Made available in DSpace on 2014-08-13T14:50:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-27Bitstream added on 2014-08-13T18:01:34Z : No. of bitstreams: 1 000744257.pdf: 1345587 bytes, checksum: 884d34a74775bc0bdd270eedb4458762 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cryptosporidium serpentis infection is common in reptiles, especially snakes, and is characterized by chronic infection with severe hypertrophic gastritis, which can be lethal. This research aimed to use the real- time polymerase chain reaction (PCR) targeting the heat shock protein gene (Hsp70) for detection of C. serpentis in fecal samples of 503 snakes, and to determine its analytical and epidemiological specificity and sensitivity using, as a gold standard, the nested PCR targeting the 18S rRNA (18S rRNA) gene followed by sequencing of the amplified fragments (nPCR/S). The real-time PCR was positive for C. serpentis in 17 samples (3.37%), and nPCR/S resulted in positive results for C. serpentis in 15 samples (2.98%). It was also observed that the nPCR/S was positive for Cryptosporidium spp. in 60 samples (11.98%). Sequencing of the fragments amplified by nPCR was possible in 38 samples, and resulted in the identification of Cryptosporidium tyzzeri, Cryptosporidium muris, Cryptosporidium varanii and C. serpentis in several species of snakes. The sensitivity and specificity of real-time PCR were, respectively, 93.8% and 99.5%. Although three samples were positive for C. serpentis only by real-time PCR, and were considered as false positive results in the estimation of the epidemiological specificity and sensitivity, the melting curve analysis indicated that these samples had the same melting temperature of the C. serpentis samples. Thus, we conclude that real-time PCR targeting the gene Hsp70 is a sensitive and specific method for the detection of C. serpentis in fecal samples from snakes / FAPESP: 07/54312-2

Resumo:A infecção por Cryptosporidium serpentis é comum em répteis, em particular em serpentes, e é caracterizada por infecção crônica com presença de gastrite hipertrófica grave, que pode ser letal. Esta pesquisa teve como objetivo utilizar a reação em cadeia da polimerase (PCR) em tempo real, tendo como alvo o gene da proteína do choque térmico (Hsp70), para detecção de C. serpentis em amostras fecais de serpentes, e determinar a sua especificidade e sensibilidade analíticas e epidemiológicas utilizando, como padrão ouro, a nested PCR para amplificação de fragmento parcial do gene da subunidade 18S do rRNA (18S rRNA) seguida de sequenciamento dos fragmentos amplificados (nPCR/S). A PCR em tempo real foi positiva para C. serpentis em 17 amostras (3,37%), enquanto a nPCR/S resultou em positividade para C. serpentis em 15 amostras (2,98%). A nPCR/S resultou em positividade para Cryptosporidium spp. em 60 amostras (11,98%). O seqüenciamento dos fragmentos amplificados pela nPCR foi possível em 38 amostras e resultou na identificação de Cryptosporidium tyzzeri, Cryptosporidium muris, Cryptosporidium varanii e C. serpentis, em diversas espécies de serpentes. Os valores de sensibilidade e especificidade epidemiológicas da PCR em tempo real foram, respectivamente, 93,8% e 99,5%. Embora três amostras tenham apresentado positividade para C. serpentis apenas pela PCR em tempo real, e foram consideradas como resultado falso positivo na estimativa da especificidade e sensibilidade epidemiológicas, a análise da curva de dissociação indicou que essas amostras apresentaram a mesma temperatura de dissociação que as amostras de C. serpentis. Assim, concluiu-se que a PCR em tempo real, visando ao gene Hsp70 é um método sensível e específico para detecção de C. serpentis em amostras fecais de serpentes / Abstract:Cryptosporidium serpentis infection is common in reptiles, especially snakes, and is characterized by chronic infection with severe hypertrophic gastritis, which can be lethal. This research aimed to use the real- time polymerase chain reaction (PCR) targeting the heat shock protein gene (Hsp70) for detection of C. serpentis in fecal samples of 503 snakes, and to determine its analytical and epidemiological specificity and sensitivity using, as a gold standard, the nested PCR targeting the 18S rRNA (18S rRNA) gene followed by sequencing of the amplified fragments (nPCR/S). The real-time PCR was positive for C. serpentis in 17 samples (3.37%), and nPCR/S resulted in positive results for C. serpentis in 15 samples (2.98%). It was also observed that the nPCR/S was positive for Cryptosporidium spp. in 60 samples (11.98%). Sequencing of the fragments amplified by nPCR was possible in 38 samples, and resulted in the identification of Cryptosporidium tyzzeri, Cryptosporidium muris, Cryptosporidium varanii and C. serpentis in several species of snakes. The sensitivity and specificity of real-time PCR were, respectively, 93.8% and 99.5%. Although three samples were positive for C. serpentis only by real-time PCR, and were considered as false positive results in the estimation of the epidemiological specificity and sensitivity, the melting curve analysis indicated that these samples had the same melting temperature of the C. serpentis samples. Thus, we conclude that real-time PCR targeting the gene Hsp70 is a sensitive and specific method for the detection of C. serpentis in fecal samples from snakes / Orientador:Marcelo Vanconcelos Meireles / Banca:Alex Akira Nakamura / Banca:Valéria de Sá Jayme / Banca: Carlos Noriyuki Kaneto / Banca:Roberta Lemos Freire / Doutor