Cryptosporidium parvum is a coccidian protozoan that colonizes epithelial cells lining respiratory and digestive tracts of animals and humans. Cryptosporidiosis is a well-recognized zoonotic disease infecting primarily neonates and immunocompromised hosts, including human immunodeficiency virus-infected patients. Clinical disease is manifested as a chronic diarrheal illness that is self-limiting in immunocompetent hosts and prolonged and often life-threatening in hosts with compromised immune systems. The lack of a suitable small animal model for screening anti-cryptosporidial drugs and for examining the pathogenicity and immunobiology of chronic cryptosporidosis was the impetus for this research effort. The objectives of the present study were three-fold: to characterize chronic Cryptosporidium parvum infections in dexamethasone-immunosuppressed mice; evaluate the effects of Cryptosporidium parvum and dexamethasone on B and T lymphocyte proliferation; and determine the effects of the immunomodulator dehydroepiandrosterone on oocyst shedding intensities of mice infected with Cryptosporidium parvum Adult C57BL/6N mice were immunosuppressed with the synthetic glucocorticoid dexamethasone, then infected with Cryptosporidium parvum (106 oocysts/mouse) and investigated for their ability to sustain a four-month chronic infection. Dexamethasone was administered intraperitoneally (125μ/mouse/day) or orally (8μ/ml) in the drinking water ad libitum. Infection chronicity was characterized by evaluating mouse mortality, oocyst excretion in the feces, tissue distribution of the parasite, and the parasite-induced pathology. A progressive infection with Cryptosporidium parvum occurred in mice immunosuppressed intraperitoneally and orally as long as dexamethsone was administered. Mice receiving dexamethasone given intraperitoneally had a shorter prepatent period and a more consistent, although cyclic, oocyst shedding pattern when compared with mice given dexamethasone orally. Mice given dexamethasone orally exhibited a delayed prepatent period, with a steady increase in oocyst shedding. All mice receiving dexamethasone orally died within three months following oocyst inoculation. Clinical signs included dehydration, icterus, and reduction in spleen and body weights. Clinical signs were more abrupt in mice receiving oral dexamethasone. Parasite colonization involved the entire intestinal tract, including the pyloric ring and Peyer's patches, but was the heaviest in the terminal ileum. Parasites were present in the lungs, gallbladder, and pancreatic ducts. Pathologic abnormalities were isolated to the terminal small intestine and included blunting and fusion of intestinal villi and crypt hyperplasia. Cryptosporidium parvum and dexamethasone administered in vivo reduced B and T lymphocyte responses to the mitogens lipopolysaccharide and concanavalin A. Dehydroepiandrosterone and dehydroepiandrosterone-sulfate resulted in no significant reductions in cryptosporidial activity as determined by oocyst shedding in the feces.

The concern of a Cryptosporidium or Giardia waterborne outbreak due to treated wastewater has had water treatment utilities using some of the highest water cleansing technologies available. Cryptosporidiosis and Giardiasis are severe diarrheal diseases which can lead to death, thus it is important that appropriate steps are taken to assure these parasites are not present in the effluent of treated wastewater. This study examined the results of 863 assays for Giardia and Cryptosporidium on the effluent of wastewater treatment facilities and found that county of collection, watershed of collection, and laboratory analyzing the sample have the most significant impact on the detection of Cryptosporidium oocysts and Giardia cysts in wastewater effluent and that there were minimal but significant differences in method of treatment and method of filtration. To date no other comprehensive analysis of this data has been done.

