Occurrence of Malassezia spp. in the external ear canals of dogs and cats with and without otitis externa

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1 Ó Medical Mycology 2002, 40, Accepted 21 February 2001 Occurrence of Malassezia spp. in the external ear canals of dogs and cats with and without otitis externa M. J. CRESPO, M. L. ABARCA & F. J. CABAN ~ ES Departament de Sanitat i d Anatomia Animals (Microbiologia), Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain We studied the lipophilic microbiota of the external ear canals of 332 animals (264 dogs and 68 cats), with and without otitis externa, over an 11-year period from 1988 to Malassezia pachydermatis was isolated from 62 2% and 50% of dogs with and without otitis externa, respectively, and from 41 2% and 17 6% of cats with and without otitis externa, respectively. In the group of animals studied for lipiddependent species, these yeasts were isolated from 4 5% of dogs with otitis externa and from 23 1% and 8 9% of cats with and without otitis externa, respectively. M. sympodialis and M. furfur were isolated from cats and M. furfur and M. obtusa from dogs. Our ndings show that lipid-dependent Malassezia species may contribute to the etiology of otitis externa in dogs and cats. Keywords cat, dog, external ear canal, Malassezia furfur, Malassezia obtusa, Malassezia pachydermatis, Malassezia sympodialis Introduction Lipophilic yeasts of the genus Malassezia are known to constitute a major proportion of the saprobic fungal microbiota of human and animal skin [1,2]. They have also been associated with a variety of pathological conditions in humans such as pityriasis versicolor, seborrheic dermatitis, folliculitis and even systemic infection [3]. In animals they may also cause disease. Carnivores in particular are affected by otitis externa and dermatitis [4]. The physiological uniqueness of Malassezia yeasts is their lipophilic nature: they have the requirement for lipids as a source of carbon. Malassezia pachydermatis is unique within the genus in that it usually grows well on routine mycological media without lipid supplementation [5]. Since the genus Malassezia was created by Baillon in 1889, its taxonomy has been a matter of controversy. A few years ago, the genus was revised on the basis of morphological, ultrastructural, physiological and genetic studies and seven species were de ned: M. furfur, Correspondence: F. J. Cabañes, Departament de Sanitat i d Anatomia Animals (Microbiologia), Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. Tel: ; Fax: ; javier.cabanes@uab.es. Downloaded Ó 2002 from ISHAM, Medical Mycology, 40, M. pachydermatis and M. sympodialis, previously described, and four new species, M. obtusa, M. globosa, M. restricta and M. sloof ae [6,7]. M. pachydermatis is usually isolated from skin and mucosae of healthy dogs and cats [8], and it may cause chronic dermatitis and otitis externa [9]. Although this species is mainly adapted to animals, it has been reported to cause nosocomial systemic infection in humans [10,11]. Human health-care workers can transmit this yeast from their pet dogs to their neonatal patients [12]. The six lipid-dependent species can be isolated from normal and diseased human skin [13]. Nevertheless, recently lipid-dependent species have been isolated from animals [14 21], but very little is known about the frequency of these species in animals and their role on animal skin. The purpose of this survey was to study the occurrence of the lipophilic microbiota in the external ear canals of dogs and cats with and without otitis externa, and to determine whether lipid-dependent Malassezia species are related to the etiology of otitis externa in dogs and cats. Materials and methods Over an 11-year period from 1988 to 1999, the external ear canals of 332 animals from the Barcelona region

