PCR-Restriction Fragment Length Polymorphism and DNA Sequencing for Identification of Malassezia species Isolated from Animals in Egypt

Abstract

Malassezia is one of the most significant yeast genera causing Malasseziosis in different animals. In the present study, the phenotypic methods, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were applied for identification of Malassezia species isolated from 160 ear swabs and skin scrapings of apparently healthy and diseased dogs, cats, horses and buffaloes (40 animals, each). Of the 82 ear swabs as well as 78 skin scrapings, 24 (29.3%) and 25 (32.1%) yielded a positive growth on mycobiotic agar, respectively. The forty-nine Malassezia isolates were subjected for phenotypic identification based on macro- and micro-morphological characters on mycobiotic agar medium, growth on Dixon`s medium at different temperatures, and the physiological characters (tween assimilation, esculin hydrolysis, tryptophan utilization, and production of catalase enzyme). Polymerase chain reaction (PCR) amplification of 26S rDNA gene, followed by restriction analysis using HhaI restriction enzyme and DNA sequencing were employed. Forty-eight and one isolates were phenotypically identified as M. pachydermatis and M. globose, respectively. The PCR-RFLP assay for 21 representative isolates revealed the identification of M. pachydermatis (n=17), M. furfur (n=1), M. globosa (n=2) and M. restricta (n=1). Furthermore, the DNA sequencing showed a maximum identity (100%) of the tested isolates to Malassezia spp. available on the Genbank database. The most frequently identified Malassezia spp. by genotypic method was M. pachydermatis (80.95%). It was isolated from 33.3%, 23.8%, 14.28% and 9.52% of examined dogs, cats, horses and buffaloes, respectively. The second frequent identified species was M. globosa (9.52%). It was isolated only from horses and buffaloes (4.76% each), meanwhile M. furfur was recovered from buffaloes and M. restricta was isolated from dogs (4.76% each). In conclusion, PCR-RFLP assay and DNA sequencing proved to be more accurate and reliable methods for Malassezia spp. identification and are complementary for phenotypic methods.