Genetic typing of Malassezia pachydermatis from different domestic animals

In the present study, random amplification of polymorphic DNA, which detects DNA polymorphism in fungal genomic DNA, was applied for genetic typing of Malassezia pachydermatis isolates. Fifty-five isolates from different domestic animals and body sites and the neotype strain CBS 1879 were characterized. Primers M13 and OPT-20 were used to analyse their genetic relatedness and similarity. This technique allowed us to distinguish four different genetic types. The predominant genetic type was observed in isolates recovered from different anatomical locations in all animals. It was the only genetic type found in cats, horse, goat and pig. The other three genetic types were observed only in isolates from external ear canals of dogs. Types II and IV were only recovered from external otitic ears and type III from healthy ears. An animal was colonised by more than one type of M. pachydermatis and different genetic types were detected in the same body site. Some genetic types were only isolated from diseased skin.     

Immunoglobulin G responses to Malassezia pachydermatis in healthy dogs and dogs with Malassezia dermatitis

Serum immunoglobulin G (IgG) responses of healthy dogs and dogs with Malasseziapachydermatis dermatitis were compared by Western immunoblotting. M pachydermatis CBS 1879 was disrupted mechanically and its proteins were separated and blotted on to nitrocellulose membranes before being incubated with sera from eight healthy beagles, eight Irish setters with gluten-sensitive enteropathy, 15 healthy basset hounds, and 30 dogs with Mpachydermatis-associated dermatitis, 20 of which were basset hounds. The mean (se) numbers of bands of immunoreactivity observed in the seborrhoeic basset hounds (10.7 [0.4]) and affected mixed-breed dogs (9.4 [0.9]) were significantly greater than in the beagles (3-0 [1.0]), Irish setters (5.5 [1.1]) and healthy basset hounds (5.6 [0.7]). The number of bands identified was correlated (r(s) = 0.76, P < 0.001) with the anti-M pachydermatis IgG values measured by ELISA in a previous study. Most of the dogs were immunoreactive towards the 132, 66 and 50 to 54 kDa proteins and the affected dogs were also usually reactive towards the 219, 110, 71 and 42 kDa proteins.     

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.     

Seborrhoeic dermatitis in a goat due to Malassezia pachydermatis

A 6-year-old female goat was presented with a seborrhoeic dermatosis of 5 months duration. The condition developed following a severe enteritis associated with weight loss. Dermatological examination showed a generalized greasy seborrhoeic dermatosis, which spared the head and extremities of the limbs. Microscopic examination of impression smears of lesional skin revealed numerous yeasts typical of Malassezia sp. Culture on Sabouraud's dextrose agar yielded Malassezia pachydermatis growth. Histopathological examination of haematoxylin/eosin and safranin (HES) stained sections of biopsies showed mild lymphocytic superficial perivascular hyperplastic dermatitis. Numerous budding yeasts were visible both on the surface, and follicular keratin, in HES and periodic acid Schiff (PAS) stained sections. A dramatic response was observed after 1 week of a topical anti-Malassezia treatment, and the resolution of the condition was complete after 4 weeks.     

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.     

Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.     

Intradermal test reactivity to Malassezia pachydermatis in healthy basset hounds and basset hounds with Malassezia dermatitis

Nineteen healthy beagles, eight healthy basset hounds and 17 basset hounds with Malassezia dermatitis were tested intradermally with two extracts of M pachydermatis. One healthy beagle and two affected basset hounds showed wheal and flare reactions 15 minutes after the injection. Delayed reactions, consisting of erythematous macules and plaques, were commonly observed 24 hours after the injection in both the healthy and affected basset hounds, but occurred infrequently in the beagles. At 24 hours the diameters of the lesions in the healthy and affected basset hounds were significantly (P<0.01) greater than those in the healthy beagles, but the diameters in the healthy and affected basset hounds did not vary significantly. Delayed reactions in six of the basset hounds with Malassezia dermatitis were characterised histologically by superficial perivascular and periadnexal infiltrates of neutrophils and lymphocytes.     

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.     

Epidermal Dysplasia And Malassezia pachydermatis Infection In West Highland White Terriers