2 116 Crespo et al. Table 1 Occurrence of Malassezia spp. in dogs with and without otitis externa Otitis externa Without otitis externa Breed Afghan Hound 2/3 0/2 0 0 Airedale Terrier 1/ Alaskan Malamute 1/1 0/1 0 0 Beagle 0 0 2/2 0/2 Belgian Shepherd Dog 4/4 0/1 0/1 0 Boxer 2/3 0/3 3/5 0/4 Brittany 0/1 1/1 0/1 0/1 Bull Terrier 2/2 1/2 0 0 Cairn Terrier 0 0 1/1 0/1 Catalan Sheepdog 0/1 0 0/1 0/1 Cocker Spaniel 16/30 0/8 3/7 0/1 Collie 1/ Dachshund 1/1 0 2/2 0/1 Doberman 0/ Drahthaar 1/ English Setter 1/1 0/1 0 0 Fox Terrier 1/3 0/1 0 0 German Shepherd Dog 24/38 0/11 0/4 0/3 Great Dane 0 0 1/1 0/1 Irish Setter 0 0 0/1 0 Labrador Retriever 1/1 0/1 0 0 Lhasa Apso 0/1 0/1 0 0 Maltese 1/1 0/1 0 0 Newfoundland 0 0 1/1 0/1 Old English Sheepdog 1/4 0 1/1 0/1 Pekinese 1/1 0 0/2 0/1 Pointer 0 0 3/3 0/2 Poodle 6/11 1/5 0/2 0/1 Pyrenean Mastiff 4/8 0 0/1 0 Pyrenean Mountain Dog 0/1 0/1 0 0 Rottweiler 2/2 0/1 0 0 Samoyed 0/ Schnauzer 2/2 0 1/2 0 Scottish Terrier 1/2 0/1 0 0 Siberian Husky 0 0 0/1 0/1 Spanish Mastiff 0 0 1/1 0/1 Spitz 1/1 0/1 0 0 Springer Spaniel 1/2 0/1 0 0 Weimaraner 0 0 1/1 0/1 West Highland white Terrier 1/ Yorkshire Terrier 3/5 0/4 1/1 0/1 Crossbred 26/37 0/17 3/14 0/10 NS 9/12 0/1 14/20 0 Age < 1 year 9/12 0/4 4/13 0/8 1 5 years 58/89 2/33 11/24 0/13 > 5 years 34/64 1/26 7/15 0/10 NS 16/23 0/3 16/24 0/4 Sex Male 61/91 1/32 14/35 0/21 Female 51/86 2/34 9/19 0/12 NS 5/ /22 0/2 Total 117 (62 2%)/188 3 (4 5%)/66 38 (50%)/76 0 (0%)/35 NS, not speci ed. *Number of dogs tested for M. pachydermatis. ynumber of dogs tested for lipid-dependent species.

3 Malassezia in the external ear canals of dogs and cats 117 were analysed in our Bacteriology and Mycology Diagnostic Service of the Faculty of Veterinary Science of Barcelona. Samples of 205 animals with otitis externa (188 house-pet dogs and 17 house-pet cats) and 127 without otitis externa (56 house-pet dogs, 20 kenneled dogs and 51 house-pet cats) were obtained by using a swab. In the group of animals with otitis externa, practitioners diagnosed clinically chronic otitis externa in all of them except in one cat with acute otitis externa. The breeds, sex and ages of these animals are detailed in Tables 1 and 2. From 1988 to 1996, samples from animals with otitis (122 dogs and four cats) were inoculated onto blood agar, MacConkey agar and Sabouraud glucose agar (SGA). Samples from animals without otitis (41 dogs and six cats) were only inoculated on SGA. In order to study the occurrence of lipid-dependent Malassezia species, from 1996 to 1999 animals with otitis (66 dogs and 13 cats) and animals without otitis (35 dogs and 45 cats) were analysed. Samples were inoculated on the media mentioned above and on SGA supplemented with olive oil (1%; Borges, Reus, Spain) and Leeming s medium [1% peptone (Biolife, Milano, Italy), 0 5% glucose, 0 01% yeast extract (Difco, Detroit, MI, USA), 0 4% desiccated ox-bile (Fluka, Steinheim, Germany), 0 1% glycerol, 0 05% glycerol monostearate (Scharlau, Barcelona, Spain), 0 05% Tween 60 (Merck, Hohenbrunn, Germany), 1% whole-fat cow s milk and 1 2% agar, ph 6 2] [22]. SGA, SGA olive oil and Leeming s medium contained 0 05% of chloramphenicol and 0 05% of cycloheximide. For cytological examination, specimens were heat- xed and stained with Gram and Diff- Quick stains. Plates of SGA, SGA olive oil and Leeming s medium were incubated at 35 o C and examined after 3, 5, 7 and 14 days. Semiquantitative colony counts were performed using the following growth categories: heavy ( > 50 CFU), moderate (between 10 and 50 CFU) and scarce ( < 10 CFU). Plates of blood agar and MacConkey agar were incubated at 37 o C with 5%CO 2 for 3 days. When growth was detected on SGA olive oil or Leeming s, ve colonies were selected from each medium and subcultured on SGA to determine their lipid-dependence. M. pachydermatis was identi ed microscopically