Thesis (M.S. in animal sciences)--Washington State University, December 2006. / Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-05Bitstream added on 2014-06-13T18:31:15Z : No. of bitstreams: 1 paulan_sc_me_araca_prot.pdf: 859556 bytes, checksum: f0e6fa2d3af33e24da91a0232d77060c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cisticercose bovina, causada pela forma metacestóide de Taenia saginata, é responsável por prejuízos econômicos devido à desvalorização de carcaças infectadas. A inspeção sanitária tem sido a principal forma de diagnóstico desta zoonose, porém apresenta baixa sensibilidade em identificar animais com infecção menos intensa. Ensaios imunoenzimáticos para detecção de anticorpos e de antígenos representam importante alternativa de diagnóstico ante mortem para a identificação de animais infectados, pois apresentam maior sensibilidade. Neste trabalho foram avaliadas a detecção de anticorpos anti-Taenia saginata e de antígenos do mesmo parasita em amostras de soro de bovinos naturalmente infectados, por meio dos testes ELISA indireto e ELISA sanduíche, respectivamente. Além das condições de realização dos testes, como o preparo das soluções e o tipo de extrato antigênico utilizados, a intensidade da infecção é um dos fatores que mais interferem nos resultados diagnósticos. Dessa forma, as características diagnósticas dos testes ELISA indireto, realizado com três diferentes extratos antigênicos, e ELISA sanduíche, realizado com o anticorpo monoclonal HP10, foram também relacionadas com a intensidade de infecção dos bovinos por Taenia saginata / Bovine cysticercosis caused by Taenia saginata metacestodes is responsible for economic losses due to depreciation of the infected carcasses. Sanitary inspection has been the main diagnostic tool but has low sensitivity in animals light infected. Immunoassays for antibodies and antigens detection are important ante mortem diagnosis, because present higher sensitivity. Antibodies anti-T. saginata and T. saginata antigens detection were evaluated in naturally infected bovine serum samples by indirect ELISA and sandwich ELISA. Besides reagents and test conditions the intensity of animal’s infection represents one of the most important interfering factors on the diagnostic results. Thus, diagnostic characteristics of indirect ELISA performed with three different antigenic extracts and sandwich ELISA, with monoclonal antibody HP10, were related to the intensity of infection in samples from Taenia saginata infected bovines

Orientador: Cáris Maroni Nunes / Banca: Valéria Marçal Félix de Lima / Banca: Hélio José Montassier / Resumo: A cisticercose bovina, causada pela forma metacestóide de Taenia saginata, é responsável por prejuízos econômicos devido à desvalorização de carcaças infectadas. A inspeção sanitária tem sido a principal forma de diagnóstico desta zoonose, porém apresenta baixa sensibilidade em identificar animais com infecção menos intensa. Ensaios imunoenzimáticos para detecção de anticorpos e de antígenos representam importante alternativa de diagnóstico ante mortem para a identificação de animais infectados, pois apresentam maior sensibilidade. Neste trabalho foram avaliadas a detecção de anticorpos anti-Taenia saginata e de antígenos do mesmo parasita em amostras de soro de bovinos naturalmente infectados, por meio dos testes ELISA indireto e ELISA sanduíche, respectivamente. Além das condições de realização dos testes, como o preparo das soluções e o tipo de extrato antigênico utilizados, a intensidade da infecção é um dos fatores que mais interferem nos resultados diagnósticos. Dessa forma, as características diagnósticas dos testes ELISA indireto, realizado com três diferentes extratos antigênicos, e ELISA sanduíche, realizado com o anticorpo monoclonal HP10, foram também relacionadas com a intensidade de infecção dos bovinos por Taenia saginata / Abstract: Bovine cysticercosis caused by Taenia saginata metacestodes is responsible for economic losses due to depreciation of the infected carcasses. Sanitary inspection has been the main diagnostic tool but has low sensitivity in animals light infected. Immunoassays for antibodies and antigens detection are important ante mortem diagnosis, because present higher sensitivity. Antibodies anti-T. saginata and T. saginata antigens detection were evaluated in naturally infected bovine serum samples by indirect ELISA and sandwich ELISA. Besides reagents and test conditions the intensity of animal's infection represents one of the most important interfering factors on the diagnostic results. Thus, diagnostic characteristics of indirect ELISA performed with three different antigenic extracts and sandwich ELISA, with monoclonal antibody HP10, were related to the intensity of infection in samples from Taenia saginata infected bovines / Mestre

