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.     

The biology of Malassezia organisms and their ability to induce immune responses and skin disease

Introduction 4 History and taxonomy of the genus Malassezia 5 Biological characteristics of Malassezia organisms 5 Structure 5 Reproduction 6 Biochemistry 6 Distribution of Malassezia organisms on the host 6 Immunological and epidermal responses to Malassezia organisms 7 The immune response to Malassezia organisms 7 Antigen release, penetration and presentation 8 Cell-mediated immune responses 8 IgG, IgM and IgA responses to Malassezia organisms 9 IgE responses to Malassezia organisms 10 Mast cell responses 11 Epidermal responses associated with Malassezia dermatitis 12 Malassezia organisms as pathogens in humans and animals 13 Diseases associated with Malassezia spp. in humans 13 Pityriasis versicolor 13 Malassezia folliculitis 13 Seborrheic dermatitis and dandruff 14 Atopic dermatitis 14 Malassezia fungaemia 14 Diseases associated with Malassezia spp. in animals 15 Malassezia dermatitis in dogs 15 Predisposing factors for overgrowth of Malassezia pachydermatis 15 Pathogenesis 16 Clinical features 16 Diagnosis 17 Treatment 18 Conclusions 19 References 19     

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.     

Otomycosis due to Aspergillus spp. in a dog: case report and literature review

This report describes the clinical findings, clinicopathology and treatment of otomycosis caused by Aspergillus spp. in an atopic dog affected by chronic unilateral purulent otitis externa unresponsive to topical and oral antibiotics and antifungal treatments. Cytology of otic exudate revealed neutrophils and septate fungal hyphae, and otic culture grew Aspergillus spp. and no bacteria. Treatments included allergen‐specific immunotherapy, topical and oral antifungal therapy and anti‐inflammatory steroid therapy. Final resolution occurred after treatment of the underlying hypersensitivity disorder, administration of topical ketoconazole and debridement of infectious ear exudate. Otomycosis due to filamentous fungi may, as in humans, occur in dogs with ear canals compromised by pre‐existing allergic or bacterial otitis, and possibly previous antibiotic therapy. Antifungal medications provided clinical improvement, but the key to successful treatment was the restoration of the normal physiology of the external auditory canal.     

Efficacy of a pimaricin suspension for treating otitis externa associated with M pachydermatis

A clinical trial was conducted to evaluate the efficacy of a 1 per cent pimaricin suspension in the treatment of canine otitis externa associated with Malassezia pachydermatis. Of 40 dogs examined, M pachydermatis was the sole infection in five ears and was commensal with other organisms in 35 ears. Pimaricin in a 1 per cent suspension was administered twice a day for two weeks. Employing continuous therapy, signs accompanying otitis externa were ameliorated steadily, and satisfactory results were achieved in 33 of the dogs. No noticeable side effects were observed with the pimaricin suspension.     

In vitro activity of an ear rinse containing tromethamine, EDTA, benzyl alcohol and 0.1% ketoconazole on Malassezia organisms from dogs with otitis externa

The purpose of this study was to evaluate the in vitro activity of an ear rinse containing tromethamine, EDTA, benzyl alcohol and 0.1% ketoconazole in purified water on Malassezia organisms from dogs with otitis externa. Malassezia organisms were collected from ear swab samples from the external ear canal of 19 dogs with otitis externa plus one control strain of Malassezia pachydermatis. Three test solutions were evaluated: ER (EDTA, tromethamine, benzyl alcohol), ER + keto (EDTA, tromethamine, benzyl alcohol, ketoconazole), and H2O (purified water). Ten-millilitre aliquots of each test solution was transferred into 20 tubes and inoculated with one of the isolates (1 tube per isolate: 19 clinical and 1 control strain). Samples were retrieved from each tube at five time points (0, 15, 30, 45 and 60 min), transferred to Petri dishes, mixed with Sabouraud dextrose agar supplemented with 0.5% Tween 80 and incubated. Following incubation, the plates were examined for growth and colonies counted as colony-forming units per millilitre. The data were analysed using a repeated measures analysis, with pair-wise comparisons of solution-time combinations. There was a significant reduction in Malassezia growth in ER + keto at all time points (P < 0.0001) compared to time zero. Neither ER nor H2O had any effect on the growth of Malassezia. ER + keto was significantly more effective in reducing Malassezia growth (P < 0.0001) at all time points compared to both ER and H2O. ER + keto may be useful in the treatment of Malassezia otitis externa. Future studies should be performed to evaluate the in vivo efficacy of ER + keto as treatment for otic infections caused by Malassezia.     

