Malassezia Species Associated with Dermatitis in Dogs and Their Antifungal Susceptibility

Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1994-2007 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 06 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.706.236 Malassezia Species Associated With Dermatitis in Dogs and Their Antifungal Susceptibility Uday Seetha, Sumanth Kumar, Raghavan Madhusoodanan Pillai, Mouttou Vivek Srinivas*, Prabhakar Xavier Antony and Hirak Kumar Mukhopadhyay Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education and Research, Pondicherry-605 009, India *Corresponding author ABSTRACT The present study was taken with the objective of isolation, characterization, molecular detection and antifungal sensitivity of Malassezia species from dermatitis cases from dogs in and around Pondicherry state. A total of 100 skin swabs were collected from the dogs K e yw or ds showing dermatological problems suggestive of Malassezia. Out of 100 swabs, 41 Malassezia isolates were successfully isolated and had good growth on Sabouraud’s Malassezia pachydermatis, Dextrose Agar (SDA) during the primary isolation from the skin swabs. Biochemical tests Sabouraud’s dextrose agar, Modified Dixon’s agar, for catalase, β- glucosidase activities and the capability to grow with three water soluble Polymerase chain reaction, lipid supplements, namely Tween 20, Tween 80 and Cremophor EL concluded that the M. Antifungal Susceptibility testing pachydermatis was the sole species isolated from the cases of canine dermatitis in Pondicherry state. Cytological examination revealed that direct skin swab smear was more Article Info sensitive than adhesive tape technique and impression smear. The frequency of isolation of Accepted: M. pachydermatis was higher in neck region (8) followed by other regions in canine. Out 18 May 2018 of 100 skin swabs screened using an M. pachydermatis species specific primers, 61 were Available Online: identified positive. The study showed a higher sensitivity of PCR (61%) in detecting 10 June 2018 Malassezia dermatitis over culture (41%). Based on invitro antifungal susceptibility studies it can be concluded that Ketoconazole, Itraconazole, Fluconazole and Amphotericin B can be advocated as the drug of choice to treat Malassezia dermatitis in this geographical location. Introduction collectively named Malassezia (Cannon, 1986). Malassezia considered as a normal Malassezia has been classified as dimorphic cutaneous inhabitant of skin in humans, fungi since it has been found to exist in both animals and birds. These lipophilic fungi yeast and mycelial phases. colonize in the stratum corneum layer of skin, which is rich in lipids. These Dimorphic The yeast form was named as Pityrosporum lipophilic fungi sometimes act as opportunistic and the mycelial form as Malassezia. pathogens and cause the dermatological However, in 1986 they were recognized to be infections such as seborrhoeic dermatitis and two forms of the same organism and were thus otitis in domestic animals. 1994 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1994-2007 The Malassezia yeast cells are round, oval or mainly Staphylococcus spp. M. pachydermatis cylindrical and may vary in size from 1 to can also be isolated from the ears of clinically 8µm diameter (Keddie, 1966). They reproduce healthy animals (Mansfield et al., 1990). asexually by budding from a broad base (Chen and Hill, 2005). The cell wall of Malassezia is Histopathologically, the lesions are very thick (0.12µm) and multilayered, characterized by inflammation, perivascular consisting mainly of sugars (70%), lipids (15- infiltration, epidermal and follicular 20%) and proteins (10%) (Ashbee and Evans, hyperkeratosis with many budding yeast cells 2002). All Malassezia species except in the stratum corneum (Mason and Evans, Malassezia pachydermatis requires an 1991). The organism is not restricted to dogs exogenous source of long chain fatty acids alone, as it has been isolated from several like C14 or C16 fatty acids for their growth. other mammalian species and birds (Scott, 1992). Zoonotic transfer has been documented The genus Malassezia belongs to the Phylum from dogs to immunocompromised patients by Basidiomycota, comprises of 14 species, health care workers who own dogs (Chang et which have been identified based on their al., 1998). morphology, biochemical features and molecular analysis. Initially, seven species (M. Dermatitis due to Malassezia species in furfur, M. obtusa, M. globosa, M. slooffiae, M. canines is recognized with increasing sympodialis, M. pachydermatis and M. frequency in veterinary practice in restricta) were described (Gueho et al., 1996). Pondicherry state (Southern India). Since no Later, seven new Malassezia spp. (M. systematic investigations were