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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
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1749 |
| Date | 27 February 2015 |
| Creators | Furtado, Tassia torres |
| Contributors | Lopes, Carlos Wilson Gomes, McIntosh, Douglas, Cardozo, Sergian Vianna, Almeida, Elan Cardozo Paes de |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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