Bovine cysticercosis is a zoonotic disease for which cattle are the intermediate hosts of the human tapeworm <i>Taenia saginata</i>. Routine inspection measures are implemented in Canada by the Canadian Food Inspection Agency (CFIA), and similarly elsewhere, for the postmortem detection of larval parasite cysts (cysticerci) in beef destined for human consumption. Detection is based on the gross examination of traditional carcass predilection sites, although it is recognized that the parasite has no true predilection for a particular tissue or site. In order to evaluate the efficacy of the inspection protocol currently implemented in Canada, a study was undertaken to determine the distribution of <i>T. saginata</i> cysticerci in tissues of experimentally infected cattle. Forty-two cross-bred beef cattle were divided into five groups of 5-12 animals each and inoculated orally with either 10000, 5000, 1000, 100 or 10 <i>T. saginata</i> eggs obtained from cases of human taeniosis in Thailand. From 47 to 376 days post-inoculation (DPI), ten animals inoculated with 5000 eggs were killed and the carcasses partitioned into 31 tissue sites. These consisted of the traditionally inspected tissue sites of heart, masseter and pterygoid muscles, tongue, oesophagus, and diaphragm (membranous and crura); as well as non-traditional sites of lung, liver and 20 additional muscles or muscle groups. After the routine inspection for cysticerci of traditional tissue sites, tissues from all sites were each cut into approximately 0.5 cm thick slices and the total number of parasitic cysts and cyst density (cysts/g of tissue) were determined for each site. Traditional sites were similarly evaluated for the remaining 32 animals that were killed between 117 and 466 DPI. Sites were ranked based on cyst density. In the animals for which non-traditional sites were also evaluated, no sites had higher cyst densities than those traditionally inspected. When only traditional sites for all animals were compared, the heart ranked highest overall, although not significantly different from masseter, and was the most frequently affected site. The traditional site of oesophagus was among the poorest of all sites for detection of cysticerci. The heart was confirmed as the site of choice for detection of bovine cysticercosis based on high cyst density and frequency of infection. There was also enhanced visibility of parasite lesions in the heart due to the relatively early degeneration and resultant gross pathology that occurs in cardiac muscle. More thorough examination of the heart is recommended during post-mortem inspection for this parasite, particularly when examining animals from an infected herd. <p>Currently, confirmation by CFIA of suspect cysticerci recovered during meat inspection relies on gross, stereomicroscopic, or standard histological examination. Although degenerating cysticerci are more likely to be detected and submitted for diagnosis, they often cannot be definitively identified by these methods. A recently developed monoclonal antibody-based immunohistochemical (IHC) assay for post-mortem diagnosis of this parasite was optimized and standardized. The IHC method was compared to the currently used histological assay using 169 degenerated known-positive <i>T. saginata</i> cysticerci collected from the experimental infections in the first study and from field submissions, and known-negative specimens and lesions of various etiologies from non-infected cattle. The use of the IHC assay identified significantly more known-positive bovine cysticerci (91.7%) than the histological method (38.5%), and non-specifically reacted only with the other cestode species examined. Since <i>T. saginata</i> is the only larval cestode typically found in the muscle of cattle, this cross-reactivity is not significant and the IHC assay will be a useful tool for the identification of lesions caused by degenerated bovine cysticerci.<p>This research provided evidence to support changes to the current post-mortem inspection, detection and diagnostic procedures and will contribute to more effective and efficient control of bovine cysticercosis.

