Isolamento e caracterização de Cyniclomyces guttulatus (Robin) Van Der Walt e Scott (1971) de cães procedentes da Barra da Tijuca, Rio de Janeiro, RJ

Abstract

The ascomycete yeast C.guttulatus, has long been recognized as a component of the normal microflora of rabbits and other herbivores. More recently, a possible association between this yeast and gastro-intestinal illness in dogs has been reported by researchers in Europe, EUA, Japan and Brazil. The current study combined morpho-phenotypic and molecular (PCR-RFLP and nucleotide sequencing), methods to examine C.guttulatus cultures recovered from Brazilian rabbits (15 isolates) and dogs (7 isolates), with the objectives of developing a means for the differential identification of C. guttulatus and to improve existing knowledge of the biology of this organism. Microscopic and macroscopic examination of isolated colonies in combination with zymogram analysis (carbohydrate fermentation tests), was unable to provide a definitive identification for the suspect cultures. In silico restriction fragment length polymorphism (RFLP) analysis of virtual PCR amplicons (corresponding to the D1-D2 domain of the gene encoding 26S ribosomal RNA of C.guttulatus and other yeast species known to be associated with dogs), indicated that digestion with the restriction enzymes DdeI, HaeIII and MspI would allow the differential identification of C. guttulatus. In vitro restriction analysis of PCR amplicons confirmed the predictions of the in silico analysis and demonstrated that differential identification of C.guttulatus could be a made using only two enzymes (DdeI and MspI). Sequencing of D1-D2 amplicons demonstrated the presence of substantial nucleotide variation within the cultures examined and divided them into three groups, denominated sequence types (ST´s). Two of these groups were closely related to each other. The third sequence type showed extensive nucleotide variation and may represent a novel species or subspecies within the genus Cyniclomyces