationship between Malassezia yeasts, their population size, and the occurrence of skin lesions from healthy and skin-diseased dogs. The efficiency of cytological examination and fungal culture for Malassezia detection was also evaluated. From March 2002 to July 2003, 33 healthy dogs and 54 dogs with pruritic localized skin diseases were examined; skin swabs (1218) were collected from 7 anatomical sites for culture and cytological examination. Malassezia prevalence according to anatomical site and the agreement between cytological results and fungal cultures were statistically analyzed. Differences in mean colony forming unit counts between positive healthy and diseased dogs were evaluated using the Bonferroni test for post hoc pair-wise comparisons. In healthy dogs, Malassezia yeasts were most frequently isolated in the perianal and perioral areas. The frequency of isolation and population size of Malassezia species were higher in dogs with localized dermatitis, especially in affected areas, indicating a role for Malassezia in the occurrence of skin lesions. Malassezia pachydermatis was the species most commonly cultured from the skin and external ear canal of healthy and diseased dogs; isolation of lipid-dependent yeasts from healthy dogs was less frequent. Using fungal culture as the gold standard, cytological examination showed good relative specificity (95%) but very low relative sensitivity (30%).     

The immunoglobulin G response to Malassezia pachydermatis extracts in atopic and non-atopic dogs

IgG immunoreactivity to Malassezia pachydermatis was compared in atopic and non-atopic dogs. Malassezia pachydermatis proteins with a molecular weight of 98 kDa were recognized at a significantly higher frequency in the sera of atopic dogs. Most of the atopic dogs with Malassezia dermatitis had a greater IgG response than did normal dogs.     

Malassezia dermatitis in dogs in Brazil: diagnosis,evaluation of clinical signs and molecular identification

Skin carriage and quantification of Malassezia yeasts were evaluated in 180 healthy dogs (group 1) and 117 dogs with clinical signs (pruritus, erythema, lichenification/seborrhoea, excoriations and alopecia) that could be related to Malassezia dermatitis (group 2) in Brazil. The lesions in the group 2 dogs were evaluated using CADESI‐03 scores. Samples were collected from five different anatomical areas. Direct examination was performed using the tape strip technique, and results were expressed as the mean number of yeasts per ×1000 microscopic field per dog. For mycological culture, a single piece of sterilized carpet was applied to the same areas sampled for cytology, and transferred onto Dixon’s modified medium. Yeast populations were expressed as mean colony forming units (CFU)/plate. Malassezia isolates were characterized by polymerase chain reaction–restriction endonuclease analysis of the large subunit (LSU) of ribosomal RNA gene. The probability of culturing Malassezia from dogs with skin lesions was significantly higher (P < 0.001) than from healthy dogs. There was a linear trend between CADESI‐03 score and mean CFU/plate. Group 2 dogs with positive cultures had higher CADESI‐03 scores than those with negative cultures (P < 0.05). Almost all isolates were identified as Malassezia pachydermatis. Only one isolate (group 2) was identified as Malassezia furfur. These data suggest that dogs with skin disorders harbouring Malassezia yeasts in quantities higher than 120 mean CFU/plate should be considered as having Malassezia dermatitis. The presence of Malassezia appears to exacerbate clinical lesions in dogs.     

Genotyping of Malassezia pachydermatis isolates from canine healthy skin and atopic dermatitis by internal spacer 1 (IGS1) region analysis

Isolates of Malassezia pachydermatis from healthy dog skin and from dogs with atopic dermatitis were molecularly characterized using internal spacer 1 (IGS1) region analyses, and their phospholipase A2 activity and pH growth profiles were then characterized in vitro. The percentage of isolates from healthy dogs that had the following IGS1 subtypes (isotype, %) were as follows: 1A, 6%; 1B, 27%; 1C, 11%; 2A, 6%; 2B, 6%; 3A, 11%; 3C, 3%; and 3D, 24%. In contrast, 9% of isolates from dogs with atopic dermatitis were isotype IB and 91% were isotype 3D, indicating that isolates of subtype 3D were the most prevalent in dogs with atopic dermatitis. Production of phospholipase A2 was statistically higher in isolates of subtype 3D than in the other subtypes. The subtype 3D isolates showed enhanced growth on alkaline medium compared with non-3D subtype isolates. The main clinical sign of canine Malassezia dermatitis is waxy exudates on the skin, which predispose the patient to development of a yeast overgrowth of the subtype 3D. Increased phospholipase A2 production may be involved in the inflammatory process associated with Malassezia dermatitis.     

