Análise genética e desenvolvimento de um método de diagnóstico, baseado em PCR, para detecção de Cyniclomyces guttulatus em fezes de cães

Abstract

Many veterinary scientists have largely overlooked the significance of fungal infections in animals. However, recent high-profile fungal disease problems e.g. sporotrichosis of felines and humans in Brazil and zoonotic cryptococcosis worldwide, have demonstrated the need to revert this long-established negative trend. The ascomycete yeast Cyniclomyces guttulatusis is the only species currently included in the genus Cyniclomyces. Traditionally, it was viewed as a commensal component of the gut microbiota of rabbits and other herbivores. Increased interest in C. guttulatus developed based on reports from Europe, Asia, North America and Brazil suggesting that C. guttulatus represented an emerging and/or opportunistic pathogen in dogs, where its presence, in large numbers, was linked to clinical signs including diarrhea, gastroenteritis and cholangiohepatitis. However, recent investigations conducted in the Netherlands and the United States have argued that the presence of this yeast in dogs suffering gastrointestinal disease is most likely circumstantial and is not of clinical relevance. Diagnosis of C. guttulatus in feces and/or in clinical material is conducted primarily using microscopic and microbiological culture-based analyses. In a minority of studies, definitive identification of yeast colonies was made using polymerase chain reaction (PCR) amplification and sequencing of a fragment of the universal yeast gene 26S (encodes for the large subunit of ribosomal RNA). The current study reports the development of innovative, molecular methods for the detection and characterization of yeast cultures and for the culture-independent detection and characterization of C. guttulatus directly from canine feces. The availability of the analyses will serve to reduce doubt over the validity of morphological diagnostics and can reduce the time for a confirmed molecular diagnosis to 48 hours, rather than the actual timescale of 7-10 days. Genotyping of a collection of isolates from guinea pigs (n=1), rabbits (n= 20) and dogs (n=8), used PCR-based amplification and sequencing of fragments of the sequences encoding the large (26S) and small (18S) subunits of ribosomal rDNA, the subunits RPB1 and RPB2 of the gene encoding RNA polymerase II, Cytochrome oxidase II and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster. Sequence alignments and evaluations of nucleotide similarity for most sequences, served to support data from earlier studies, which had suggested the possible existence of two species of Cyniclomyces. Intriguingly, the use of the ITS region indicated that there may exist at least three species of Cyniclomyces. The identification of the new species of Cyniclomyces calls into question much of the data produced until now in relation to the pathogenicity of this organism. It is envisaged that the findings of this research will serve to stimulate an expansion in the study of the genus Cyniclomyces, and as such will make meaningful contributions to our comprehension of its role in canine health.