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.

Memoria para optar al Título Profesional de Médico Veterinario / La cisticercosis muscular bovina (Cysticercus bovis) es una zoonosis con importancia económica y para la salud pública. En el presente estudio se dan a conocer antecedentes sobre la presencia de C. bovis en predios de las regiones de Coquimbo, Valparaíso y Metropolitana y su relación espacial con asentamientos humanos y cursos de agua superficiales que actuarían como factores de riesgo. Para esto se realizó un seguimiento a 457 bovinos diagnosticados post-mortem con cisticercosis muscular, beneficiados durante el año 2010 en dos plantas faenadoras que cuentan con inspección veterinaria oficial del Servicio Agrícola y Ganadero en la Región de Valparaíso. Del total indicado, 424 casos correspondieron a cisticercosis leve (92,78%) y 33 casos a cisticercosis masiva (7,22%). De los 26.167 bovinos beneficiados en la región, por categoría, las vacas fueron las que presentaron un mayor porcentaje de hallazgos de C. bovis (2,38%) diferencia estadísticamente significativa (α=0,05), comparado con novillos, bueyes, toros, vaquillas y terneros. Según la distribución mensual, se observó una mayor proporción de casos en los meses de verano, también diferente significativamente comparado con las otras tres estaciones (α=0,05). Se descartaron 48 animales de procedencia distinta a las regiones objetivo y 113 animales (24,7%), que no presentaban todos los antecedentes necesarios para determinar su predio de origen. De esta manera, se obtuvo un grupo de 296 bovinos, pertenecientes a 112 predios, de los cuales 12 se encontraron en la Región de Coquimbo, 78 en la Región de Valparaíso y 22 en la Región Metropolitana.. Estos predios se georreferenciaron y situaron en un mapa digital donde se registró la distancia que presentaban con asentamientos humanos y cursos de agua superficiales, obteniéndose en ambos casos, que la mayor cantidad de predios positivos a esta parasitosis, se ubicaban en áreas cercanas (menos de 300 metros) de dichos factores de riesgo

Made available in DSpace on 2015-03-26T13:46:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1867425 bytes, checksum: f26d8b051d4ec39dd9de144658e3edf1 (MD5) Previous issue date: 2008-07-09 / The in vitro action of the nematophagous fungi Pochonia chlamydosporia (VC1 and VC4), Paecilomyces lilacinus, Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34), on eggs of Taenia saginata and capsules eggs of Dipylidium caninum was evaluated. A hundred fifty thousand eggs of T. saginata (experimental assay 1) and a hundred fifty thousand eggs capsules of D. caninum (experimental assay 2) were used. These eggs were maintained in plates of Petri containing the isolated fungi and without fungi (controls), in medium water-agar 2% (AA2%), where they grew for 10 days, being made 25 repetitions for each group. In of five, 10 and 15 days, about a hundred eggs were removed of each plate and placed in glass sheets with a drop of Amam blue 1%, and evaluated in agreement with the following parameters: type 1, physiologic and biochemical effect without morphological damage to the eggshell, with hyphae adhered to the eggshell; type 2, lytic effect with morphological change of the embryo and eggshell, without hyphal penetration through eggshell; and type 3, lytic effect with morphological change of the embryo and eggshell, besides hyphal penetration and internal egg colonization. In the experimental assay 1, the isolated fungi P. chlamydosporia (VC1 and VC4) and P. lilacinus presented ovicidal activity for the effects of the types 1, 2 and 3 on eggs of T. saginata, while the isolated fungi D. flagrans (AC001) and M. thaumasium (NF34) only presented effect of the type 1. In the experimental assay 2, the isolated fungi D. flagrans (AC001) and M. thaumasium (NF34) only presented effect of the type 1 for all the analyzed intervals. The isolated fungi of the P. chlamydosporia (VC1 and VC4) and P. lilacinus, presented effects of the type 1, 2 and 3 in the intervals of five, 10 and 15 days. At the end of the experiments 1 and 2, was demonstrated that the isolated P. chlamydosporia (VC1 and VC4) and P. lilacinus acted in a negative effect on eggs of T. saginata and capsules eggs of D. caninum, and the effect can be considered promising candidates in the biological control of those helminths. / Com o objetivo de avaliar a ação in vitro dos fungos nematófagos Pochonia chlamydosporia (VC1 e VC4), Paecilomyces lilacinus, Duddingtonia flagrans (AC001) e