Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-06-07T14:26:09Z No. of bitstreams: 1 2015 - Tassia Torres Furtado.pdf: 14895763 bytes, checksum: 7d611b6398d83fa875abc58ca88acab3 (MD5) / Made available in DSpace on 2017-06-07T14:26:09Z (GMT). No. of bitstreams: 1 2015 - Tassia Torres Furtado.pdf: 14895763 bytes, checksum: 7d611b6398d83fa875abc58ca88acab3 (MD5) Previous issue date: 2015-02-27 / 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 / A levedura, Cyniclomycesguttulatus ? um ascomiceto e tem sido reconhecida como um componente da microbiota natural de coelhos e outros herb?voros. Mais recentemente, uma poss?vel associa??o entre esta levedura e doen?as gastrintestinais em c?es tem sido relatada por pesquisadores na Europa, EUA, Jap?o e Brasil. O presente estudo combinou m?todos de an?lises morfol?gica e molecular (PCR-RFLP e sequenciamento de nucleot?deos), para examinar culturas de C. guttulatus recuperados de coelhos brasileiros (15 isolados) e c?es (7 isolados), com o objetivo de desenvolvimento de um m?todo para a identifica??o diferencial de C. guttulatus e melhorar o conhecimento existente sobre a biologia desse organismo. O exame microsc?pico e macrosc?pico das col?nias isoladas em combina??o com an?lise do zimograma (testes de fermenta??o de carboidratos), n?o foi capaz de fornecer uma identifica??o definitiva para as culturas suspeitas. As an?lises in silico de polimorfismo de comprimento de fragmentos de restri??o (RFLP) de produtos de PCR virtuais (correspondentes ao dom?nio D1/D2 do gene 26S RNA ribossomal de C.guttulatus e outras esp?cies de levedura conhecidas como associadas a c?es), indicou que a digest?o com a enzimas de restri??o Ddel, HaeIII e MspI permitiria a identifica??o diferencial de C. guttulatus. As an?lises das digest?es in vitro de produtos de PCR confirmaram as previs?es das an?lises in silico e demonstrou que a identifica??o diferencial de C.guttulatus poderia ser feita usando apenas duas enzimas (DdeI e MspI). O sequenciamento dos produtos de PCR do dom?nio D1/D2 demonstraram a presen?a de varia??es substanciais de nucleot?deos nas culturas analisadas e estas foram divididas em tr?s grupos, denominados de sequ?ncias tipos (ST's). Dois destes grupos estavam estreitamente relacionados uns aos outros. O terceiro tipo de sequ?ncia mostrou grande varia??o de nucleot?deos e pode representar uma nova esp?cie ou subesp?cie dentro do g?nero Cyniclomyces