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Favor colocar o Certificado e fazer as alterações quanto à bolsa FAPESP. on 2018-01-08T13:19:20Z (GMT) / Submitted by BRUNA NICOLETI SANTANA null (brunanicoleti.ata@hotmail.com) on 2018-01-08T16:35:49Z No. of bitstreams: 1 Defesa Final (revisado) - Bruna 2017.docx: 2115998 bytes, checksum: d53869153af6d87bd313087bb51809fe (MD5) / Approved for entry into archive by Ederson Vasconcelos Pereira null (edersonpereira@fmva.unesp.br) on 2018-01-08T19:37:16Z (GMT) No. of bitstreams: 1 santana_bn_me_araca_int.pdf.docx: 2115998 bytes, checksum: d53869153af6d87bd313087bb51809fe (MD5) / Made available in DSpace on 2018-01-08T19:37:16Z (GMT). No. of bitstreams: 1 santana_bn_me_araca_int.pdf.docx: 2115998 bytes, checksum: d53869153af6d87bd313087bb51809fe (MD5) Previous issue date: 2017-12-18 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivou-se determinar a ocorrência de Cryptosporidium spp. em amostras de fezes de galinha doméstica em criações extensivas, semiextensivas e intensivas, no Estado de São Paulo, e avaliar três protocolos da reação em cadeia pela polimerase (nested PCR) para diagnóstico de Cryptosporidium spp. A purificação e concentração dos oocistos presentes em amostras fecais provenientes de 190 aves foram realizadas por meio de centrífugo-flutuação em solução de Sheather. As amostras foram submetidas à extração do DNA genômico dos oocistos e submetidas à pesquisa de Cryptosporidium spp. utilizando três protocolos de nested PCR para amplificação de fragmento parcial do gene da subunidade 18S do rRNA (18S rRNA), seguida de sequenciamento dos fragmentos amplificados. Os resultados obtidos pelos três protocolos de nested PCR foram analisados pelo teste de McNemar e pelo índice de correlação Kappa. As amostras que foram identificadas como Cryptosporidium meleagridis ou Cryptosporidium sp., pela análise do gene 18S rRNA, foram submetidas à caracterização adicional por meio de subgenotipagem de C. meleagridis pela nested PCR e sequenciamento de fragmento parcial do gene GP60 ou pela nested PCR e sequenciamento de fragmento parcial do gene da actina, respectivamente. A positividade total para Cryptosporidium (total de amostras positivas em pelo menos um método diagnóstico) obtida pela nested PCR foi de 12,6% (24/190), com identificação de Cryptosporidium baileyi (9,47%; 18/190), C. meleagridis (0,53%; 1/190), Cryptosporidium parvum (2,1%; 4/190) e Cryptosporidium sp. (0,53%; 1/190). A subgenotipagem de C. meleagridis revelou a presença do subtipo zoonótico IIIgA23G3R1. A análise do gene da actina permitiu a identificação de um novo genótipo de Cryptosporidium, em uma ave de criação extensiva, relacionado geneticamente com Cryptosporidium bovis e Cryptosporidium xiaoi. A análise de regressão logística não revelou diferenças significativas nas taxas de detecção de Cryptosporidium associadas aos diferentes sistemas de produção (extensivo, semi-intensivo e intensivo). Em comparação com frangos de corte, houve maior probabilidade de que as aves de postura e as aves de criações mistas fossem positivas para Cryptosporidium. Não houve diferença significativa na frequência de resultados positivos obtidos pelos três protocolos de nested PCR (p> 0,05); a concordância obtida pelo índice Kappa variou de substancial (0,70) a quase perfeita (0,9). / The objective of this study was to determine the occurrence of Cryptosporidium spp. in domestic chickens raised in different chicken production systems in Brazil using three nested PCR protocols. The purification and concentration of oocysts present in 190 fecal samples from chickens raised in extensive, semi-intensive and intensive production systems were accomplished by centrifugal flotation in Sheather's solution and were followed by the extraction of genomic DNA. The detection and molecular characterization of Cryptosporidium species and genotypes were performed using three nested polymerase chain reaction (nested PCR) protocols targeting the 18S rRNA gene followed by sequencing of the amplified fragments. The results obtained by the three nested PCR reactions were analyzed using the McNemar test and the Kappa correlation index. Subgenotyping of Cryptosporidium meleagridis was performed using a nested PCR reaction targeting the gp60 gene. Samples identified as Cryptosporidium sp. genetically similar to Cryptosporidium xiaoi and Cryptosporidium bovis by 18S rRNA gene sequencing were further analyzed by nested PCR targeting the actin gene and subsequent sequencing of the amplified fragment. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the nested PCR results was 12.6% (24/190), including Cryptosporidium baileyi (9.47%; 18/190), C. meleagridis (0.53%, 1/190), Cryptosporidium parvum (2.1%; 4/190) and Cryptosporidium sp. (0.53%; 1/190). Subgenotyping of C. meleagridis revealed the presence of the zoonotic subtype IIIgA23G3R1. Sequencing of the 18S rRNA gene and the actin gene fragments revealed a new Cryptosporidium genotype in an extensive poultry system genetically related to C. xiaoi and C. bovis. Logistic regression analysis did not reveal significant differences in the rates of Cryptosporidium detection associated with the variable production system (extensive, semi-intensive and intensive). In comparison to broiler chickens, there were higher odds for layer chickens and mixed chickens to be positive for Cryptosporidium. There was no significant difference in the frequency of positive results obtained by the three nested PCR protocols (p> 0.05); additionally, the agreement obtained by Kappa index ranged from substantial (0.70) to almost perfect (0.9). / FAPESP: 15/26334-8.

Orientador: Marcelo Vasconcelos Meireles / Banca: Flávia Lombardi Lopes / Banca: Alex Akira Nakamura / Resumo: O objetivo deste estudo foi avaliar a ocorrência e os métodos de diagnóstico para Cryptosporidium spp. em psitacídeos exóticos de cativeiro provenientes das regiões sul e sudeste do Brasil. A purificação dos oocistos nas amostras fecais de 463 psitacídeos foi realizada por meio de centrifugo-flutuação em solução de Sheather. Para análise microscópica, nós utilizamos a coloração negativa de verde malaquita. A amplificação de um fragmento parcial do gene 18S rRNA de Cryptosporidium spp. foi feita usando-se nested PCR seguida de sequenciamento dos fragmentos amplificados (nPCR/S). As amostras também foram testadas por meio de PCR duplex em tempo real, visando-se amplificar um fragmento do gene 18S rRNA de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A ocorrência de Cryptosporidium spp. pela microscopia e nested PCR (nPCR) foi de 3, 02% (14/463) e 4, 97% (23/463), respectivamente. A nPCR/S demonstrou positividade de 1, 73% (8/463) para Cryptosporidium genótipo III de aves, 0, 86% (4/463) para Cryptosporidium parvum e 0, 22% (1/463) para Cryptosporidium canis. A PCR duplex em tempo real demonstrou positividade de 9, 50% (44/463) para as criptosporidiose gástrica, sendo 1, 94% (9/463) para C. galli, 5, 83% (27/463) para Cryptosporidium genótipo III de aves e 1, 73% (8/463) para infecções mistas. Não houve diferença estatística significante entre a positividade pela nPCR e microscopia (p = 0. 1237) e houve concordância justa entre elas (Kappa = 0. 242). Diferença es... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the prevalence and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. The oocyst purification in fecal samples from 463 psittacines was performed by centrifugal-flotation in Sheather's sugar solution. For microscopic analysis, we used malachite green negative staining. Amplification of a partial fragment of the 18S rRNA gene of Cryptosporidium spp. was accomplished using nested PCR (nPCR) followed by sequencing of the amplified fragments (nPCR/S). Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. by microscopy and nPCR was 3. 02% (14/463) and 4. 97% (23/463), respectively. The nPCR/S showed positivity of 1. 73% (8/463) for Cryptosporidium avian genotype III, 0. 86% (4/463) for Cryptosporidium parvum and 0. 22% (1/463) for Cryptosporidium canis. Duplex real-time PCR showed a positivity of 9. 50% (44/463) for gastric cryptosporidiosis, 1. 94% (9/463) for C. galli, 5. 