Higher incidence of Malassezia pachydermatis in the eyes of dogs with corneal ulcer than in healthy dogs

Malassezia pachydermatis is usually associated with otitis and dermatitis in dogs but it can also cause diseases in other species, including humans. In a human neonatal intensive care unit, M. pachydermatis was isolated from an infant's ocular discharge. Therefore, the aim of this study was to ascertain the presence of Malassezia spp. and its possible consequences in dogs' eyes. This research included 19 dogs with unilateral or bilateral corneal ulcers and 60 healthy dogs. A total of 158 clinical specimens from both the groups were obtained from the conjunctival sac of each eye by a calibrated platinum loop. The samples were placed on Dixon and blood agar, incubated at 35 degrees C, and examined daily for 15 days. Then, the strains were subcultured on Sabouraud agar. Of 22 clinical specimens collected from the eyes with corneal ulcers, five cultures (23%) were positive for M. pachydermatis. Of 16 samples collected from the contralateral healthy eye, cultures were positive in three samples (19%). Three animals had unilateral corneal ulcer and positive cultures for M. pachydermatis in both the eyes. Two dogs had unilateral corneal ulcer and positive cultures for M. pachydermatis in the same eye. However, from the 120 samples of 60 healthy dogs, only four clinical specimens (3%) had positive cultures for M. pachydermatis. The findings of M. pachydermatis, in a considerable percentage of clinical specimens from dogs with corneal ulcer, suggest its possible role at least as an aggravating factor in the pathophysiology of this disease.     

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 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.     

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.     

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.     

Isolation of Malassezia species from healthy cats and cats with otitis

Lipid-dependent Malassezia species have recently been cultured from veterinary specimens. The identification of Malassezia species isolates from animals is important to clarify the epidemiology of these lipophilic yeasts. Malassezia species were cultured from the external ear canals of 63 out of 99 cats with otitis and 12 of 52 (23%) healthy control cats. The rate of isolation in affected animals versus controls was highly significant (P<0.01). Malassezia pachydermatis was isolated as a pure culture in 33 (45.2%) cats, associated with Malassezia globosa and Malassezia furfur in 20 (50%) and 17 (42.5%) animals, respectively. Three different species were isolated simultaneously in three cats (two cats with M pachydermatis, M globosa and M furfur, one subject with M pachydermatis, M furfur and Malassezia sympodialis). M globosa was isolated as the sole species in two animals. The present work confirms the presence of some lipid-dependent species of Malassezia in both healthy and otitic cats.     

Bacteriology and mycology of otitis externa in dogs]

The bacterial and fungal flora of 1118 ears of dogs with otitis externa and 100 ears of healthy control dogs were studied in order to isolate the causative agents. The yeast Malassezia pachydermatis (56%) was by far the most common organism in otitic dogs followed by the bacteria Staphylococcus intermedius (23%), Pseudomonas aeruginosa (12%), Proteus spp. (6%) and Streptococcus canis (5%). A statistical analysis of observed results showed that the incidence of these organisms is significant in otitic dogs. Many strains of S.intermedius, P.aeruginosa and Proteus spp. are resistant to antimicrobial agents commonly used to treat otitis externa. Therefore an antimicrobial susceptibility testing was performed using "Cobas Bact" for these bacterias. Furthermore, 80 strains of M.pachydermatis were submitted to identification-kits (API 20 CAUX, API STAPH, Cobas Micro). The observed results showed that an identification with these tests was not possible.     

Isolation of Malassezia pachydermatis and M. sympodialis from the external ear canal of cats with and without otitis externa

The objective of this study was to determine the presence of Malassezia spp. in the external ear canal of cats with and without otitis. Forty-five animals were studied, 20 with and 25 without otitis externa (OE). Cerumen or secretion from external ear canal samples was cultured on modified Mycosel agar and sterile olive oil was added to the surface of the medium before specimen seeding. The isolates were analysed for macro- and micromorphology and identified by catalase tests and on the basis of growth on Tween 20, 40, 60 and 80. Malassezia spp. were isolated from 15 out of 20 (75%) animals with otitis and from 7 out of 25 (28%) cats without OE; the difference between the two groups was statistically significant (P < or = 0.05). Malassezia pachydermatis and M. sympodialis were isolated from 60% (12/20) and 40% (8/20) of cats with otitis, respectively, with no significant difference in the frequency of isolation between the two species. In the microflora of the healthy ear canal M. pachydermatis was significantly more common (6/25, 24%) than M sympodialis (1/25, 4%). The present investigation confirms that M. sympodialis can also act as an aetiological agent of feline OE, and if commercial veterinary laboratories do not use media with added lipids for the isolation of Malassezia spp., this might lead to false-negative results.     

Otitis externa induced with Malassezia pachydermatis in dogs and the efficacy of pimaricin.