by morphology and ability to grow on SGA. The identi cation of lipid-dependent yeasts was based on the ability to use certain polyoxyethylene sorbitanesters [Tweens 20 (ICN, Aurora, IL, USA), 40 (Sigma, St. Louis MO, USA), 60 (Merck, Hohenbrunn, Germany) and 80 (ICN)], following the identi cation of species described by Guého et al. [6] and the Tween diffusion test proposed by Guillot et al. [7]. The Table 2 Occurrence of Malassezia spp. in cats with and without otitis externa Otitis externa Without otitis externa Breed Angora 1/2 1/2 0 0 Domestic shorthaired 3/6 1/4 7/34 3 /33 Persian 1/4 1/4 0/2 0/1 Siamese 1/2 0/1 1/9 0/7 Charteaux 0 0 0/1 0/1 Crossbred 1/3 0/2 1/5 1/3 Age < 1 year 2/2 0/2 4/12 0/ years 5/9 1 /5 3/27 4/24 > 5 years 0/6 2/6 0/9 0/7 NS 0 0 2/3 0/3 Sex Male 5/9 0/7 4/23 2/21 Female 2/8 3/6 5/28 2/24 Total 7 (41 2%)/17 3 (23 1%)/13 9 (17 6%)/51 4 (8 9%)/45 NS, not speci ed. *Number of cats tested for M. pachydermatis. ynumber of cats tested for lipid-dependent species. Downloaded Ó 2002 from ISHAM, Medical Mycology, 40,

4 118 Crespo et al. Cremophor EL (Sigma) assimilation test and the splitting of esculin (Sigma) were used as additional key characters for the differentiation of the species [13,23]. Bacteria were identi ed for diagnostic purposes but are not dealt with in this paper. Statistical analysis Data were analysed by using the chi-square test. A P value of <0 05 was considered signi cant. Statistical analyses were performed using the SPSS version 8 0 software (SPSS Inc., Chicago, IL, USA). Results We examined 188 dogs with otitis externa (48 4% male, 45 7% female, 5 8% not speci ed) and 76 dogs without otitis externa (46% male, 25% female, 29% not speci ed) (Table 1). Thirty-two and 22 breeds were represented in the dogs with and without otitis, respectively. In the group with otitis, German Shepherd dogs were the most common (20 2%) followed by crossbred dogs (19 7%) and Cocker Spaniels (15 9%). In the group without otitis, crossbred dogs (18 4%) were the most common. We examined, 17 cats with (52 9% male, 47 1% female) and 51 cats without otitis (45 1% male, 54 9% female) (Table 2). Domestic short-haired cats were predominant in both groups (35 3% and 66 6%, respectively). Of the dogs with otitis, 172 were examined by cytology and typical Malassezia cells were evident in 110 (63 9%; Table 3). Isolates were obtained from 119 dogs (63 3%), including M. pachydermatis from 117 (62 2%). The growth of this yeast was heavy in 89 (76 1%), moderate in 16 (13 7%) and scarce in three (2 5%) (7 7% not recorded). Of the 66 dogs with otitis tested for lipiddependent species (Table 4), M. pachydermatis was isolated in 46 (69 7%) and lipid-dependent species were obtained from three dogs. In one dog, M. pachydermatis co-occurred with M. furfur. M. furfur was isolated in two dogs and M. obtusa in one. In the 76 dogs without otitis externa, cytology revealed Malassezia in 36 (47 4%). M. pachydermatis was the only yeast isolated from 38 dogs (50%), manifesting heavy growth in 14 (36 8%), moderate in eight (21 1%) and scarce in 13 (34 2%) (7 9% not recorded). Malassezia was observed in cytology for nine of the 17 cats with otitis (53%) and was isolated from nine cats (Table 3). M. pachydermatis was isolated from seven cats (41 2%). Growth was heavy in three cats, moderate in two and scarce in two. M. sympodialis was isolated from three cats with otitis (Table 4). In one, it grew along with M. pachydermatis. In cats without otitis externa, Malassezia cells were observed in 16 cats and Malassezia spp. were isolated from 12 cats. M. pachydermatis was isolated from nine cats (17 6%), with growth heavy in seven cases, moderate in one and scarce in one. In one animal, M. sympodialis was obtained in association with M. pachydermatis. M. sympodialis and M. furfur were the lipid-dependent species