83% (27/463) for Cryptosporidium avian genotype III and 1. 73% (8/463) for mixed infections. There was no statistically significant difference between positivity for nPCR and microscopy (p = 0. 1237) and fair agreement between them (Kappa = 0. 242). A significant statistical difference (p <0. 0001) and fair agreement (Kappa = 0. 317) were obtained between nPCR and duplex real-time PCR. We found out that duplex real-time PCR is the best option for the diagnosis of gastric cryptosporidiosis and that Cryptosporidium avian genotype III is the most common Cryptosporidium species /genotype in psittacines / Mestre

Orientador: Marcelo Vasconcelos Meireles / Coorientador: Alex Akira Nakamura / Banca: Gisele Fabrino MAchado / Banca Weslen Fabricio Pires Teixeira / Resumo: Objetivou-se determinar a ocorrência de Cryptosporidium spp. em amostras de fezes de galinha doméstica em criações extensivas, semiextensivas e intensivas, no Estado de São Paulo, e avaliar três protocolos da reação em cadeia pela polimerase (nested PCR) para diagnóstico de Cryptosporidium spp. A purificação e concentração dos oocistos presentes em amostras fecais provenientes de 190 aves foram realizadas por meio de centrífugo-flutuação em solução de Sheather. As amostras foram submetidas à extração do DNA genômico dos oocistos e submetidas à pesquisa de Cryptosporidium spp. utilizando três protocolos de nested PCR para amplificação de fragmento parcial do gene da subunidade 18S do rRNA (18S rRNA), seguida de sequenciamento dos fragmentos amplificados. Os resultados obtidos pelos três protocolos de nested PCR foram analisados pelo teste de McNemar e pelo índice de correlação Kappa. As amostras que foram identificadas como Cryptosporidium meleagridis ou Cryptosporidium sp., pela análise do gene 18S rRNA, foram submetidas à caracterização adicional por meio de subgenotipagem de C. meleagridis pela nested PCR e sequenciamento de fragmento parcial do gene GP60 ou pela nested PCR e sequenciamento de fragmento parcial do gene da actina, respectivamente. A positividade total para Cryptosporidium (total de amostras positivas em pelo menos um método diagnóstico) obtida pela nested PCR foi de 12,6% (24/190), com identificação de Cryptosporidium baileyi (9,47%; 18/190), C. meleagridis ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to determine the occurrence of Cryptosporidium spp. in domestic chickens raised in different chicken production systems in Brazil using three nested PCR protocols. The purification and concentration of oocysts present in 190 fecal samples from chickens raised in extensive, semi-intensive and intensive production systems were accomplished by centrifugal flotation in Sheather's solution and were followed by the extraction of genomic DNA. The detection and molecular characterization of Cryptosporidium species and genotypes were performed using three nested polymerase chain reaction (nested PCR) protocols targeting the 18S rRNA gene followed by sequencing of the amplified fragments. The results obtained by the three nested PCR reactions were analyzed using the McNemar test and the Kappa correlation index. Subgenotyping of Cryptosporidium meleagridis was performed using a nested PCR reaction targeting the gp60 gene. Samples identified as Cryptosporidium sp. genetically similar to Cryptosporidium xiaoi and Cryptosporidium bovis by 18S rRNA gene sequencing were further analyzed by nested PCR targeting the actin gene and subsequent sequencing of the amplified fragment. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the nested PCR results was 12.6% (24/190), including Cryptosporidium baileyi (9.47%; 18/190), C. meleagridis (0.53%, 1/190), Cryptosporidium parvum (2.1%; 4/190) and Cryptosporidium sp.... (Complete abstract click electronic access below) / Mestre