P‐74 Evaluation of the in vivo effects of Tris‐EDTA and chlorhexidine digluconate 0.15% solution in chronic bacterial otitis externa: 11 cases

The objectives of this study were to evaluate in vivo tolerance, and antimicrobial and clinical activities of a topical otic preparation containing EDTA tromethamine (Tris) and chlorhexidine digluconate 0.15% solution (Otodine^®) in dogs with chronic bacterial otitis externa. Eleven dogs were included. The affected ears were filled with the solution once daily during a 2‐week period. Dogs were evaluated on days 0, 14 and 28. Three clinical parameters (exudate, erythema, pain) and three cytologic parameters (Malassezia, cocci, rods) were scored (0–4 scale) by otoscopic and cytological examinations of otic exudate. Bacterial cultures were performed at each time point. If there were bacteria on cytological examination on day 14, the dogs were treated with the original product, with the addition of enrofloxacin (5%) applied 10 min after the original product, for a further 2 weeks. All 11 cases yielded isolates of resistant gram‐negative bacteria; gram‐positive bacteria were also isolated from six of 11 dogs. On day 14, six of 11 dogs were negative on culture examination; on day 28, 10 of 11 were negative and only one case had a positive culture. On day 14, clinical and microbial scores (cytology) were reduced by 54.6 and 71.1%, respectively, and by 85.7 and 94% on day 28. All cases reported good tolerance of the treatment. The results show that this ear solution was helpful in the management of chronic bacterial otitis externa in dogs and was well tolerated. There seems to be a synergistic effect of the combination of Tris‐EDTA/chlorhexidine digluconate 0.15% solution, and an antimicrobial agent (enrofloxacin) against resistant gram‐positive and gram‐negative bacteria. Funding: Self‐funded.     

Factors associated with elevated cutaneous Malassezia p a chyderm a tis populations in dogs with Pruritic skin isease

The prevalences of breeds and concurrent diseases in a group of 40 dogs with pruritic skin disease associated with elevated cutaneous Malassezia pachydermatis populations were compared with samples of a dermatological hospital population. The ages and genders of the affected dogs were comparable to those of the dermatology population. Basset hounds, cocker spaniels and West Highland white terriers were significantly overrepresented. Concurrent diseases were diagnosed in 27 dogs, of which 15 were atopic. However, the prevalences of atopic disease, primary keratinisation defects and endocrinopathies in dogs with elevated cutaneous M pachydermatis populations were comparable to those in the dermatology population as a whole. These results indicate that certain breeds are predisposed to the development of elevated cutaneous M pachydermatis populations and that concurrent skin diseases can frequently be identified in affected dogs. However, the relationship between these concurrent diseases and abnormal M pachydermatis populations remains unclear.     

Malassezia pachydermatis isolated from normal and diseased external ear canals in dogs: a comparative analysis

To investigate the role of Malassezia pachydermatis as a pathogenic agent in canine otitis, a comparative analysis of isolates from normal and diseased external ear canals in dogs was undertaken. Specimens were collected from the ears of dogs with unilateral or bilateral otitis and from healthy dogs. Mycological analysis was by direct microscopy and fungal culture on Sabouraud's dextrose agar and Dixon's agar. Of the otitis specimens, 63.7% showed typical Malassezia cells on cytological examination. In samples taken from the healthy ears of dogs with unilateral otitis, only 21.43% (P<0.05) showed evidence of Malassezia. M. pachydermatis was identified cytologically and culturally in 57.53% (P<0.05), 14.29% and 30.0% of samples from the ears of dogs with otitis, from the healthy ears of dogs with unilateral otitis and from the ears of healthy dogs with no otitis. In the group with otitis associated with M. pachydermatis, the poodle was the most common breed (39.29%; P<0.05), whereas in the group without otitis, the German Shepherd breed was prominent (although this observation was not statistically significant). In both groups, the majority of dogs with M. pachydermatis were aged between 1 and 3 years (P<0.05). The higher incidence of M. pachydermatis isolated from the ears of dogs with otitis externa suggests a putative pathogenic role of this yeast in this condition.     