Monacrosporium thaumasium (NF34), sobre ovos de Taenia saginata e cápsulas ovígeras de Dipylidium caninum, foram utilizados cento e cinqüenta mil ovos de T. saginata (ensaio experimental 1) e cento e cinqüenta mil cápsulas ovígeras de D. caninum (ensaio experimental 2). Esses ovos foram vertidos em placas de Petri contendo os isolados fúngicos e sem fungo (controle), em meio ágar-água 2% (AA2%), onde cresceram por 10 dias, sendo feitas 25 repetições para cada grupo. Nos intervalos de cinco, 10 e 15 dias, cerca de cem ovos foram retirados de cada placa e colocados em lâminas de vidro com uma gota de azul de Amam 1%, para serem avaliados de acordo com os seguintes parâmetros: efeito do tipo 1: efeito fisiológico e bioquímico sem prejuízo morfológico à casca, onde são visualizadas as hifas aderidas à casca do ovo; efeito do tipo 2: efeito lítico com alteração morfológica da casca e embrião; e efeito do tipo 3: efeito lítico com alteração morfológica do embrião e da casca, além de penetração de hifas e colonização interna do ovo. No ensaio experimental 1, os isolados fúngicos P. chlamydosporia (VC1 e VC4) e P. lilacinus, apresentaram atividade ovicida para todos os efeitos dos tipos 1, 2 e 3 sobre ovos de T. saginata, enquanto os isolados fúngicos D. flagrans (AC001) e M. thaumasium (NF34), só apresentaram efeito do tipo 1. No ensaio experimental 2, os isolados fúngicos D. flagrans (AC001) e M. thaumasium (NF34), também só apresentaram efeito do tipo 1, para todos os intervalos analisados. Já os isolados fúngicos do fungo P. chlamydosporia (VC1 e VC4) e P. lilacinus, apresentaram efeitos do tipo 1, 2 e 3 nos intervalos de cinco, 10 e 15 dias. Ao final dos ensaios experimentais 1 e 2, ficou demonstrado que os isolados P. chlamydosporia (VC1 e VC4) e P. lilacinus, agiram de forma negativa sobre ovos de T. saginata e cápsulas ovígeras de D. caninum, e desta forma, podem ser considerados candidatos promissores no controle biológico desses helmintos.

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

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-09-06T11:51:20Z No. of bitstreams: 1 texto completo.pdf: 929936 bytes, checksum: a92c4c289b141c7d1bcc147b944ab7c6 (MD5) / Made available in DSpace on 2018-09-06T11:51:20Z (GMT). No. of bitstreams: 1 texto completo.pdf: 929936 bytes, checksum: a92c4c289b141c7d1bcc147b944ab7c6 (MD5) Previous issue date: 2018-05-25 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A cisticercose bovina é uma enfermidade de distribuição cosmopolita e de caráter zoonótico, causada pela forma larval Taenia saginata. A prevalência dessa zoonose em estabelecimentos de abate é responsável por consideráveis perdas econômicas para o país devido à condenação e ao aumento do custo de processamento das carcaças submetidas a aproveitamento condicional de bovinos infectados. Apesar da importância da cisticercose bovina para a saúde pública, para a saúde animal e para economia, a realidade epidemiológica da ocorrência dessa zoonose no Brasil é pouco conhecida. Dessa forma, esta pesquisa teve por objetivo realizar um levantamento epidemiológico e avaliar os principais fatores de risco relacionados com a transmissão da cisticercose bovina em animais enviados para abate provenientes de propriedades localizadas na região do Triângulo Mineiro, com base no rastreamento dos casos de cisticercose bovina detectados em um estabelecimento de abate do município de Uberlândia-MG. Foi realizado um estudo de corte transversal em 87 propriedades. A partir da Guia de Trânsito Animal (GTA) foram obtidos os endereços das propriedades incluídas no estudo e realizadas as coletas de sangue de 1024 bovinos. O diagnóstico sorológico realizado por triagem pelo teste Elisa indireto e Immunoblot para confirmação. Foi aplicado também um questionário epidemiológico sobre as condições sanitárias das propriedades, em relação ao sistema de criação animal, como por exemplo, manejo, origem dos animais, atividade predominante, fonte de abastecimento de água, higiene pessoal, padrão de alimentação e moradia das propriedades, a fim de determinar os possíveis fatores de risco associados a transmissão T. saginata. A prevalência encontrada foi de 5,1% (IC 95%= 3,74-6,42). Os fatores de risco identificados na área de estudo foram a origem dos bovinos (RC= 4,9 IC 95%= 1,5 – 15,8), sistema de pastejo (RC= 6,4 IC 95%= 2,0-20,2) e destino do esgoto das propriedades (RC= 3,6 IC 95%= 2,3-5,6). A partir da análise espacial foi identificado um aglomerado na região de estudo, com risco de infecção 2,85 vezes maior