Orientador: Cáris Maroni Nunes / Banca: Marcelo Vasconcelos Meireles / Banca: Eliana Reiko Matushima / Resumo: A manutenção de animais silvestres em cativeiro domiciliar como animais de estimação é bastante comum no Brasil e os papagaios tem sido preferidos por serem considerados curiosos, inteligentes e divertidos, além de serem excelentes imitadores e faladores. Entretanto, os papagaios-verdadeiros (Amazona aestiva) podem ser fontes de infecção de algumas zoonoses. Neste trabalho foram estudados 50 papagaios-verdadeiros mantidos em cativeiro domiciliar no município de Araçatuba, São Paulo. As condições sócio-econômicas e os manejos sanitário e nutricional das aves, bem como o contato com os residentes foram avaliados por meio de visitas às casas. Os resultados revelaram manejos sanitário e nutricional inadequados na maioria dos casos, estreito contato com os papagaios e falta de conhecimento sobre enfermidades dos mesmos. Não foi isolada Salmonella sp. nas amostras de fezes, enquanto Escherichia coli estava presente em três animais e estruturas leveduriformes foram encontradas na maioria deles. Cryptosporidium sp. foi encontrado em uma das amostras. Pode-se concluir que o estreito contato dos residentes com as aves e as condições sanitárias inadequadas podem favorecer a ocorrência de zoonoses nas residências avaliadas. A presença de Cryptosporidium sp., caso se trate de uma espécie zoonótica, indica a possibilidade da transmissão de criptosporidiose de papagaios para o homem em condições de cativeiro domiciliar. / Abstract: The maintenance of wild animals in domiciliary captivity as pets has been common in Brazil and parrots are preferred because they are considered curious, intelligents, amusing, excellent talkative and mimics. However, the blue- fronted amazon parrot (Amazona aestiva) can be source of some zoonosis infections. In the present study the sanitary and nutritional management of 50 blue-fronted amazon parrots kept in domiciliary captivity in Araçatuba city, SP, as well as the occurrence of zoonosis agents in stools samples, social-economic conditions and residents-birds contact were evaluated. Results showed inadequate sanitary and nutritional management in the majority of the cases, strait contact with the parrots and lack of knowledge about parrots diseases. Salmonella was not found in stool samples while Escherichia coli was present in three samples and levedures-like structures were found in the majority them. Cryptosporidium was found in one sample. We can conclude that the close contact with the birds and the uncorrect management can favour occurrence of zoonosis in evaluated residences. The presence of Cryptosporidium sp. Indicates transmition possibility of cryptosporidiosis, in case of zoonotic specie, from parrots to humans in domiciliary captivity conditions. / Mestre

Submitted by Elís Domingos Ferrari null (elisd.ferrari@yahoo.com.br) on 2017-10-31T11:41:02Z No. of bitstreams: 1 FINAL-Defesa Mestrado CORRIGIDO.pdf: 1177720 bytes, checksum: 7a08f0cef1e5a124653c8190f8178822 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-11-10T18:35:51Z (GMT) No. of bitstreams: 1 ferrari_ed_me_araca_par.pdf: 606404 bytes, checksum: 87114f79e3b3cffb7d3ab08cd9009eb6 (MD5) / Made available in DSpace on 2017-11-10T18:35:51Z (GMT). No. of bitstreams: 1 ferrari_ed_me_araca_par.pdf: 606404 bytes, checksum: 87114f79e3b3cffb7d3ab08cd9009eb6 (MD5) Previous issue date: 2017-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar a ocorrência e os métodos de diagnóstico para Cryptosporidium spp. em psitacídeos exóticos de cativeiro provenientes das regiões sul e sudeste do Brasil. A purificação dos oocistos nas amostras fecais de 463 psitacídeos foi realizada por meio de centrifugo-flutuação em solução de Sheather. Para análise microscópica, nós utilizamos a coloração negativa de verde malaquita. A amplificação de um fragmento parcial do gene 18S rRNA de Cryptosporidium spp. foi feita usando-se nested PCR seguida de sequenciamento dos fragmentos amplificados (nPCR/S). As amostras também foram testadas por meio de PCR duplex em tempo real, visando-se amplificar um fragmento do gene 18S rRNA de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A ocorrência de Cryptosporidium spp. pela microscopia e nested PCR (nPCR) foi de 3, 02% (14/463) e 4, 97% (23/463), respectivamente. A nPCR/S demonstrou positividade de 1, 73% (8/463) para Cryptosporidium genótipo III de aves, 0, 86% (4/463) para Cryptosporidium parvum e 0, 22% (1/463) para Cryptosporidium canis. A PCR duplex em tempo real demonstrou positividade de 9, 50% (44/463) para as criptosporidiose gástrica, sendo 1, 94% (9/463) para C. galli, 5, 83% (27/463) para Cryptosporidium genótipo III de aves e 1, 73% (8/463) para infecções mistas. Não houve diferença estatística significante entre a positividade pela nPCR e microscopia (p = 0. 