Identification of major allergens of Malassezia pachydermatis in dogs with atopic dermatitis and Malassezia overgrowth

Abstract We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis . The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.     

Occurrence, distribution and population size of Malassezia pachydermatis on skin and mucosae of atopic dogs

Aim of the present study was to determine the distribution and quantification of Malassezia yeasts on a wide number of cutaneous sites in atopic dogs by means of a semiquantitative swab technique. A possible relationship between the presence of clinical signs and the occurrence and population size of yeasts was attempted. Forty-one privately owned atopic dogs of different age and breed were sampled. Results were expressed as colony forming units per swab. Malassezia colonies obtained from each plate were counted, scored and typed. All dogs yielded Malassezia pachydermatis from at least one skin area. Yeast population mean size by site was 6.98 (S.D.=3.47) as compared to other body areas. The frequence of isolation was higher from interdigital areas (70.7%), ears (63.4%), nail folds (35.7%), mouth (33.3%), groin (30.9%), conjunctiva and axillae (23.8%), perineum and anus (19%), perianal glands (9.5%). Ears, anus, interdigital areas, perianal glands and groin yielded the largest mycotic amount. M. pachydermatis was the sole species of yeast to colonize canine skin in examined animals. No statistical correlation between the presence of cutaneous alterations and Malassezia isolation was detected. Highest scores were not exclusively found on affected areas, but also on lesion-free sites, demonstrating that atopic animals can be heavily colonized also in apparently healthy areas.     

Comparative analysis of the frequency, distribution and population sizes of yeasts associated with canine seborrheic dermatitis and healthy skin

The purpose of this study was to investigate the diversity of yeast associated with the degree of canine seborrheic dermatitis (SD) by anatomical sites. Fifty-seven samples were divided as 17 healthy skin, 20 with primary seborrheic dermatitis (PSD), and 20 with secondary seborrheic dermatitis (SSD). Yeast isolation and characterization were carried out based on microscopical features and biochemical properties. DNA analysis at the internal transcribed spacer I of 26S rDNA region was utilized for species confirmation. Four species of yeast consisting Malassezia pachydermatis, Malassezia furfur, Candida parapsilosis and Candida tropicalis recovered from examined dogs. M. pachydermatis and C. parapsilosis were isolated from all dogs, but C. tropicalis and M. furfur were recovered from 3 healthy dogs and one diseased dog, respectively. The number of M. pachydermatis and C. parapsilosis in diseased dogs was higher than that of healthy specimens (P<0.01). High frequency and population size of C. parapsilosis were closely associated to PSD, while those of M. pachydermatis were associated with both PSD and SSD (P<0.01). C. parapsilosis were predominant at the perianal area. This study demonstrated the co-colonization of M. pachydermatis and C. parapsilosis in large amounts and frequency associated with stage of disease and anatomical site.     

Putative Malassezia dermatitis in six goats

Histopathology submissions from 28 goats with dermatological disease were identified in an archival search of pathology files. Microscopic sections of skin biopsy specimens were examined for the presence of Malassezia spp. organisms. Six cases with many Malassezia yeasts were identified histopathologically. Based on the extent of clinical disease, three cases were regarded as localized and three were generalized infections. Clinical findings included alopecia with dry seborrhoea (four cases), greasy seborrhoea (one case), and no clinical findings specific to localized Malassezia infection when concurrent bacterial infection was present (one case). Mild pruritus was reported in two cases of generalized infection. No breed predilection was apparent. Three cases were male and three were female. Malassezia dermatitis occurred in goats from 10 months to 13 years of age. Three of six cases had concurrent bacterial infection. Skin lesions resolved following topical antifungal therapy in the two goats that were treated. Histopathological findings in all cases were severe follicular and epidermal orthokeratotic hyperkeratosis with minimal epithelial change and mild superficial perivascular to interstitial nonsuppurative inflammation. Numerous budding yeasts were visible within the stratum corneum of all cases; however, Malassezia was not isolated in the three cases in which culture was attempted. Based upon these findings, the authors suggest that the diagnosis Malassezia dermatitis in goats is most likely to be made by cytological examination of skin impressions or by examination of skin biopsy samples.     