nas propriedades localizados dentro desse aglomerado quando comparado com as propriedades localizadas fora dessa área. Esses resultados mostram que a região do Triângulo mineiro é endêmica para a doença e medidas preventivas devem ser aplicadas em relação ao sistema de manejo e a estrutura infra sanitária das propriedades. / Bovine cysticercosis is a zoonotic, cosmopolitan disease caused by the larval form of Taenia saginata. The prevalence of this zoonosis in slaughterhouses is responsible for considerable economic losses to the country due to the condemnation and increase of the cost of processing the carcasses subject to the conditional use of infected cattle. Despite the importance of bovine cysticercosis to public health, animal health and economics, the epidemiological reality of the occurrence of this zoonosis in Brazil is poorly understood. Thus, this research aimed to carry out an epidemiological survey and evaluate the main risk factors related to the transmission of bovine cysticercosis in animals sent to slaughter from farms located in the Triângulo Mineiro region, based on the tracing of cases of bovine cysticercosis detected in a slaughter establishment in the municipalitiy of Uberlândia-MG. Conducted a cross-sectional epidemiological study on 87 farms. From the Animal Transit Guide (GTA), obtained the addresses of the farms included in the study and blood samples collected from 1024 cattle. The serological diagnosis performed by screening by the indirect Elisa test and Immunoblot for confirmation. Was also applied an epidemiological questionnaire on the sanitary conditions of farms, in relation to the animal husbandry system, such as management, origin of animals, predominant activity, source of water supply, personal hygiene, and feeding pattern and housing of the farms, in order to determine the possible risk factors associated with the transmission of T. saginata. The prevalence was 5.1% (95% CI = 3.74-6.42). The risk factors identified in the study area were the cattle origin (OR= 4.9/ 95% CI = 1.5 – 15.8), grazing system (OR= 6.4/ 95% CI = 2.0-20.2) and the destination of the farms' sewage (OR= 3,6/ 95% CI = 2.3-5.6). From the spatial analysis, in the study region identified a cluster, with a risk of infection 2.85 times higher in farms located inside this cluster when compared to farms located elsewhere. These results show that the Triângulo Mineiro region is endemic to the disease and preventive measures should be applied in relation to the management system and the infra-sanitary structure of the farms.

Orientador: Cáris Maroni Nunes / Resumo: O complexo teníase-cisticercose é causado pelos cestódeos Taenia saginata e Taenia solium, que têm o homem como hospedeiro definitivo e obrigatório e os bovinos e os suínos, respectivamente, como hospedeiros intermediários. A presença da forma adulta destes cestódeos no intestino delgado causa pequenos danos ao homem, enquanto a presença das formas larvárias nos tecidos e órgãos causam lesões nos hospedeiros intermediários, resultando na cisticercose. O homem pode também ser hospedeiro ocasional da T. solium e desenvolver a cisticercose, quando da ingestão acidental de seus ovos. Uma das medidas de controle desta zoonose é a inspeção sanitária de bovinos e suínos ao abate, evitando assim que os parasitas completem seu ciclo biológico. Assim, o complexo teníase-cisticercose, além de ser um grave problema de saúde pública, é também um problema econômico na agropecuária, visto que as carcaças podem ser condenadas quando da presença de formas larvares ou terem seu valor reduzido. A neurocisticercose é a forma mais grave desta zoonose no homem e seu diagnóstico é realizado por meios imunológicos e/ou por exames de imagens, que permitem a visualização dos das lesões no sistema nervoso central. Já a teníase é diagnosticada por meio da pesquisa de proglotes ou de ovos das tênias nas fezes, os quais são indistinguíveis morfologicamente. O objetivo do presente estudo foi avaliar a ocorrência de teníase humana por meio da análise dos resultados de exames coproparasitológicos rotineirame... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The taeniasis-cysticercosis complex is caused by the cestodes Taenia saginata and Taenia solium, which have man as their definitive and mandatory host and cattle and swine, respectively, as intermediate hosts. The presence of the adult form of these cestodes in the small intestine causes minor damage to humans, while the presence of the larval forms in tissues/organs causes damage to the intermediate hosts, resulting in cysticercosis. Man can also be an occasional host of T. solium and develop cysticercosis when ingesting its eggs. One of the measures to control this zoonosis is sanitary inspection of cattle and pigs at slaughter in order to prevent the parasites from completing their biological cycle. Thus, in addition of being a serious public health problem the taeniasiscysticercosis complex is also an economic problem once the carcasses with the larval forms can be condemned or have a reduced value. Neurocysticercosis is the most severe form of this zoonosis in man and its diagnosis is done by immunological method and/or by imaging exams, which allow the visualization of the lesions in the central nervous system. Taeniasis is diagnosed by searching for proglottids and/or tapeworm eggs in feces, which are morphologically indistinguishable. The objective of the present study was to evaluate the occurrence of human taeniasis by analyzing the results of the coproparasitological exams routinely performed in a private clinical analysis laboratory at a city in São Paulo state, B... (Complete abstract click electronic access below) / Mestre

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-11-29T18:34:51Z No. of bitstreams: 1 texto completo.pdf: 1306405 bytes, checksum: 1458047941be6c1d95930520f19df28c (MD5) / Made available in DSpace on 2016-11-29T18:34:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1306405 bytes, checksum: 1458047941be6c1d95930520f19df28c (MD5) Previous issue date: 2016-09-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A cisticercose bovina é uma doença de caráter zoonótico que acarreta prejuízos tanto de ordem sanitária quanto de ordem econômica. Tradicionalmente seu diagnóstico é realizado durante a inspeção post mortem em matadouros-frigoríficos sob fiscalização sanitária, e consiste basicamente na visualização macroscópica através de cortes realizados em grupos musculares preditos como preferenciais para a implantação dos cisticercos. Ressalta-se que este método apresenta deficiências de desempenho, deixando passar despercebidas formas discretas da doença. Essa pesquisa foi desenvolvida com a meta de aprimorar o diagnóstico da cisticercose bovina, e sobretudo, dos testes sorológicos atualmente desenvolvidos, aumentando a confiabilidade do diagnóstico. A pesquisa caracterizou-se pela seleção de oito peptídeos sintéticos e desenvolvimento de uma proteína quimera recombinante com alto potencial no diagnóstico da cisticercose bovina. Foram selecionadas três diferentes proteínas de Taenia saginata (GenBank) (18 kDa oncosphere antigen [Taenia saginata]; Antigen TSA16 [Taenia saginata]; Antigen TSA36 [Taenia saginata] Heat shock protein 20 homolog [Taenia saginata]), e realizada a predição de oito peptídeos (EP1-EP8) selecionados por diferentes programas de bioinformática. Após sintetização e análise do desempenho dos peptídeos pelo ELISA, foram selecionados os três peptídeos que alcançaram melhores resultados (EP1, EP4 e EP6) para compor uma proteína recombinante quimérica, que posteriormente foi denominada de rqTSA-25. A proteína rqTSA-25 obteve as seguintes taxas de desempenho no ELISA: sensibilidade de 88% (IC=76-94%), especificidade de 96% (IC=84-99%), valor preditivo positivo 96% (IC=86-99%), valor preditivo negativo 87% (IC=74-94%) e acurácia de 92%, sendo que, ao submeter diferentes amostras de soro grupo controle ao teste imunoblot, não foi verificada nenhuma reação falso positiva ou falso negativa. Dessa forma, destaca-se a inovação na produção da proteína rqTSA-25 e seu alto desempenho relatado, sendo um importante aliado na melhoria do teste diagnóstico sorológico para o diagnóstico da cisticercose bovina. / Bovine cysticercosis is a zoonotic disease that causes losses both for health and economic order. Traditionally the diagnosis is performed during the post-mortem inspection in slaughterhouses under sanitary inspection, and consistent basically in macroscopic visualization by means of cuts carried out in predicted muscle groups such as preferred for the implementation of cysticercosis. It is emphasized that this method has performance deficiencies, leaving pass unnoticed discrete forms of the disease. This research was developed with the goal of improving the performance of the diagnosis of bovine cysticercosis and serologic tests currently developed, allowing the presence of cysticercosis in live animals and increasing the reliability of the diagnosis. The research was characterized by the selection of synthetic