1237) e houve concordância justa entre elas (Kappa = 0. 242). Diferença estatística significante (p <0. 0001) e concordância justa (Kappa = 0. 317) foram obtidas nas comparações entre nPCR e PCR duplex em tempo real. Nós concluímos que a PCR duplex em tempo real é a melhor opção para o diagnóstico de criptosporidiose gástrica e que Cryptosporidium genótipo III de aves é o mais comum dentre as espécies/ genótipos de Cryptosporidium que acometem psitacídeos. / The aim of this study was to evaluate the prevalence and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. The oocyst purification in fecal samples from 463 psittacines was performed by centrifugal-flotation in Sheather's sugar solution. For microscopic analysis, we used malachite green negative staining. Amplification of a partial fragment of the 18S rRNA gene of Cryptosporidium spp. was accomplished using nested PCR (nPCR) followed by sequencing of the amplified fragments (nPCR/S). Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. by microscopy and nPCR was 3. 02% (14/463) and 4. 97% (23/463), respectively. The nPCR/S showed positivity of 1. 73% (8/463) for Cryptosporidium avian genotype III, 0. 86% (4/463) for Cryptosporidium parvum and 0. 22% (1/463) for Cryptosporidium canis. Duplex real-time PCR showed a positivity of 9. 50% (44/463) for gastric cryptosporidiosis, 1. 94% (9/463) for C. galli, 5. 83% (27/463) for Cryptosporidium avian genotype III and 1. 73% (8/463) for mixed infections. There was no statistically significant difference between positivity for nPCR and microscopy (p = 0. 1237) and fair agreement between them (Kappa = 0. 242). A significant statistical difference (p <0. 0001) and fair agreement (Kappa = 0. 317) were obtained between nPCR and duplex real-time PCR. We found out that duplex real-time PCR is the best option for the diagnosis of gastric cryptosporidiosis and that Cryptosporidium avian genotype III is the most common Cryptosporidium species /genotype in psittacines. / FAPESP: 2015/26334-8

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-09Bitstream added on 2014-06-13T18:31:12Z : No. of bitstreams: 1 aquino_mcc_me_araca.pdf: 684677 bytes, checksum: 7318d697f23b509c8008184e6a67c272 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / om o objetivo de determinar a ocorrência e caracterizar molecularmente a infecção por Cryptosporidium spp. em bezerros bubalinos do Estado de São Paulo, Brasil, foram colhidas 222 amostras fecais de animais da raça Murrah, com até seis meses de idade. As amostras foram avaliadas pela técnica de Ziehl-Neelsen modificada e por meio da reação em cadeia da polimerase tipo nested (nPCR) para amplificação de fragmentos de DNA da subunidade 18S do gene do RNA ribossômico e sequenciamento dos fragmentos amplificados. Pela técnica de Ziehl-Neelsen modificada, foi detectada positividade de 8,1% (18/222), e pela nPCR, foi observada amplificação em 48,2% (107/222) das amostras, das quais 63 foram sequenciadas. A análise das sequências obtidas mostrou que a espécie mais frequente nesses animais foi Cryptosporidium ryanae, em bezerros bubalinos a partir de cinco dias de idade. A espécie zoonótica Cryptosporidium parvum foi detectada em apenas um animal e um genótipo incomum, similar a Cryptosporidium sp. W20486, foi encontrado, pela primeira vez em búfalos / With the aim of determining the occurrence and molecularly characterizing infection by Cryptosporidium spp. in buffalo calves from São Paulo State, Brazil, 222 fecal samples were collected from Murrah animals aged up to six months. The samples were assessed by means of modified