Evaluation of serum obtained from atopic dogs with dermatitis attributable to Malassezia pachydermatis for passive transfer of immediate hypersensitivity to that organism

OBJECTIVE: To determine the functionality of canine anti-Malassezia IgE via the passive transfer of immediate hypersensitivity localized to the skin (ie, cutaneous anaphylaxis) from atopic dogs with dermatitis attributable to overgrowth of Malassezia pachydermatis (Malassezia dermatitis [MD]) to healthy recipient dogs by use of the Prausnitz-Küstner (P-K) technique. ANIMALS: 7 clinically normal dogs, 32 atopic dogs with MD, serum from 11 atopic dogs with MD, and 3 healthy dogs without prior sensitization to M pachydermatis. PROCEDURE: Serum from atopic dogs with MD was used for P-K tests in 3 clinically normal recipient dogs. Serial dilutions of untreated, heat-inactivated, IgE-absorbed, and bovine serum albumin (BSA)-absorbed (control) aliquots of serum were injected ID in triplicate for dermal sensitization. Twenty-four, 48, and 72 hours later, a crude extract of M pachydermatis was injected ID into the sites used for sensitization injections, and immediate hypersensitivity reactions were graded on a 4-point scale. RESULTS: Untreated serum caused P-K reactivity beginning 24 hours after passive sensitization and persisting through 72 hours (titers, 1:32 to 1:64). Heat inactivation and IgE-absorption of serum eliminated P-K reactivity, whereas treatment of serum with BSA did not. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of P-K test results supports the passive transfer of cutaneous anaphylaxis by anti-Malassezia IgE and indicates it is functional in type-1 hypersensitivity reactions of atopic dogs with MD. Reduction or blockade of anti-Malassezia IgE in atopic dogs with MD may provide better clinical control of the disease.     

Adherence of Staphylococcus intermedius to corneocytes of healthy and atopic dogs: effect of pyoderma, pruritus score, treatment and gender

Staphylococcal pyoderma occurs commonly in atopic dogs. Some studies have suggested that adherence of staphylococci to corneocytes of atopic dogs and humans is higher than to corneocytes of healthy individuals. This hypothesis and possible differences resulting from the presence or absence of pyoderma, the severity of pruritus or the effect of treatment or gender, were studied. Adherent bacteria (Staphylococcus intermedius) were quantified by computerized image analysis on corneocytes collected from healthy or atopic dogs using double-sided adhesive tape. The adherence of S. intermedius to the corneocytes of atopic dogs was significantly greater than to those of healthy dogs (P=0.005). Furthermore, adherence was significantly greater in dogs with high levels of pruritus compared to those with low scores. No significant differences were found between atopic dogs with no history of pyoderma, atopic dogs with a history of pyoderma and atopic dogs with pyoderma at the time of sampling (P=0.068), suggesting that factors other than adherence are necessary for clinical pyoderma to develop. Treatment did not generally influence the adherence of S. intermedius to corneocytes of atopic dogs and there was no gender difference in adherence in either healthy or atopic dogs.     

Use of contact plates for the quantitative culture of Malassezia pachydermatis from canine skin

The Malassezia pachydermatis populations of the axilla and groin of 12 normal and 12 atopic dogs were compared using tape-strips and contact plates. When assessed by either method, the mean density of yeasts in the groin of the atopic dogs was significantly greater (P<0.05) than that of the normal dogs, suggesting that the cutaneous microenvironment of the groin region of the atopic dogs favoured colonisation by this yeast. Differences between the counts from the axilla were not significant. The frequency of isolation of yeasts from both dogs and sites was significantly higher (P<0.05 and P<0.001, respectively) in the atopic group. There was a very highly significant correlation (P<0.001) between the tape-strip counts and contact plate counts in the atopic group only. This study suggests that isolation of numerous M pachydermatis colonies from the axilla and groin of dogs using contact plates is indicative of elevated skin surface populations. The simplicity of the contact plate method makes it suitable for the routine quantitative culture of cutaneous M pachydermatis populations in dogs with dermatological disease.     