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%).     

Population sizes and frequency of Malassezia pachydermatis at skin and mucosal sites on healthy dogs

Skin and mucosal carriage of Malassezia pachydermatis was studied in 20 healthy pet dogs of various breeds and in 20 kennelled beagles. Using swabs, anal carriage was detected in 10 pet dogs and 11 beagles and the nose, mouth, prepuce and vulva were shown to be infrequently colonised. M pachydermatis was isolated from the external ear canal of 11 beagles and two pet dogs; both the population sizes and frequency of isolation were significantly (P<0·05) greater in the beagles. The yeast was infrequently isolated from the axilla and groin in low numbers using contact plates and detergent scrub samples but was often cultured from the lower lip and the dorsal interdigital spaces; isolation frequencies and population sizes in the two groups of dogs were not significantly different. These results demonstrate that the anus, external ear canal and lip and interdigital skin of healthy dogs are frequently colonised by M pachydermatis.     

Comparative study of the microbial profile from bilateral canine otitis externa

Fifty dogs with bilateral otitis externa were studied over a 10-month period. The exudates of both external ears were obtained, using sterile swabs, and microorganisms were isolated according to standard microbiological techniques. Antimicrobial susceptibility testing of Staphylococcus intermedius was done by the agar diffusion method. There was bacterial and/or fungal growth in all of the samples. These were all polymicrobial infections. Anaerobic bacteria were not isolated in any sample. The most common pathogens isolated were S. intermedius and Malassezia pachydermatis. A statistically significant difference (P < 0.05) was observed in the isolation pattern between the right and left ears in 34 of the 50 animals (68%). High resistance rates of S. intermedius strains to penicillin, ampicillin, erythromycin, tetracycline, and clindamycin were found. The results suggest that in bilateral canine otitis externa, each ear should be cultured separately and considered as separate units.     

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.     

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.     

Malassezia spp. overgrowth in allergic cats

A series of 18 allergic cats with multifocal Malassezia spp. overgrowth is reported: atopic dermatitis was diagnosed in 16, an adverse food reaction in another and one was euthanized 2 months after diagnosis of Malassezia overgrowth. All the cats were otherwise healthy and those tested (16 out of 18) for feline leukaemia or feline immunodeficiency virus infections were all negative. At dermatological examination, multifocal alopecia, erythema, crusting and greasy adherent brownish scales were variably distributed on all cats. Cytological examination revealed Malassezia spp. overgrowth with/without bacterial infection in facial skin (n = 11), ventral neck (n = 6), abdomen (n = 6), ear canal (n = 4), chin (n = 2), ear pinnae (n = 2), interdigital (n = 1) and claw folds skin (n = 1). Moreover, in two cats Malassezia pachydermatis was isolated in fungal cultures from lesional skin. Azoles therapy alone was prescribed in seven, azoles and antibacterial therapy in eight and azoles with both antibacterial and anti-inflammatory therapy in three of the cats. After 3-4 weeks of treatment, substantial reduction of pruritus and skin lesions was observed in all 11 cats treated with a combined therapy and in five of seven treated solely with azoles. Malassezia spp. overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those presented for dermatological examination displaying greasy adherent brownish scales. The favourable response to treatment with antifungal treatments alone suggests that, as in dogs, Malassezia spp. may be partly responsible for both pruritus and cutaneous lesions in allergic cats.     

Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa

The bacterial and fungal flora of the external ear canal of dogs with otitis externa and of healthy dogs were studied. The most frequently isolated microorganism from otitic ears was Staphylococcus intermedius (58.8%), followed by Malassezia pachydermatis (30.9%), Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%). A statistical analysis of our results showed that the prevalence of these microorganisms is significant in dogs with otitis externa. Furthermore, the antimicrobial susceptibility patterns of isolated strains were determined. Majority of all bacterial isolates were most susceptible to gentamicin. Malassezia pachydermatis, the most prevalent yeast in this study, showed an excellent level of susceptibility to all antifungal agents tested.     