Ziehl-Neelsen technique and nested polymerase chain reaction (nPCR) for amplification of DNA fragments from subunit 18S of the gene of ribosomal RNA and sequencing of the amplified fragments. The modified Ziehl-Neelsen technique indicated 8.1% positivity (18/222), while nPCR revealed amplification for 48.2% (107/222) samples, of which 63 were sequenced. The analysis of the obtained sequences showed that the most frequent species in these animals was Cryptosporidium ryanae, present in buffalo calves from five days of age. The zoonotic specie Cryptosporidium parvum was detected in only one animal, and an uncommon genotype, similar to that of Cryptosporidium sp. W20486, was found for the first time in buffaloes

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-12Bitstream added on 2014-06-13T18:55:51Z : No. of bitstreams: 1 oliveira_fp_me_araca.pdf: 209460 bytes, checksum: 16f297129399701b5967432ef69fe128 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Um total de 196 amostras fecais de bezerros leiteiros mestiços, de 7 a 30 dias de idade de ambos o sexos foram colhidas em 31 propriedades com o objetivo de determinar a ocorrência de Cryptosporidium na região Noroeste do Estado de São Paulo, assim como caracterizar molecularmente as espécies envolvidas nesta parasitose. Realizou-se a análise microscópica pela técnica de coloração negativa com verde malaquita em todas as amostras de fezes. Para identificação molecular de Cryptosporidium, utilizou-se a reação de nested PCR, utilizando primers específicos para amplificação de fragmentos do gene codificador da subunidade 18S do RNA ribossômico e do gene da glicoproteína GP 60, submetendo o produto da PCR a sequenciamento e análise filogenética. Por meio de exame de fezes, foi observada positividade de 2% (4/196), por meio de microscopia, e de 10,7% (21/196) pela nested PCR. Como resultado do sequenciamento, foram identificadas quatro espécies de Cryptosporidium: C. parvum (subtipo IIa15G2R1) C. ryanae, C. bovis e C. andersoni. Esta descoberta de C. parvum subtipo IIaA15G2R1 na região estudada ilustra a utilidade da subtipagem. Embora estes resultados iniciais baseados em subtipos com GP60 sejam úteis para compreender a dinâmica de transmissão das espécies de Cryptosporidium, o número de amostras analisadas é relativamente pequeno e muito mais ainda pode ser pesquisado. É interessante notar que as informações de tipagem molecular com o gene 18S rRNA e a subtipagem com o gene GP60, permitem que os epidemiologistas possam rastrear as fontes de surtos das diferentes espécies de Cryptosporidium. / A total of 196 fecal samples from crossbred calves, 7-30 days old of both sexes were collected in 31 properties with the objective of determining the prevalence of Cryptosporidium in the northwestern region of São Paulo, as well as characterizing the molecular species involved in this parasite. We performed microscopic analysis by the technique of negative staining with malachite green in all stool samples. For molecular identification of Cryptosporidium, we used the reaction of nested PCR using specific primers for amplification of gene fragments coding for the 18S subunit ribosomal RNA gene and the glycoprotein GP 60 by subjecting the PCR product sequencing and phylogenetic analysis. By stool examination, positivity was observed 2% (4 / 196) by means of microscopy, and 10.7% (21/196) by nested PCR. As a result of sequencing, we identified four species of Cryptosporidium: C. parvum (subtype IIa15G2R1) C. Ryanair, C. bovis and C. andersoni. This discovery of C. IIaA15G2R1 parvum subtype in the studied region illustrates the usefulness of subtyping. Although based on these initial results with GP60 subtypes are useful for understanding the transmission dynamics of the species of Cryptosporidium, the number of samples is relatively small and much more can still be searched. It is interesting to note that the information of molecular typing with 18S rRNA gene and subtyping with the GP60 gene, allow epidemiologists can track sources of outbreaks of different species of Cryptosporidium.