Eight beagles were experimentally inoculated intraotally with Malassezia pachydermatis to induce acute otitis externa. Three or 4 days after the inoculation, the animals showed the symptoms of otitis externa. All ear canals were erythematous and the dogs were shaking their heads. A large number of M. pachydermatis was noticed in exudate taken from every ear canal. Clinical signs of otitis externa were reduced after treatment with 0.1 ml (per canal) of 1% pimaricin suspension twice a day for 3 days. The amount of exudate decreased gradually and 12 of the 16 ear swabs examined, thereafter, were found to be negative for M. pachydermatis within 10 days. No side effects were observed in all the treated cases. These results suggested that M. pachydermatis could induce the canine otitis externa, and that pimaricin is effective agent for M. pachydermatis infection in ear canals.     

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.     

In vitro antifungal susceptibility of Malassezia pachydermatis from dogs with and without skin lesions

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections.     

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%).     

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.     

Assessment of the ability of Malassezia pachydermatis to stimulate proliferation of canine keratinocytes in vitro

OBJECTIVE: To investigate the direct interaction between canine keratinocytes and live Malassezia pachydermatis and thereby determine the role of these organisms in the pathogenesis of epidermal hyperplasia associated with Malassezia dermatitis in dogs. SAMPLE POPULATION: Primary canine keratinocyte cultures established from skin samples obtained from clinically normal dogs. PROCEDURE: The proliferative response of keratinocytes co-cultured with Malassezia organisms for 1, 2, or 3 days was assessed by use of direct manual counting (to determine the number of keratinocytes in both the monolayer and the medium) and immunohistochemical staining techniques involving antibodies against proliferating cell nuclear antigen (PCNA) and another cellular proliferation marker, Ki-67. The potential cytotoxic effect of Malassezia organisms was investigated by use of an apoptosis detection kit to label keratinocytes co-cultured with M. pachydermatis that underwent apoptosis. RESULTS: No stimulatory effect of Malassezia organisms on canine keratinocyte proliferation was detected via cell counting and immunohistochemical techniques. However, there was a significant increase in dead keratinocytes in the medium with increasing numbers of Malassezia organisms in the co-culture. More apoptotic cells were observed in keratinocyte monolayers co-cultured with high numbers of M. pachydermatis than there were in monolayers cultured without Malassezia organisms, and the number increased after prolonged incubation. CONCLUSIONS AND CLINICAL RELEVANCE: M. pachydermatis did not stimulate canine keratinocyte proliferation in vitro. The results suggested that the epidermal hyperplasia observed in dogs with Malassezia dermatitis is unlikely to be caused by a direct effect of the organism on the keratinocyte cell cycle, but is likely to involve other mechanisms.     

Study of lipid in the ear canal in canine otitis externa with Malassezia pachydermatis

An epidemiological investigation of 120 canine otitis externa cases in 1,370 dogs was done on the incidence rate, ear pinna shapes, breeds and their relationships. Eighty-five cases (12.6%) in 672 dogs with pendulous ears and 35 cases (5.0%) in 698 dogs with erect ears had otitis externa, and the difference between them was significant (P<0.05). Ninety-five auditory cerumen specimens were cultured for Malassezia pachydermatis (M. pachydermatis) and analyzed for concentrations of major fatty acids. Although rates of cases positive for M. pachydermatis in both ear pinna shapes were almost the same, i.e. 55.2% in the pendulous group and 53.6% in the erect group, the average total fatty acid level of the pendulous ear group was significantly (P<0.05) higher than that in the erect ear group after dismissing extraordinary levels in the Siberian husky. Isolated M. pachydermatis strains were examined for the effects of fatty acid supplementation on their growth. The majority of the strains utilized fatty acids and grew faster in fatty acid supplemented broth. These results suggest that M. pachydermatis, the predominant causative agent of canine otitis externa, prefers the auditory canal of dogs with lipid-rich earwax and grows fast, but growth strongly depends upon the canine breed.     

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.     

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.     

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.     

Carriage of Malassezia spp. yeasts in Cornish Rex, Devon Rex and Domestic short-haired cats: a cross-sectional survey

Carriage of Malassezia spp. yeasts in healthy Cornish Rex cats (CRC) was compared with that in Devon Rex (DRC) and Domestic short-haired (DSH) cats. Samples obtained from the left external ear canal, anus and claw fold of digit III of the left fore foot by swabbing, and the axilla and groin using contact plates, were incubated for yeasts on modified Dixon's agar at 32 degrees C for 7 days. Malassezia species were isolated from 90% of the DRC, but from only 39% of the CRC and 50% of the DSH cats. M. pachydermatis accounted for 121 of 141 Malassezia spp. isolates. Five CRC were colonized by M. pachydermatis alone, one CRC yielded only M. nana, and one cat yielded only M. slooffiae, whereas five CRC were colonized by both M. pachydermatis and M. nana and another yielded M. pachydermatis, M. slooffiae and M. nana. M. nana was primarily isolated from the ear canal, whereas M. slooffiae was most often isolated from the claw. Both the frequencies of isolation and the population sizes of M. pachydermatis at all sites sampled in the CRC were comparable to those of 10 healthy DSH cats. Populations of M. pachydermatis in the left axilla and left and right groin in the CRC were significantly lower when compared with counts in a group of 21 healthy DRC, a breed with very similar coat characteristics but prone to seborrheic dermatitis caused by M. pachydermatis.     

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.