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.     

An azole-resistant isolate of Malassezia pachydermatis

Canine Malassezia dermatitis (MD) is frequently treated with systemic ketoconazole (KTZ) and itaconazole (ITZ). However, the antifungal susceptibility of clinical isolates of M. pachydermatis from dogs and cats to the azoles has not been well investigated. In the present study, the in vitro susceptibility of the standard strain (CBS1879: the neotype strain of M. pachydermatis) and 29 clinical isolates of M. pachydermatis to the azoles was measured by a modified CLSI M27-A2 test using modified Dixon medium as well as by the E-test. The minimum inhibitory concentrations (MICs) of the 30 isolates of M. pachydermatis (including the neotype strain) against KTZ and ITZ were <0.03 μg/ml by the two methods. The MICs of 1 clinical isolate (ASC-11) were 1 and 2 μg/ml against KTZ, and 2 and 8 μg/ml against ITZ, by the modified CLSI M27-A2 test and the E-test, respectively. Thus, isolate ASC-11 may be resistant to these azoles, making this the first report of a resistant isolate of M. pachydermatis to KTZ and ITZ.     

In Vitro Activities of Antifungal Agents against Clinical Isolates of Dermatophytes from Animals

Although the susceptibility of dermatophytes to antifungal drugs is well documented in humans, the effectiveness in animals has not been previously investigated. The in vitro susceptibility of 54 clinical isolates from animal dermatophytoses to ketoconazole (KTZ), itaconazole (ITZ) and terbinafine (TFN) was measured using microdilution assay (CLSI M38-A2 test) and by the E-test (KTZ and ITZ). All 3 drugs showed antifungal activity, while KTZ displayed the broadest minimum inhibition concentration (MIC) range (0.125-16 μg/ml) against M. canis and M. gypseum. The MIC of KTZ and ITZ was almost the same for human and animal isolates of T. mentagrophytes and T. rubrum. The MIC of TFN was almost the same for dermatophytes isolated from humans and animals.     

P-19 Sporotrichosis in São Paulo (Brazil): clinical and epidemiological features

The aim of this study was to determine the in vitro antifungal activity of several antifungal drugs (posaconazole, nystatin, miconazole and clotrimazole) against Malassezia pachydermatis with microdilution and agar dilution techniques. Malassezia pachydermatis isolates were obtained from the skin and ears of dogs. Tests on solid media were performed using 25-well Petri dishes (2 mL/well containing Sabouraud's dextrose agar and diluted antifungal drug) inoculated with 5 μL suspensions of M. pachydermatis . Microtitre broth dilution used 96-well microtitre plates containing Sabourauds dextrose broth and appropriate dilutions of antifungal drugs, inoculated with 10 μL standard suspensions of M. pachydermatis . Plates were inoculated in duplicate and incubated at 30°C for 5 days and growth assessed. The four antifungal drugs were tested in 10 dilutions (4.0-0.007 μg/mL for posaconazole, and 32--0.06 μg/mL for clotrimazole, miconazole and nystatin). Results obtained for 83 strains of M. pachydermatis and a control reference strain (CBS 1879) exhibited the same pattern. Results of the MIC between microtitre and agar methodologies showed no significant differences (≤ 2-fold) across all drugs. For both solid and liquid methods, posaconazole was the most effective antifungal drug of the four tested with MIC₉₀ of 1–2 μg/mL for posaconazole, 16–32 μg/mL for clotrimazole, and ≥ 32 μg/mL for miconazole and nystatin.
Funding: Schering-Plough.     

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.     

Isolation of antifungal-resistant Candida from the blowholes of captive dolphins

In this study, we isolated eight strains of Candida albicans from the blowhole air cultures of eight dolphins (one Pacific white-sided dolphin and seven bottlenose dolphins) housed at the Enoshima Aquarium. The minimum inhibitory concentrations of antifungals for these isolates were determined by conducting E-test and broth microdilution assays using the CLSI M27-A3 protocol antifungal susceptibility testing method. Only one of the eight dolphins from which Candida had been isolated had been treated with amphotericin B (AMB), and four had been treated with itraconazole (ITZ). All isolates were identified as Candida albicans, and all were resistant to both ITZ and voriconazole, though the isolates exhibited susceptibility to AMB and micafungin. Based on our findings, we suspect that the frequency of occurrence of azole-resistant Candida species is increasing in captive dolphins as well as in their aquarium environments.     

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.     

Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro

Abstract Suspensions of Malassezia pachydermatis adhered to canine corneocytes attached io adhesive tape in a dose (P < 0.001) and time-dependent (P < 0.01) manner; adherence was maximal after 2 h. M. pachydermatis cells were approximately 10 times more adherent than Saccharomyces cerevisiae (P < 0.001) cells after 2 h incubation. The adherence of formalin-treated and frozen-thawed M. pachydermatis cells was comparable with untreated controls. Stationary-phase cells adhered better (P < 0.05) than exponential-phase cells. Pretreatment of the yeasts, or corneocytes, with 0.1% trypsin for 30 min reduced (P < 0.01) the adherence of four, and two, out of five strains, respectively, whereas incubation with 300 mM solutions of D(+) mannose, sucrose and N-acetyl D-glucosamine had no consistent effect. These results suggest that trypsin-sensitive proteins or glycoproteins on the yeast cell wall, and on the corneocyte surface, play an important role in the adherence of M. pachydermatis to canine corneocytes in vitro, whereas a role for carbohydrate receptors was not demonstrated. Résumé— Des suspensions de Malassezia pachydermatis adhérant à des cornéocytes des chiens sont attachées à des rubans adhésifs de façon significative en function du nombre (P < 0,001) et de la durée (P < 0,01). L'adhérence est maximale après 2 heures. Les levures du genre Malassezia pachydermatis sont approximativement dix fois plus adhérentes que les levures du genre Saccharomyces cerevisiae (P < 0,001) après une incubation de 2 heures. L'adhérence des Malassezia pachydermatis traitées par le formol et congelées - décongelées, est comparable à celle des témoins. Les levures qui ne sont pas en phase de croissance adhérent mieux (P < 0,05) que celles qui le sont. Le traitement préalable des levures ou des cornéocytes avec une solution de trypsine pendant 30 minutes réduit (P < 0,01) l'adhérence tandis que l'incubation avec des solutions à 300 mM de D + mannose, sucrose et de N acétyl D glucosamine n'a pas d'effets. Ces résultats suggèrent que des protéines sensibles à la trypsine ou des glycoprotéines sur la paroi des levures et à la surface des cornéocytes jouent un rôle important in vitro dans l'adhérence des Malassezia pachydermatis aux cornéocytes du chien, alors que le rôle des récepteurs glucidiques n'a pas été démontré. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine cornéocytes in vitro (Facteurs influençant l'adhérence de Malassezia pachydermatis aux cornéocytes du chien in vitro). Veterinary Dermatology 1996; 7 : 49–56.] Resumen Las suspensiones de Malassezia pachydermatis se adherian a cinta adhesiva de forma dependiente de la dosis (P < 0.001) y del tiempo (P < 0.01); su adherencia fue máxima a las 2 h. Las células de M. pachydermatis fueron aproximadamente 10 veces más adherentes que las de Saccharomyces cerevisiae (P < 0.001) a las 2 h de incubación. La adherencia de células de M. pachydermatis tratadas con formalina y congeladas-descongeladas fue comparable con los controles no tratados. Las células en estadio estacionario se adherian mejor (P < 0.05) que las de fase exponencial. El tratamiento previo de las levaduras o los corneocitos con 0.1% de tripsina durante 30 min redujo (P < 0.01) la adherencia de cuatro, y dos, de cinco cepas, respectivamente, mientras que su incubación con soluciones 300 mM de D(+) manosa, sucrosa y N-acetil D-glucosamina no tuvieron un efecto constante. Estos resultados sugieren que proteinas o glieoproteinas sensibles a la tripsina en la pared de la levadura, y en la superficie del corneocito, juegan un papel importante en la adherencia de M. pachydermatis a los corneocitos caninos in vitro, mientras que no se pudo demostrar un papel por parte de los receptores de carbohidratos. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro (Facto res que afectan la adherencia de Malassezia pachydermatis a los corneocitos caninos in vitro). Veterinary Dermatology 1996; 7 : 49–56.] Zusammenfassung— Suspensionen von Malassezia pachydermatis zeigten eine Adhärenz an kanine Korneozyten, die an einem Klebeband befestigt waren, in dosisabhängiger (P < 0,001) und zeitabhängiger Weise (P < 0,01). Die Adhärenz erreichte ein maximum nach 2 Stunden. M. pachydermatis-Zellen waren ungefähr 10 mal stärker adhärent als Saccharomyces cervisiae-Zellen nach Zstündiger Inkubation (P < 0,001). Die Adhärenz von formalinbehandelten und gefrorenen/aufgetauten M. pachydermatis-Zellen war vergleichbar mit unbehandelten Kontrollzellen. Zellen der stationären Phase waren besser adhärent (P < 0,05) als Zellen der exponentiellen Phase. Eine Vorbehandlung der Hefen oder Korneozyten mit 0,1% igem Trypsin über 30 Minuten reduzierte die Adhärenz (P < 0,01) von 4 bzw. 2 aus 5 Linien, während eine Inkubation mit 300 mm Lösungen von D(+)Mannose, Sucrose und N-Acetyl-D-Glukosaminen keinen entsprechenden Effekt hatte. Diese Ergebnisse legen nahe, daß Trypsin-sensible Proteine oder Glykoproteine an der Zellwand der Hefe und auf der Korneozytenoberfläche eine wichtige Rolle für die Adhärenz von M. pachydermatis an kanine Korneozyten in-vitro spielen, während eine Bedeutung für Kohlenhydrat-Rezeptoren nicht demonstriert werden konnte. [Bond, R., Lloyd, D. H. Factors affecting the adherence of Malassezia pachydermatis to canine corneocytes in vitro (Einflußfaktoren auf die Adhärenz von Malassezia pachydermatis an kanine Korneozyten in vitro). Veterinary Dermatology 1996; 7 : 49–56.]     