Bovine cysticercosis is a zoonotic disease for which cattle are the intermediate hosts of the human tapeworm <i>Taenia saginata</i>. Routine inspection measures are implemented in Canada by the Canadian Food Inspection Agency (CFIA), and similarly elsewhere, for the postmortem detection of larval parasite cysts (cysticerci) in beef destined for human consumption. Detection is based on the gross examination of traditional carcass predilection sites, although it is recognized that the parasite has no true predilection for a particular tissue or site. In order to evaluate the efficacy of the inspection protocol currently implemented in Canada, a study was undertaken to determine the distribution of <i>T. saginata</i> cysticerci in tissues of experimentally infected cattle. Forty-two cross-bred beef cattle were divided into five groups of 5-12 animals each and inoculated orally with either 10000, 5000, 1000, 100 or 10 <i>T. saginata</i> eggs obtained from cases of human taeniosis in Thailand. From 47 to 376 days post-inoculation (DPI), ten animals inoculated with 5000 eggs were killed and the carcasses partitioned into 31 tissue sites. These consisted of the traditionally inspected tissue sites of heart, masseter and pterygoid muscles, tongue, oesophagus, and diaphragm (membranous and crura); as well as non-traditional sites of lung, liver and 20 additional muscles or muscle groups. After the routine inspection for cysticerci of traditional tissue sites, tissues from all sites were each cut into approximately 0.5 cm thick slices and the total number of parasitic cysts and cyst density (cysts/g of tissue) were determined for each site. Traditional sites were similarly evaluated for the remaining 32 animals that were killed between 117 and 466 DPI. Sites were ranked based on cyst density. In the animals for which non-traditional sites were also evaluated, no sites had higher cyst densities than those traditionally inspected. When only traditional sites for all animals were compared, the heart ranked highest overall, although not significantly different from masseter, and was the most frequently affected site. The traditional site of oesophagus was among the poorest of all sites for detection of cysticerci. The heart was confirmed as the site of choice for detection of bovine cysticercosis based on high cyst density and frequency of infection. There was also enhanced visibility of parasite lesions in the heart due to the relatively early degeneration and resultant gross pathology that occurs in cardiac muscle. More thorough examination of the heart is recommended during post-mortem inspection for this parasite, particularly when examining animals from an infected herd. <p>Currently, confirmation by CFIA of suspect cysticerci recovered during meat inspection relies on gross, stereomicroscopic, or standard histological examination. Although degenerating cysticerci are more likely to be detected and submitted for diagnosis, they often cannot be definitively identified by these methods. A recently developed monoclonal antibody-based immunohistochemical (IHC) assay for post-mortem diagnosis of this parasite was optimized and standardized. The IHC method was compared to the currently used histological assay using 169 degenerated known-positive <i>T. saginata</i> cysticerci collected from the experimental infections in the first study and from field submissions, and known-negative specimens and lesions of various etiologies from non-infected cattle. The use of the IHC assay identified significantly more known-positive bovine cysticerci (91.7%) than the histological method (38.5%), and non-specifically reacted only with the other cestode species examined. Since <i>T. saginata</i> is the only larval cestode typically found in the muscle of cattle, this cross-reactivity is not significant and the IHC assay will be a useful tool for the identification of lesions caused by degenerated bovine cysticerci.<p>This research provided evidence to support changes to the current post-mortem inspection, detection and diagnostic procedures and will contribute to more effective and efficient control of bovine cysticercosis.

No description available.

Thesis (doctoral)--Rijksuniversiteit te Utrecht, 1980. / Dutch summary. Includes vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:29:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-02Bitstream added on 2014-06-13T18:39:10Z : No. of bitstreams: 1 oliveira_jcv_me_botfmvz.pdf: 876846 bytes, checksum: 0964099d5611eb445b184c0c142d8ec3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cisticercose é uma das principais causas de perdas econômicas na pecuária de corte brasileira devido às condenações da carne para o controle do complexo teníase-cisticercose. O tratamento quimioterápico pode ser utilizado para o controle da infecção nos animais, mas sua eficácia deve ser monitorada por testes de diagnóstico que detectem parasitas vivos ou seus produtos. Na presente pesquisa, anticorpos monoclonais contra antígenos totais (TAEB) e de fluido vesicular (TAEF) de metacestóides de Taenia saginata foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados. Cinco fusões TAEB e quatro TAEF foram realizadas e os sobrenadantes de cultura foram triados por teste ELISA indireto. Dez e nove híbridos pertencentes aos protocolos TAEB e TAEF, respectivamente, foram selecionados e clonados. As linhagens celulares clonadas foram mantidas em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 18 clones IgG1 e 32 IgM reativos para TAEB e 9 IgG1 e 9 IgM para TAEF. Dentre esses clones, 5 do protocolo TAEB e 5 TAEF foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. A avaliação da identificação antigênica dos anticorpos produzidos aos antígenos homólogos foi realizada por meio de western blotting, resultando em reatividade com frações protéicas de baixo peso molecular (< 18kDa), 43, 55, 66 e 100kDa. A imunofluorescência indireta demonstrou que os anticorpos monoclonais avaliados reconhecem antígenos presentes tanto do escólex quanto da membrana vesicular de metacestóides de bovinos naturalmente infectados. Os anticorpos monoclonais produzidos poderão ser utilizados para detecção de antígenos circulantes na avaliação da eficácia do tratamento de bovinos infectados, evitando assim perdas econômicas e danos à saúde pública. / Cysticercosis is a major cause of economic losses in the Brazilian bovine chain production due to meat condemnation for the taeniasis-cysticercosis complex control. Chemotherapy can be used to control infection in cattle but treatment efficacy should be monitored by diagnostic tests that can detect live parasites or their products. Monoclonal antibodies against Taenia saginata metacestode crude (TAEB) and cyst fluid (TAEF) antigens were produced by cell fusion procedures using spleen cells from immunized BALB / c mice. Five TAEB and four TAEF fusions were performed and the culture supernatants from these cell cultures were screened by indirect ELISA assay. Ten TAEB and nine TAEF hybrids were selected and cloned. Cloned cell lines were grown in culture medium to collect the monoclonal antibodies, resulting in 18 IgG1 and 32 IgM clones reactive to TAEB and 9 IgG1 and 9 IgM clones reactive to TAEF. Five TAEB and five TAEB clones were selected for ascitic fluid production by injection in BALB/c mice and further harvesting. Western blotting was performed to test the antigenic identification by the antibodies produced resulting in reactivity to protein fractions of low molecular weight (<18kDa) and to 43, 55, 66 and 100kDa. The indirect immunofluorescence test showed that monoclonal antibodies recognize antigens from both the scolex and the bladder wall of metadestodes from naturally infected bovine. The monoclonal antibodies obtained can be useful for detection of circulant antigens in treatment efficacy evaluation preventing economic losses and public health injury.