isolated from three cats and one cat, respectively. Statistical analysis of all M. pachydermatis samples and of cultures of lipid-dependent species showed no signi cant differences related to the breed, age or sex of Table 3 Cytological and culture results in relation to all diseased and healthy dogs and cats tested Cytological examination Microbiological culture Malassezia spp., Only bacteria, Negative, Malassezia spp Positive/totalz Only bacteria, Negative, Dogs with otitis 110/188* 57/188* 5/188* 117/188 3/66 (2, 1}) 54/188 15/188 Dogs without otitis 36/76 2/76 38/76 38/76 0/35 NT 38/76 Cats with otitis 9/17 4/17 4/17 7/17 3/13 (2, 1}) 4/17 4/17 Cats without otitis 16/51 2/51 33/51 9/51 4/45 (3, 1}) NT 39/51 NT, not tested. *In sixteen dogs, the results of cytological examination were not available. ynumber of animals tested for M. pachydermatis. znumber of animals tested for lipid-dependent species. Lipid-dependent species. }One isolate each of M. pachydermatis and a lipid-dependent species were isolated from a single animal.

5 Malassezia in the external ear canals of dogs and cats 119 Table 4 Identi cation of Malassezia species from the animals tested for lipid-dependent species Number of animals Malassezia spp. Total/positive* M. pachydermatis Lipid-dependent species Dogs with otitis 66/48 46y 3y (1Mo, 2Mfy) Dogs without otitis 35/ Cats with otitis 13/7 5y 3y (3 Ms) Cats without otitis 45/12 9y 4 (3Msy, 1Mf) *Number of animals in which Malassezia spp. were isolated. yone isolate each of M. pachydermatis and a lipid-dependent species were isolated from a single animal. Mf, M. furfur; Mo, M. obtusa; Ms, M. sympodialis. either dogs or cats. In Table 5, the statistical analysis of the differences between dogs and cats within each category in relation to Malassezia spp isolation is summarized. Discussion M. pachydermatis was more frequently isolated from dogs than cats as other authors have shown [8]. This yeast was also more frequently isolated from dogs with otitis (62 2%) than without otitis (50%). However, although this apparent difference was not statistically signi cant, more dogs with (76 1%) than without otitis (47 4%) yielded a heavy outgrowth. The high incidence of M. pachydermatis in canine otitis externa and especially in chronic otitis is well established [24 26]. The pathogenic role of this yeast in otitis externa has been controversial [8]. The yeast appears able to induce in ammatory changes in the canine external ear canal in the presence of moisture [27], and it was shown to cause the disease when a large inoculum was experimentally introduced [28]. It seems to have an opportunistic nature and may become pathogenic with any alteration in the skin surface microclimate or host defense [8,9]. Thus, the frequency of isolation in dogs can increase from 15 49% in healthy ears up to 50 83% in otitic ears [4,9,26,29,30]. Primary diseases that cause in ammation and increased sebum production provide a cutaneous microenvironment (increased moisture and surface lipids, compromised stratum corneum barrier function) that encourages overgrowth of this yeast [9,31,32]. M. pachydermatis is the most common yeast that contributes to otitis externa as a perpetuating factor in dogs [9]. In the group of dogs with otitis, M. pachydermatis was isolated more often from German shepherds (12 8%), cocker spaniels (8 5%) and poodles (3 7%). These breeds are predisposed to recurrent otitis [31], and this yeast can be also isolated from their external ear canals more frequently. M. pachydermatis was isolated relatively frequently in dogs between one- and ve-year old and in male dogs. Although the age and sex are not considered as predisposing factors for otitis externa in dogs, other authors have pointed out that male dogs and those aged one- to ve-year old are more affected than others [33]. The frequencies of