peptides and development of a recombinant chimera protein with high potential for diagnosis of bovine cysticercosis. First, three different proteins were selected from Taenia saginata (GenBank) (18 kDa oncosphere antigen [Taenia saginata]; Antigen TSA16 [Taenia saginata]; Antigen TSA36 [Taenia saginata] Heat shock protein 20 homolog [Taenia saginata]), being carried out the prediction of eight peptides (EP1-EP8) with antigenic power by different bioinformatic programs. After synthesis and performance analysis of peptides by ELISA test, we selected three peptides that have reached the best performance rates (EP1, EP4 and EP6) to compose a chimeric recombinant protein, subsequently named rqTSA-25, which was inserted into pET 29a (+) for subsequent expression in bacterial system. The rqTSA-25 protein allowed the following performance ratios by ELISA: sensitivity 88% (CI = 76- 94%), specificity 96% (CI = 84-99%), positive predictive value 96% (CI = 86- 99%), negative predictive value 87% (CI = 74-94%) and 92% accuracy, and subjecting different samples of serum control group to immunoblotting, it was not found any false positive reaction or false negative. Thus, first it stands out of innovation in the production of rqTSA-25 protein and its reported performance, combined with important improvement in in the serologic test for the diagnosis of bovine cysticercosis.

Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T18:48:34Z : No. of bitstreams: 1 menosso_jc_me_botfmvz.pdf: 1568332 bytes, checksum: 5f6742d0d4281777a48557024d7871e0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cisticercose bovina é resultante da ingestão de ovos de T. saginata proveniente de pastagens e águas contaminadas fornecidas aos animais. É causa de preocupações na área de saúde pública por ocasionar a teníase em humanos, mas causa sérios prejuízos na indústria agropecuária e aos pecuaristas, devido às condenações ou tratamentos de frio ou salga pelas quais as carcaças consideradas positivas são submetidas. O método de diagnóstico rotineiramente utilizado para a cisticercose bovina tem sido a inspeção post-mortem. Muitos pesquisadores têm desenvolvido métodos de diagnóstico antemortem, com especificidade e sensibilidade variadas. O presente estudo teve como objetivo avaliar a aplicação da amplificação de DNA circulante de T. saginata, através da Reação em Cadeia pela Polimerase (PCR), como ferramenta diagnóstica para cisticercose bovina. Para a realização da pesquisa foram utilizadas amostras de sangue de bovinos abatidos em frigoríficos sob S.I.F., segundo apresentarem ou não cisticercose. Inicialmente avaliaram-se cinco pares de oligonucleotídeos iniciadores através da determinação do limiar de detecção da PCR de cada um deles. Objetivou-se ainda estabelecer qual a fração do sangue experimentalmente contaminado resultaria em amplificação de fragmentos de DNA através da PCR, para posteriormente avaliar a aplicabilidade da PCR em amostras de sangue de bovinos naturalmente infectados com T. saginata. O melhor limiar de detecção foi observado com o uso do par de oligonucleotídeos iniciadores SCAR K, nas frações de sangue denominadas pellet e sobrenadante. As amostras de sangue de animais positivos para cisticercose na inspeção de rotina e submetidos a PCR não resultaram em amplificação do fragmento de DNA desejado, sugerindo que a baixa carga parasitária possa ter influenciado os resultados. / Bovine cysticercosis is caused by the ingestion of T. saginata eggs from contaminated pastures and water provided to animals. It is a public health problem by causing the taeniasis in human beings, but it also causes serious damages in the agro-cattle industry and to the cattle raisers by de condemnation or treatment by coldness or salting of carcass detected as positive. Routinely bovine cysticercosis diagnosis has been the post-mortem inspection. Several researchers have developed ante-mortem diagnosis methods, with variable specificity and sensitivity. The present study aimed at evaluating the amplification of circulating DNA of T. saginata, by the Polymerase Chain Reaction (PCR), as a diagnostic tool to bovine cysticercosis. Blood samples from bovines slaughtered under Federal Meat Inspection abattoirs were taken according to the presence or absence of cyst forms. Initially, we evaluated five primers and established which blood fraction experimentally contaminated resulted in DNA fragment amplification by PCR. The best detection limit was observed with the use of the SCAR K primers, in the blood fractions denominated “pellet” and “supernatant”. Blood samples of naturally infected animals considered positive in the routine inspection didn’t result in the desired DNA fragment amplification by PCR.