Resumo: A cisticercose é uma das principais causas de perdas econômicas na pecuária de corte brasileira devido às condenações da carne para o controle do complexo teníase-cisticercose. O tratamento quimioterápico pode ser utilizado para o controle da infecção nos animais, mas sua eficácia deve ser monitorada por testes de diagnóstico que detectem parasitas vivos ou seus produtos. Na presente pesquisa, anticorpos monoclonais contra antígenos totais (TAEB) e de fluido vesicular (TAEF) de metacestóides de Taenia saginata foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados. Cinco fusões TAEB e quatro TAEF foram realizadas e os sobrenadantes de cultura foram triados por teste ELISA indireto. Dez e nove híbridos pertencentes aos protocolos TAEB e TAEF, respectivamente, foram selecionados e clonados. As linhagens celulares clonadas foram mantidas em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 18 clones IgG1 e 32 IgM reativos para TAEB e 9 IgG1 e 9 IgM para TAEF. Dentre esses clones, 5 do protocolo TAEB e 5 TAEF foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. A avaliação da identificação antigênica dos anticorpos produzidos aos antígenos homólogos foi realizada por meio de western blotting, resultando em reatividade com frações protéicas de baixo peso molecular (< 18kDa), 43, 55, 66 e 100kDa. A imunofluorescência indireta demonstrou que os anticorpos monoclonais avaliados reconhecem antígenos presentes tanto do escólex quanto da membrana vesicular de metacestóides de bovinos naturalmente infectados. Os anticorpos monoclonais produzidos poderão ser utilizados para detecção de antígenos circulantes na avaliação da eficácia do tratamento de bovinos infectados, evitando assim perdas econômicas e danos à saúde pública. / Abstract: Cysticercosis is a major cause of economic losses in the Brazilian bovine chain production due to meat condemnation for the taeniasis-cysticercosis complex control. Chemotherapy can be used to control infection in cattle but treatment efficacy should be monitored by diagnostic tests that can detect live parasites or their products. Monoclonal antibodies against Taenia saginata metacestode crude (TAEB) and cyst fluid (TAEF) antigens were produced by cell fusion procedures using spleen cells from immunized BALB / c mice. Five TAEB and four TAEF fusions were performed and the culture supernatants from these cell cultures were screened by indirect ELISA assay. Ten TAEB and nine TAEF hybrids were selected and cloned. Cloned cell lines were grown in culture medium to collect the monoclonal antibodies, resulting in 18 IgG1 and 32 IgM clones reactive to TAEB and 9 IgG1 and 9 IgM clones reactive to TAEF. Five TAEB and five TAEB clones were selected for ascitic fluid production by injection in BALB/c mice and further harvesting. Western blotting was performed to test the antigenic identification by the antibodies produced resulting in reactivity to protein fractions of low molecular weight (<18kDa) and to 43, 55, 66 and 100kDa. The indirect immunofluorescence test showed that monoclonal antibodies recognize antigens from both the scolex and the bladder wall of metadestodes from naturally infected bovine. The monoclonal antibodies obtained can be useful for detection of circulant antigens in treatment efficacy evaluation preventing economic losses and public health injury. / Orientador: Cáris Maroni Nunes / Coorientador: Elenice Deffune / Banca: Valéria Marçal Félix de Lima / Banca: Rosana Rossi Ferreira / Mestre

Made available in DSpace on 2015-03-26T13:47:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 414918 bytes, checksum: 9cdf78f58a88854b69201c78bf0c0899 (MD5) Previous issue date: 2011-07-29 / Bovine cysticercosis is a disease of cosmopolitan distribution and zoonotic feature caused by the larval form of Taenia Saginata. Severe infections are uncommon in cattle, the vast majority of cases found in slaughterhouses are mild infection, and however, it leads to the condemnation of the organ and, in some cases, the conditional avail of the carcass. Considering the risk to public health and economic losses caused by bovine cysticercosis, measures of sanitary control of this zoonosis should be taken not just at slaughterhouses, but also during the production phase. The aim of this study was to evaluate the performance of serological diagnosis in the detection of bovine cysticercosis in field