carried out dermatis, M. equina, M. japonica, M. nana, with regards to the occurrence and treatment M. yamatoensis, M. caprae and M. cunicoli) of Malassezia infections in this region, the were identified using biochemical, present study was aimed with the Isolation, morphological, biological and molecular characterization followed by PCR-based analysis (Sugita et al., 2004; Hirai et al., 2004; detection and Antifungal sensitivity of Cabanes et al., 2007). Malassezia species from dermatitis dogs cases in and around Pondicherry state. M. pachydermatis produces proteolytic enzymes that can damage the epithelium Materials and Methods leading to hyperplasia with enlargement of the ceruminous glands (Nicklas and Mumme, Sample collection and Processing 1979). Malassezia produces keratinase and other enzymes capable of digesting the keratin A total of 100 skin swabs were sampled from protein complex, allowing the organism to the dog cases came to the Teaching Veterinary burrow deeper into the stratum corneum in the Clinical Complex, RIVER, Pondicherry (33 host and elicit an inflammatory reaction nos) and Animal husbandry department, (Guillot, 1995). Pondicherry (67 nos) showing dermatological problems suggestive of Malassezia dermatitis The lesions are usually first seen on the like erythema, hyperpigmentation, greasy abdominal skin, but may spread to the entire exudates, scaling etc (Supplementary Figure abdomen, the axilla and the inguinal region 1). (Larsson et al., 1988). In diseased ears, however, M. pachydermatis can be readily Sterile cotton swabs moistened with sterile found alone or associated with bacteria, distilled water were used to collect the 1995 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1994-2007 specimen. The cotton swab was rolled and Isolation and identification rubbed firmly against the entire skin area for 10 seconds from the dogs showing The skin swabs were plated on Sabouraud’s dermatological problems suggestive of dextrose agar (SDA) and Modified Dixon’s Malassezia dermatitis. The swab was then agar (MDA) and incubated at 37°C for 7 days transferred onto the sterile test tube and for the isolation of Malassezia yeasts. The transported to laboratory within 2 to 4 hours of colonies showing cultural characters of the collection. Malassezia were examined microscopically for the typical morphology and were further Cytological studies subjected for a) Catalase activity, b) β glucosidase test, c) Tween and Cremophor EL Impression smear examination utilization biochemical tests to identify them upto species level (Cafarchia et al., 2011). The impression Smears were made in a clean glass slide directly from the surface of the Catalase activity lesion. Then the heat fixed impression smears were stained with Methylene blue stain to A loopfull of test culture on SDA agar was detect the presence of Malassezia yeasts under mixed with 2-3 drops of 3 per cent H2O2 on a oil immersion objective. More than one yeast clean glass slide and examined for the release cell per field is considered positive for of nascent oxygen in the form of gas bubbles. Malassezia dermatitis (Nardoni et al., 2008). A positive reaction was indicated by the effervescence of oxygen within 1-2 min. Adhesive tape examination (Scotch test) (Gueho et al., 1996). Here the impression is made with the adhesive β glucosidase test surface of the cellophane tape placed onto the affected skin surface firmly for 2 - 3 sec and β glucosidase test was done by inoculating a then sticked onto a clean microscopic slide loop full of fresh culture into esculin iron agar containing 2-3 drops of lactophenol cotton medium and incubated at 37°C for 5 days. A blue (LCB) mount. The tape strip stained with positive reaction was indicated by the LCB mount for 2 minutes was examined blackening of the medium which reveals that under high power objective microscope to Malassezia organism possess a β glucosidase detect the presence of Malassezia yeasts. that is able to hydrolyse the glucosidic bond of esculin, thus liberating glucose and esculin. Skin swab smear The phenol moiety reacts with the iron to give a black colour (Kaneko et al., 2006). The smears are made by rolling / rubbing the wet cotton swab firmly against the entire skin Tween and Cremophor EL utilization lesions area for 10 seconds and then rolled / rubbed over the microscopic glass slide. Then Two loops of a 4 to 5 day old Malassezia the heat fixed impression smears were stained culture were suspended in 3.0ml of sterile with Methylene blue stain to detect the demineralised water. This inoculum was presence of Malassezia yeasts under oil added to molten SDA maintained at 50°C, and immersion objective. More than one yeast cell the mixture was poured immediately in a

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