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-13Bitstream added on 2014-06-13T19:55:43Z : No. of bitstreams: 1 teixeira_wfp_me_araca.pdf: 257034 bytes, checksum: 74c2e0dcd5f52341aac3382521691e4d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi padronizar a reação de Imunofluorescência direta (IFD) para detecção de oocistos de Cryptosporidium parvum em amostras fecais de bezerros. Os anticorpos policlonais anti-C. parvum foram produzidos em dois coelhos adultos da raça Nova Zelândia, e titulados por meio do ensaio imuno-enzimático indireto (ELISA). A fração de imunoglobulina G (IgG) foi purificada a partir do soro do coelho imunizado, utilizando-se diálise em sulfato de amônio seguida de cromatografia em coluna de DEAE celulose e conjugação com isotiocianato de fluoresceína. Por meio da IFD padronizada, foi testada a reatividade cruzada do conjugado produzido contra outras espécies de Cryptosporidium (C. andersoni e C. serpentis) e com outros agentes etiológicos presentes em fezes de bovinos (Escherichia coli, Eimeria sp. e Candida sp.). A IFD foi comparada à microscopia com contraste de fase em solução de Sheather (MCF), avaliando a capacidade de detecção de oocistos de Cryptosporidium sp. em alíquotas de fezes de bezerro inoculadas com oocistos de C. parvum, e em amostras fecais de bezerros naturalmente infectados provenientes de 37 propriedades leiteiras da região de Araçatuba, SP. Nas amostras comprovadas como positivas, foi ainda feita uma análise semi-quantitativa de oocistos de Cryptosporidium sp., visualizados por campo de microscopia em ambas as técnicas. O conjugado anti-C. parvum apresentou reatividade com C. andersoni e C. serpentis. A IFD foi utilizada com sucesso para detecção de oocistos de C. parvum em fezes de bezerros, apresentando sensibilidade para detecção de até 104 oocistos em 3 g de fezes. Entre as 300 amostras fecais de bezerros naturalmente infectados, 19,67% (59/300) foram positivas para a presença de oocistos de Cryptosporidium sp. pela IFD, apresentando diferença estatisticamente significante (p<0.05) em relação às amostras positivas pela MCF / The objective of this experiment was to standardize the direct immunofluorescence assay (DIF) for detection of Cryptosporidium parvum oocysts in fecal samples of calves. The anti-C. parvum polyclonal antibodies were produced in two adult rabbits of New Zealand breed, and titrated by an indirect enzyme-linked immunosorbent (ELISA). The immunoglobulin G (IgG) was purified from the serum of immunized rabbits by dialysis in ammonium sulfate followed by column chromatography on DEAE cellulose, and conjugated with fluorescein isothiocyanate. Using DIF the anti-C. parvum conjugate was tested for the presence of cross-reactivity against other species of Cryptosporidium (C. andersoni and C. serpentis), and other infectious agents present in cattle fecal samples (Escherichia coli, Eimeria sp. and Candida sp.). A comparison between the DIF and phase contrast microscopy in Sheather solution (MCF) was accomplished for evaluation of the efficiency of both techniques for detection of Cryptosporidium sp. oocysts in fecal samples of calves seeded with C. parvum oocysts, and in fecal samples from naturally infected calves from 37 dairy farms in the region of Araçatuba, SP. A semi-quantitative analysis of Cryptosporidium sp. oocysts per microscopic field was accomplished for both techniques. The anti-C. parvum conjugate showed cross-reactivity with C. andersoni and C. serpentis oocysts. The DIF has been used successfully in the detection of C. parvum oocysts in fecal samples of calves, with sensitivity for detecting 104 oocysts in 3 g of fecal samples. Among the 300 fecal samples from naturally infected calves, 19.67% (59/300) were positive for Cryptosporidium oocysts by DIF, with significant statistical difference (p <0.05) when compared to the number of positive samples by MCF