Abstract— A chronic, severely pruritic, seborrhoeic skin disorder of West Highland White terriers is reported. The dermatosis has no apparent sex predilection, often begins in animals less than one year of age and is probably genetically programmed. This syndrome is characteristically refractory to standard therapeutic regimens. Histologically the dermatosis is characterized by variable degrees of hyperplastic superficial perivascular dermatitis with a keratinization defect, epidermal dysplasia and the presence of budding yeast in surface and follicular keratin. Malassezia pachydermatis is isolated in pure culture from the skin lesions. Because of the unique histological findings in this syndrome, we propose that the disorder be called “epidermal dysplasia and Malassezia pachydermatis infection in the West Highland White terrier”. Résumé— Une affection cutanée chronique, sévèrement, prurigineuse, séborrhéique, est décrite chez des West Highland White terriers. La dermatose, sans prédisposition apparente de sexe, débute souvent sur des animaux agés de moins d'un an et reconnait probablement une origine héréditaire. La caractéristique de ce syndrome est d'étre résistant aux thérapeutiques habituelles. A l'histologie, la dermatose est caractérisée par des degrés variables de dermatite hyperplasique superficielle périvasculaire avec un défaut de kératinisation une dysplasie épidermique et la présence de levures bourgeonnantes dans la kératine superficielle et folliculaire. Malassezia pachydermatis est isolée en culture pure à partir des lésions cutanées. En raison de l'aspect histologique univoque dans ce syndrome, nous proposons que cette affection soit appelée: “Dysplasie épidermique et infection àMalassezia pachydermatis du West Highland White terrier”. Zusammenfassung— Eine chronische, hochgradig pruriginÖse, seborrhoische Hauterkrankung des Westhighland-White-Terriers wird beschrieben. Die Dermatose zeigt keine deutliche Geschlechtsdisposition, beginnt oft im Alter unter einem Jahr und ist wahrscheinlich genetisch bedingt. Dieses Syndrom spricht auf die üblichen therapeatischen Maßnahmen nicht an (Diagnostik durch Biopsie). Histologisch ist diese Dermatose gekennzeichnet durch unterschiedliche Grade einer hyperplastischen, superfiziellen, perivaskulären Dermatitis mit Keratinisierungsdefekt, durch epidermale Dysplasie und die Anwesenheit von Sproßpilzen (Hefen) im oberflächlichen und follikulären Keratin. Aus den Hautveränderungen wird Malassezia pachydermatis in Reinkultur isoliert. Wegen der einzigartigen histologischen Befunde bei diesem Syndrom schlagen wir vor, diese Erkrankung “Epidermale Dysplasie und Malassezia pachydermatis—Infektion beim Westhighland-White-Terrier” zu nennen.     

Epidermal Dysplasia And Malassezia pachydermatis Infection In West Highland White Terriers

Abstract— A chronic, severely pruritic, seborrhoeic skin disorder of West Highland White terriers is reported. The dermatosis has no apparent sex predilection, often begins in animals less than one year of age and is probably genetically programmed. This syndrome is characteristically refractory to standard therapeutic regimens. Histologically the dermatosis is characterized by variable degrees of hyperplastic superficial perivascular dermatitis with a keratinization defect, epidermal dysplasia and the presence of budding yeast in surface and follicular keratin. Malassezia pachydermatis is isolated in pure culture from the skin lesions. Because of the unique histological findings in this syndrome, we propose that the disorder be called “epidermal dysplasia and Malassezia pachydermatis infection in the West Highland White terrier”. Résumé— Une affection cutanée chronique, sévèrement, prurigineuse, séborrhéique, est décrite chez des West Highland White terriers. La dermatose, sans prédisposition apparente de sexe, débute souvent sur des animaux agés de moins d'un an et reconnait probablement une origine héréditaire. La caractéristique de ce syndrome est d'étre résistant aux thérapeutiques habituelles. A l'histologie, la dermatose est caractérisée par des degrés variables de dermatite hyperplasique superficielle périvasculaire avec un défaut de kératinisation une dysplasie épidermique et la présence de levures bourgeonnantes dans la kératine superficielle et folliculaire. Malassezia pachydermatis est isolée en culture pure à partir des lésions cutanées. En raison de l'aspect histologique univoque dans ce syndrome, nous proposons que cette affection soit appelée: “Dysplasie épidermique et infection àMalassezia pachydermatis du West Highland White terrier”. Zusammenfassung— Eine chronische, hochgradig pruriginÖse, seborrhoische Hauterkrankung des Westhighland-White-Terriers wird beschrieben. Die Dermatose zeigt keine deutliche Geschlechtsdisposition, beginnt oft im Alter unter einem Jahr und ist wahrscheinlich genetisch bedingt. Dieses Syndrom spricht auf die üblichen therapeatischen Maßnahmen nicht an (Diagnostik durch Biopsie). Histologisch ist diese Dermatose gekennzeichnet durch unterschiedliche Grade einer hyperplastischen, superfiziellen, perivaskulären Dermatitis mit Keratinisierungsdefekt, durch epidermale Dysplasie und die Anwesenheit von Sproßpilzen (Hefen) im oberflächlichen und follikulären Keratin. Aus den Hautveränderungen wird Malassezia pachydermatis in Reinkultur isoliert. Wegen der einzigartigen histologischen Befunde bei diesem Syndrom schlagen wir vor, diese Erkrankung “Epidermale Dysplasie und Malassezia pachydermatis—Infektion beim Westhighland-White-Terrier” zu nennen.     

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.]     

A unique isolate of Malassezia from a cat

An isolate of Malassezia from a cat with otitis externa was examined mycologically as well as molecularly. The isolate was similar to M. sympodialis in morphological and biochemical characteristics. In molecular analysis, however, it differed from the 7 species of Malassezia previously reported. Therefore, this clinical isolate from a cat might be a new species of Malassezia.     

Evaluation of the effect of pH on in vitro growth of Malassezia pachydermatis

The purpose of this study was to evaluate the effects of pH on the growth of canine Malassezia pachydermatis isolates in vitro. Yeast growth was monitored by measuring the optical density with a spectrophotometer. The growth of American Type Culture Collection and field strains of M. pachydermatis was optimal between the pH values of 4.0 and 8.0, and inhibited at the ranges of 1.0 to 3.0 and 9.0 to 10.0. An analysis of covariance showed no significant differences among the growth curves at pH levels 5.0 to 8.0. Although specific contrast tests showed that the growth slope at pH 4.0 was significantly different from that at pH 5.0 to 8.0, only small, random differences were found when the growth slope at pH 4.0 was compared to the individual slopes at pH 5.0, 6.0, 7.0, and 8.0. The findings of this study suggest that topical acidifying products could be beneficial therapeutic options for cutaneous yeast infections in dogs.