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neurocysticercosis (NC) caused by the Taenia solium metacestodes usually affects the central nervous system and can be confused with other brain pathologies, which present similar clinical and image findings. This parasitose is endemic in Brazil and the serological diagnosis of NC is of great importance in routine clinical management of patients. The search for alternative antigens for NC immunodiagnosis is importance because of the difficulty of obtaining parasites from naturally infected pigs for the preparation of metacestodes T. solium homologous antigen. Once that purified antigen is more sensitive and specific than crude saline extracts, the objective of this study was to evaluate the performance of enzyme linked immunosorbent assay (ELISA) and Immunoblotting (IB) for the serological diagnosis of human NC, using the crude saline extract (Crude) obtained from T. saginata metacestodes and two fractions of this extract, hydrophobic or detergent fraction (D) and hydrophilic or aqueous fraction (A), purified by extraction with Triton X-114 as alternative purified antigens for the diagnosis of human NC. Serum samples were obtained from 115 individuals: 40 patients with a definitive diagnosis of NC, 45 patients with Taenia sp. and with other parasitic diseases and 30 apparently healthy individuals. The serum samples were diluted 1:200 for ELISA but only the samples positives were diluted 1:100 and evaluated in IB. ELISA sensitivity and specificity were 95% and 73.3% when using Crude, 95% and 82.6% for D phase and 65% and 61.3% for A phase, respectively. The diagnostic efficiency and Youden index were: 83.9% and 0.74 (Crude), 88.5% and 0.80 (D phase) and 72.8 % and 0.46 (A phase), respectively. IB of the antigens confirmed the results obtained by ELISA, and the D phase antigen proved to be more specific than other extracts, showing only one immunodominant protein of 70-64 kDa that was the most frequent (92.5%) among patients with NC. Our results suggest that the D phase obtained with Triton X-114 can be used as heterologous purified antigen for the serological diagnosis of human NC in the IB. / A Neurocisticercose (NC), resultado do acometimento do sistema nervoso central pelas formas metacestódeas de Taenia solium, é uma doença polimórfica podendo ser confundida com outras síndromes neurológicas que apresentam achados clínicos e de imagem similares. Uma vez que esta parasitose é endêmica no Brasil o diagnóstico sorológico é de grande importância, pois auxilia na rotina laboratorial de pacientes com suspeita clínica. A busca por antígenos alternativos se faz necessária diante da dificuldade de obtenção de suínos naturalmente infectados com metacestódeos de T. solium, para a produção do antígeno homólogo. Uma vez que antígenos purificados apresentam sensibilidade e especificidade superiores aos extratos totais, o objetivo deste estudo foi avaliar os testes enzyme linked immunosorbent assay (ELISA) e Immunoblotting (IB) no diagnóstico sorológico da NC humana utilizando o extrato salino total (S) de metacestódeos de Taenia saginata e suas frações purificadas com Triton X-114 (TX-114). Uma alíquota do extrato S foi fracionada com TX-114, para obtenção das frações antigênicas: hidrofóbica ou detergente (D) e hidrofílica ou aquosa (A). Os extratos antigênicos foram analisados em SDS-PAGE 12% e utilizados no ELISA na concentração de 10&#956;g/mL. Foram analisadas 115 amostras de soro: 40 de pacientes com diagnóstico definitivo de NC, 45 de pacientes com Taenia sp. e por outros parasitos e 30 de indivíduos saudáveis. As amostras foram diluídas a 1:200 para o ELISA, sendo apenas as positivas submetidas ao IB, na diluição de 1:100. A sensibilidade e especificidade do ELISA foram: 95% e 73,3% (S), 95% e 82,6% (D) e 65% e 61,3% (A), respectivamente. O cálculo de eficiência do diagnóstico e Youden Index foram: 83,9% e 0,74 (S); 88,5% e 0,80 (D) e 72,8 % e 0,46 (A), respectivamente. No IB a fração D apresentou com exclusividade a banda imunodominante de 70-64 kDa, sendo detectada em 92,5% dos pacientes com NC. A fração D foi significativamente mais eficiente que a fração A, tanto no ELISA quanto no IB, sendo assim, recomendada como antígeno heterólogo purificado no diagnóstico sorológico da NC humana no IB. / Mestre em Imunologia e Parasitologia Aplicadas