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.     

Effects of beta-thujaplicin on anti-Malassezia pachydermatis remedy for canine otitis externa

The antifungal activity of beta-thujaplicin was evaluated against 51 Malassezia pachydermatis strains isolated from canine ear canals with or without otitis externa. For comparison, sensitivity tests were performed on M. pachydermatis isolates for nystatin, ketoconazole, and terbinafine HCl, all clinically available antifungal agents. The minimal inhibition concentrations over 50% of the tested isolates (MIC50) were 3.13 microg/ml for beta-thujaplicin and nystatin, 0.016 microg/ml for ketoconazole, and 1.56 microg/ml for terbinafine HCl. The antifungal effect for M. pachydermatis of beta-thujaplicin compared favorably with commercial antifungal agents. None of the 51 M. pachydermatis isolates showed resistance against any of the tested antibiotics investigated in this study. Ten representative isolates of M. pachydermatis were subcultured for 30 generations at concentrations close to the MIC levels of beta-thujaplicin, nystatin, ketoconazole, and terbinafine HCl, and examined to determine whether they had acquired resistance to each drug. As a result, M. pachydermatis was found to achieve resistance more easily for ketoconazole and terbinafine HCl than for beta-thujaplicin or nystatin. The MIC50 of beta-thujaplicin did not change during the course of subculture, and it is thought that the potential development of a resistant strain is low, even with continuous infusion for otitis externa therapy. beta-Thujaplicin is an inexpensive and safe treatment with anti-inflammatory and deodorant effects that can be recommended as an effective remedy for canine otitis externa.     

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.     

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.     

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.     

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.     

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.     

P‐17 In vitro activity of posaconazole and other antifungals against Malasseziapachydermatis isolated from dogs

The aim of this study was to determine the in vitro antifungal activity of several antifungal drugs (posaconazole, nystatin, miconazole and clotrimazole) against Malassezia pachydermatis with microdilution and agar dilution techniques. Malassezia pachydermatis isolates were obtained from the skin and ears of dogs. Tests on solid media were performed using 25‐well Petri dishes (2 mL/well containing Sabouraud's dextrose agar and diluted antifungal drug) inoculated with 5 μL suspensions of M. pachydermatis. Microtitre broth dilution used 96‐well microtitre plates containing Sabourauds dextrose broth and appropriate dilutions of antifungal drugs, inoculated with 10 μL standard suspensions of M. pachydermatis. Plates were inoculated in duplicate and incubated at 30°C for 5 days and growth assessed. The four antifungal drugs were tested in 10 dilutions (4.0‐0.007 μg/mL for posaconazole, and 32‐‐0.06 μg/mL for clotrimazole, miconazole and nystatin). Results obtained for 83 strains of M. pachydermatis and a control reference strain (CBS 1879) exhibited the same pattern. Results of the MIC between microtitre and agar methodologies showed no significant differences (≤ 2‐fold) across all drugs. For both solid and liquid methods, posaconazole was the most effective antifungal drug of the four tested with MIC₉₀ of 1–2 μg/mL for posaconazole, 16–32 μg/mL for clotrimazole, and ≥ 32 μg/mL for miconazole and nystatin. Funding: Schering‐Plough.     

Homogeneous cell suspension of Malassezia pachydermatis obtained with an ultrasonic homogenizer

It is difficult to produce homogeneous cell suspensions of Malassezia pachydermatis, since yeast cells paste up and form many clumps. However, homogeneous fungal suspensions are required for susceptibility examinations and biochemical analyses. Although several types of trials have been carried out using glass homogenizers and many types of agents to obtain homogeneous fungal suspension. They have not yielded good results. We therefore attempted to use an ultrasonic homogenizer to separate clumps of yeast cells into separate individual cells. We succeeded in this fashion in producing homogeneous cell suspensions of M. pachydermatis. These results indicate that an ultrasonic homogenizer can be used to prepare homogeneous fungal suspensions of M. pachydermatis.     

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.     

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 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.