Medycyna Wet. 64 (6), 822-827, 2008
Nowakiewicz A., Ziółkowska G.
Intraspecies differentiation of Malassezia pachydermatis strains with PCR-REA technique
The aim of the study was to make an attempt at showing the intraspecies heterogenecity of Malassezia pachydermatis strains with regards to their origin (strains isolated from healthy dogs and with otitis externa symptoms). The study included 41 strains of Malassezia pachydermatis species isolated in a pure culture from dogs with clinical otitis externa symptoms (n=20), clinically healthy dogs (n=20) and a reference strain, M. pachydermatis (CBS7925). In order to isolate the genetic material from the fungal cells, the following four procedures were selected: mechanical, enzymatic, thermal and chemical. Considering the yield and repeatability of a method for the genomic DNA extraction, a mechanical method was applied. The genetic material research of each strain was performed according to PCR-REA technique with the amplification of three genome regions: ITS, LSU rRNA and a gene encoding beta-tubuline. The ITS and LSU rRNA regions were amplified employing the standard PCR reagents, whereas the region coding beta-tubuline with the so called touch down. The obtained amplification products were subjected to restrictive analysis by means of the following enzymes: EcoRI, Ncol, Hinfl, Alul, and Eco881 (Aval). The performed investigations made it possible to reveal the genotypic differentiation within M.pachydermatis species as well as some correlation between a genotypic profile and the origin of a strain (from healthy animals or with otitis externa symptoms), which may imply the existence of genetic conditioning of the Malassezia strains’ pathogenicity.
Key words: Malassezia pachydermatis, PCR-REA, otitis externa