P-76 The effect of omega-3 fatty acid supplementation on cutaneous and plasma fatty acid concentrations in dogs with atopic dermatitis

The objectives of this study were to evaluate in vivo tolerance, and antimicrobial and clinical activities of a topical otic preparation containing EDTA tromethamine (Tris) and chlorhexidine digluconate 0.15% solution (Otodine^®) in dogs with chronic bacterial otitis externa. Eleven dogs were included. The affected ears were filled with the solution once daily during a 2-week period. Dogs were evaluated on days 0, 14 and 28. Three clinical parameters (exudate, erythema, pain) and three cytologic parameters ( Malassezia , cocci, rods) were scored (0–4 scale) by otoscopic and cytological examinations of otic exudate. Bacterial cultures were performed at each time point. If there were bacteria on cytological examination on day 14, the dogs were treated with the original product, with the addition of enrofloxacin (5%) applied 10 min after the original product, for a further 2 weeks. All 11 cases yielded isolates of resistant gram-negative bacteria; gram-positive bacteria were also isolated from six of 11 dogs. On day 14, six of 11 dogs were negative on culture examination; on day 28, 10 of 11 were negative and only one case had a positive culture. On day 14, clinical and microbial scores (cytology) were reduced by 54.6 and 71.1%, respectively, and by 85.7 and 94% on day 28. All cases reported good tolerance of the treatment. The results show that this ear solution was helpful in the management of chronic bacterial otitis externa in dogs and was well tolerated. There seems to be a synergistic effect of the combination of Tris-EDTA/chlorhexidine digluconate 0.15% solution, and an antimicrobial agent (enrofloxacin) against resistant gram-positive and gram-negative bacteria.
Funding: Self-funded.     

A semiquantitative cytological evaluation of normal and pathological samples from the external ear canal of dogs and cats

Abstract  Numbers of desquamated epithelial cells, yeast cells and bacterial organisms were counted in samples collected from the external ear canal of 37 normal dogs and 16 normal cats, and from 24 dogs and 22 cats with otitis externa. The aims of the study were to establish quantitative reference ranges and to correlate these data with the clinical status of the dogs and cats. Numbers of yeast cells and bacterial organisms were significantly increased in dogs ( P  = 0.05; P  = 0.0001) and cats ( P  = 0.0001; P  = 0.0001) with otitis externa, and in most cases high counts were correlated with clinical signs. Mean Malassezia counts per high-power dry field of ≥ 5 in the dog and ≥ 12 in the cat were considered abnormal. Mean bacterial counts per high-power dry field of ≥ 25 in the dog and ≥ 15 in the cat were considered abnormal. When used to differentiate normal from inflamed external ear canals, these figures provided a low sensitivity but a specificity of ≥ 95%.     

Clinical evaluation of an antiinflammatory and antioxidant diet effect in 30 dogs affected by chronic otitis externa: preliminary results

The aim of this evaluation study was to assess the possible role of a specific nutraceutical diet in relieving main clinical symptoms of chronic bilateral otitis externa (occlusion of ear canal, erythema, discharge quantity, and odor) in 30 adult dogs. Thirty dogs of different breeds (mean age?±?SEM; 6.03?±?0.15 years and mean weight?±?SEM; 32.01?±?1.17 Kg; 53.3 % males, 46.6 % females) with evident chronic clinical otitis symptoms were equally divided and randomly assigned to receive either the nutraceutical diet (ND group) or a standard diet (SD group) over a period of 90 days. In all cases a topical pharmacological treatment was given. The nutraceutical diet, also endowed with anti-inflammatory and antioxidant activities, significantly decreased the mean score intensity of all symptoms after 90 days of intervention (P?<?0.0001) with the exception of Malassezia pachydermatis infection which was only slightly reduced. Our investigation is one of the few evidence-based results where a commercial nutraceutical diet has been proven effective, in combination with drugs, in relieving otitis externa-related symptoms. This study opens new insights into otitis externa clinical management providing evidence of efficacy of a combined therapy with drugs and a specific nutraceutical diet.     

Malassezia otitis externa in the dog: the effect of heat-fixing otic exudate for cytological analysis

This study was conducted on 32 dogs with Malassezia otitis externa to determine the effect of heat-fixing otic exudate on cytological analysis. Malassezia infection was confirmed by cytological examination of otic exudate. Otic discharge collected with cotton swabs was then rolled onto glass slides. One slide per dog was heat-fixed prior to staining; the other slide was not heat-fixed. The number of yeast in 10 oil-immersion fields (1000 x magnification) was counted for both slides from each dog. Heat-fixing did not systematically cause either increased or decreased numbers of Malassezia on cytology of otic exudate.