samples, track properties in the city of Colatina-ES that sent animals to slaughtering and to assess the likely risk factors for the animals to be contaminated with Taenia eggs. The study was conducted from the animals slaughter and from the data of Official Inspection Services in three slaughterhouses in Colatina. Three hundred sixteen samples were collected, in which one there were a hundred eighty-three positive samples, sixty-two negative samples from healthy animals and seventy-one negative samples from animals with other diseases such as fascioliasis, hydatidosis and tuberculosis. The samples were analyzed by ELISA and immunoblot methods, and the results were organized in contingency table to calculate the performance of tests determined by the rates of sensitivity and specificity, positive and negative predictive values and agreement. From the Animal Transit Guide (GTA) the addresses of the properties included in the study were obtained, the tracing allowed the realization of the epidemiological survey to assess the risk factors related to the presence of cysticercosis in the region, mapping of the studied region distribution and analysis of cases and controls. The ELISA test did not show sufficient reliability to be used in detecting animals with cysticercosis in mild level, due to the high rate of false-negatives results. On the other hand, the immunoblot showed to be a more reliable method to detect positive samples. The main risk factors associated with cysticercosis in the properties in Colatina were: properties size, animal origin, age of the animals acquired and sewer destination. The diagnosis of cysticercosis in the refrigerator,added to the source information of the animal, allowed to define the areas of disease occurrence and its quantification. / A cisticercose bovina é uma enfermidade de distribuição cosmopolita de caráter zoonótico, causada pela forma larval da Taenia saginata. Infecções graves em bovinos são pouco frequentes, a grande maioria dos casos encontrados em matadouros consiste em infecção discreta, mesmo assim, levam a condenção do órgão e, em alguns casos, aproveitamento condicional da carcaça. Levando em consideração o risco para a saúde pública e os prejuízos econômicos provocados pela cisticercose bovina, medidas de controle sanitário dessa zoonose devem ser tomadas não só no momento do abate, mas também na fase de produção dos bovinos. O objetivo dessa pesquisa foi avaliar o desempenho do diagnóstico sorológico na detecção da cisticercose bovina em amostras de campo, rastrear propriedades localizadas no município de Colatina-ES que enviaram animais para abate e avaliar os prováveis fatores de risco de contaminação dos animais com larvas de T. saginata. Foi realizado estudo a partir do abate de animais e de dados dos Serviços Oficiais de Inspeção em três matadouros localizados no município de Colatina-ES. Foram coletadas 316 amostras de sangue, sendo 183 amostras positivas, 62 negativas provenientes de animais sadios e 71 negativas provenientes de animais com outras patologias como fasciolose, hidatidose e tuberculose. As amostras foram analisadas através das técnicas ELISA e immunoblot e os resultados foram organizados em tabela de contingência para cálculo do desempenho dos testes determinado pelas taxas de sensibilidade e especificidade, valores preditivos positivo e negativo e concordância. A partir da Guia de Trânsito Animal (GTA) foram obtidos os endereços das propriedades incluídas no estudo, a rastreabilidade permitiu a realização de inquérito epidemiológico, para avaliação dos fatores de risco relacionados à presença da cisticercose na região, mapeamento da região estudada e análise de distribuição dos casos e controles. O teste ELISA não apresentou confiabilidade suficiente para ser utilizado na detecção de animais portadores de cisticercose com apresentação discreta da doença, em decorrência da alta taxa de falso-cegativos. Por outro lado, o immunoblot demonstrou ser um método mais confiável na detecção de amostras positivas. Os principais fatores de risco associados às propriedades com cisticercose no município de Colatina foram tamanho das propriedades,origem dos animais, idade dos animais adquiridos e destino do esgoto. O diagnóstico da cisticercose no frigorífico, somado a informação de origem do animal, possibilitou definir as áreas de ocorrência da doença e sua quantificação.