isolation of M. pachydermatis were 17 6% in healthy cats and 41 2% in cats with otitis externa. In healthy cats, reported percentages of this species ranges from less than 10% [15,34] to 20 23% [4,20,29]. The only reported gure found for the occurrence of this yeast in cats with otitis externa [4] was 19%, which is considerably lower than the proportion obtained in our study. There is no known predisposition for otitis related to breed, age or sex in cats. We isolated lipid-dependent species from dogs with otitis externa and cats with and without otitis externa. Table 5 Statistical analysis of the differences between dogs and cats within each category in relation to Malassezia spp. isolation Downloaded Ó 2002 from ISHAM, Medical Mycology, 40, M. pachydermatis Lipid-dependent species % P % P Dogs/cats 58 7/23 5 y 3/11 9 * Dogs with otitis/dogs without otitis 62 2/50 NS 4 5/0 NS Cats with otitis/cats without otitis 41 2/17 6 * 23 1/8 9 NS NS, not signi cant. *P< 0 05, yp< 0 01.

6 120 Crespo et al. Lipid-dependent species were not isolated from dogs without otitis. Classically, lipid-dependent species have only been reported in association with human skin, and M. pachydermatis was considered the only species that could be isolated from animals. The isolation of lipiddependent species from the skin of various animals changed this idea [20]. The presence of lipid-dependent species in healthy carnivores was recently demonstrated, initially in felines. M. globosa was isolated in the cheetah [6,20] and both M. sympodialis [14] and M. globosa [15] in healthy cats. More recently, lipid-dependent species were also reported from dogs [21]. According to our data, lipid-dependent species seem to be more frequent in cats than in dogs. We isolated lipid-dependent species M. sympodialis and M. furfur in cats without otitis. The isolation of M. furfur, reported previously, seems to be the rst for this species in cats [16]. M. sympodialis seems to be the most common lipid-dependent species isolated from healthy cats. This species could be more common in this microenvironment and could have a similar role to M. pachydermatis in otitis externa. Although the skin of animals can also be colonized by lipid-dependent species in addition to M. pachydermatis, very little is known about their role in animal skin. There is a suggestion that Malassezia cells morphologically different from M. pachydermatis were involved in a skin disorder in milking goats [35]. Nevertheless, the lipiddependence of the yeast in question could not be investigated because isolation was unsuccessful. More recently, lipid-dependent species have been isolated in bovines with otitis externa [19] and M. sympodialis was isolated from affected skin of a horse [36]. In our study, lipid-dependent species were associated with otitis externa. M. sympodialis was isolated in cats with otitis externa [17] and M. furfur and M. obtusa in dogs with otitis externa [18]. Our ndings show that lipid-dependent Malassezia species can be isolated from, and may contribute to the etiology of, at least a small number of cases of otitis externa in dogs and cats. However, more studies is required to discern their role in animal skin. For this reason, to isolate lipid-dependent species and to determine their prevalence in this microenvironment, culture media with lipid sources such as SGA olive oil, Leeming s medium or modi ed Dixon s agar should be used in addition to media without lipid supplements. Acknowledgements We thank the Autonomous University of Barcelona Veterinary Teaching Hospital and different veterinary clinics from the Barcelona region for the samples kindly provided for this investigation. References 1 Dufait R. Presence de Malassezia pachydermatis (syn. P. canis) sur les poils et les plumes d animaux domestiques. 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