Identificação molecular e caracterização genotípica de isolados de Malassezia sp. de cães com otite externa

Abstract

Malassezia is a genus of fat-dependent yeasts that live on the surface of the skin and mucous membranes of several mammals and birds. The Malassezia pachydermatis species is the most attributed in the series of fungal otitis externa in dogs, being considered a multifactorial skin disorder, characterized mainly by inflammation in the ear canal of dogs. In this sense, this work aimed to characterize the Malassezia sp. obtained from dogs with otitis externa, in relation to their biochemical characteristics, molecular identification and phylogenetic analysis, treated at the University Veterinary Hospital “Fancisco Edilberto Uchôa Lopes” (HVU – UEMA) in the city of São Luís/MA. For direct identification, fungal isolation and biochemical tests, 110 samples were collected from the auditory canal of 55 dogs, through sterile swabs in the proximal third of the external portion of the acoustic meatus, with two collections being carried out in each ear, one to prepare the smear for examination. direct and another used for fungal cultivation. For molecular analysis, 32 samples were used, using the phenol/chloroform/isoamyl DNA extraction protocol and conventional PCR for the amplification of the D1/D2 region of the larger subunit of the 26S rRNA gene, followed by sequencing. The editing and phylogenetic analysis of the sequences were performed in specific programs. In the direct examination of the 110 samples, it was observed that in 100 (90.9%) there was the presence of some microbial agent (yeasts and bacteria), in 71 only structures similar to Malassezia sp. mixed infection, with bacteria and yeast. Malassezia sp. was isolated in 83 (75.45%) of the 110 swab samples collected, all of which were positive in the biochemical catalase test, while for the urease test, positivity was found in 80 (96.4%) of the isolated samples, It can be inferred that the species under study was M. pachydermatis. As for the molecular analyses, of the 32 isolated samples suggestive of Malassezia sp. submitted to conventional PCR, 24 showed a band pattern of 600bp. BLAST analysis of 15 sequences from the D1/D2 region of the major subunit of the 26S rRNA gene from the isolates revealed that 14 were identical to several ribosomal DNA sequences from Malassezia pachydermatis and one from Malassezia furfur, showing 100% coverage and 100% identity. In the phylogenetic analysis, the sequences formed two well-supported clades, the first grouped Malassezia pachydermatis sequences with boostrap indices of 100 and 99% in the two analyzes performed, clustering of neighbors/NJ and maximum likelihood/ML, the second clade gathered the only Malassezia furfur sequence obtained in this study forming a well supported group with sequences from Genbank (NJ/99% and ML/94%,). The Malassezia pachydermatis clade also formed two subgroups with strong boostrap values in the analyses, indicating an intraspecific variation and the occurrence of two lineages, one of European origin and another of South American origin. The importance of studies on Malassezia sp. in dogs with otitis externa is observed through the frequency of detection of this pathogen in most cases evaluated. The importance of studies on Malassezia sp. in dogs with otitis externa is observed through the frequency of detection of this pathogen in most cases evaluated. The identification of M. furfur through sequencing demonstrates the importance of molecular analysis in order to correctly identify the etiologic agent and detect the interspecific genetic variability of the Malassezia genus