The ACVD task force on canine atopic dermatitis (XII): the relationship of cutaneous infections to the pathogenesis and clinical course of canine atopic dermatitis.

Dogs and human beings with atopic dermatitis (AD) frequently exhibit concurrent skin infections with Staphylococcus sp. bacteria or Malassezia yeast, and treatment of such infections is an important facet of managing these patients. Staphylococci appear to colonize atopic skin readily, and bacterial products on the skin could augment cutaneous inflammation via immediate hypersensitivity responses to the bacteria, by superantigen-mediated lymphocyte activation, or other non-specific mechanisms. Similarly, skin colonization by Malassezia yeast could contribute to clinical signs of AD; yeast components could induce inflammation via non-specific mechanisms, such as alteration in mediator release, or via antigen-specific hypersensitivity reactions. Clinical and experimental evidence exists that secondary microbial infections can both initiate and perpetuate episodes of AD in dogs and humans, and could even participate in promotion of pro-allergic immunologic responses. Mechanistic details of these complex interactions are under extensive investigation in human beings; only a few observations have been extended to include dog with AD.     

Epidermal dysplasia and Malassezia infection in two West Highland White Terrier siblings: an inherited skin disorder or reaction to severe Malassezia infection?

Two 9-month-old West Highland White Terrier siblings were referred to our clinic with pruritus, alopecia and lichenification. Cytological examination of Scotch tape strippings revealed Malassezia organisms and cocci. Skin biopsy specimens showed epidermal dysplasia. Treatment included bathing with a 2% miconazole/chlorhexidine-containing shampoo, orally administered ketoconazole (5 mg kg-1, every 12 h) and cloxacillin (25 mg kg-1 every 8 h). Six weeks later, the dermal infection had resolved and there was hair regrowth. However, the dogs were still moderately pruritic. Intradermal allergy testing was positive for house dust mites, storage mites and Malassezia. Immunotherapy was initiated, and treatment with ketoconazole and cloxacillin was stopped. Skin biopsies, which were performed in both dogs 4 months after the first presentation, revealed mild superficial perivascular dermatitis. The remaining mild facial pruritus was easily controlled with topical treatment. These two cases indicate that epidermal dysplasia might be an inflammatory or hypersensitivity reaction to the Malassezia infection or a result of excessive self-trauma, rather than a congenital keratinization disorder.     

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.     

Frequency of yeasts and dermatophytes from healthy and diseased dogs.

The aim of this study was to investigate the presence of dermatophytes and yeasts in healthy and diseased dogs. A total of 633 samples were collected from 26 healthy animals (104 samples), 131 with dermatitis (343 samples), 74 with otitis (148 samples), and 19 with ocular diseases (38 samples). Cultures from healthy animals were positive for Malassezia pachydermatis in 13.5% (7/52) of samples from skin, 42.3% (11/26) from ear, and 3.8% (1/26) from eye. Fungal growth was observed in 20.4% (70/343) samples from animals with dermatitis. Microsporum canis was the most isolated fungus (n = 39), followed by M. pachydermatis (n = 30) and Malassezia sp. (n = 3). Of the 148 samples from dogs with otitis, 90 (60.8%) were positive for M. pachydermatis, and of the clinical specimens from the conjunctiva of animals with ophthalmic disease, 2.6% (1/38) presented positive cultures for M. pachydermatis. Only 14.3% (2/14) of the positive cultures for M. pachydermatis and 40.9% (9/22) of those for M. canis were positive in the direct exam. Direct exams were positive in 84.3% (70/83) of the culture positive samples from affected ears of dogs with otitis. Malassezia pachydermatis may act as an aggravating factor in the occurrence of cutaneous diseases, or the isolation of M. canis may be associated with the onset of dermatophytosis. Fungal culture, rather than microscopic examination, should be used as the definitive diagnostic test for dermatomycoses and otitis.