Descrevem-se os aspectos epidemiológicos e clínico-patológicos da paratuberculose em um rebanho de búfalos no Município de Guaíba no Rio Grande do Sul. A propriedade possuía cento e noventa e quatro búfalos principalmente da raça mediterrâneo e destinava-se a produção de leite e carne. No período de abril de 2009 a setembro de 2010 seis búfalos apresentaram diarreia crônica intermitente, perda de peso e apetite normal. Na neccropsia observaram-se vasos linfáticos proeminentes no mesentério e na mucosa do intestino. Os linfonodos mesentéricos estavam moderadamente aumentados, com áreas escuras entremeadas com focos multinodulares brancacentos, principalmente na região cortical. A mucosa do intestino se encontrava espessa e cerebriforme. Ao exame histológico, desde o duodeno até o intestino grosso havia infiltrado composto principalmente por macrófagos epitelióides e, em menor escala, linfócitos, plasmócitos e células gigantes tipo Langhans. Outros achados observados foram dilatação dos vasos linfáticos, linfangite granulomatosa e linfangiectasia. Nos linfonodos mesentéricos verificaram-se lesões histológicas semelhantes. A coloração de Ziehl-Neelsen e a imuno-histoquimica revelaram bacilos álcool-ácido resistentes no interior de macrófagos, de células gigantes de Langhans e livres na mucosa e submucosa do intestino e dos linfonodos mesentéricos. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano de amostras de fezes, raspado de mucosa intestinal e de linfonodos mesentéricos. A identificação do agente foi realizada pela técnica de PCR IS900. Foi realizado, também, levantamento sorológico de 136 búfalos da propriedade através da técnica de ELISA, na qual foram observados 15,45 % reagentes. O diagnóstico da paratuberculose foi baseado nos dados clínicopatológicos, sorologia, isolamento e identificação do agente. / It´s described the epidemiologic, clinical and pathologic aspects of paratuberculosis in a bubaline herd in Guaiba city, southern Brazil. The property had one hundred and ninety animals for milk and meet production. Six adults Mediterranean breed bubalines showed chronic intermittent diarrhea, lost of weight and normal appetite. At necropsy, the lymphatic vessels were prominent in the serosa, the mesenteric lymphnodes were slightly enlarged with dark areas interwoven by multinodular white spots mainly in the cortical region. The intestine mucosa were thickened and of corrugated aspect. The microscopic exam revealed severe diffuse granulomatous inflammation, marked dilatation of the lymphatic vessels, granulomatous lymphangitis and lymphangioectasia from the duodenum to the large bowl. The mesenteric lymphnodes showed similar histological lesions. Acid-fast bacilli in macrophages, in Langhans giant cells and freely in the mucosa and submucosa of the small intestine and lymphnodes were shown at Ziehl-Neelsen staining and immunohistochemistry. Mycobacterium avium subsp. paratuberculosis was isolated through bacterial cultivation of samples taken from feces, intestinal mucosa, mesenteric lymphnodes, and identified through IS900 PCR. In addition, a serologic survey was performed in the property by ELISA test. 15.45 percent of the animals reacted positive to the test. The diagnosis of paratuberculosis was based on clinical, pathological and epidemiological data, serology exam, bacterial isolation and identification of the agent.

Descrevem-se os aspectos epidemiológicos e clínico-patológicos da paratuberculose em um rebanho de búfalos no Município de Guaíba no Rio Grande do Sul. A propriedade possuía cento e noventa e quatro búfalos principalmente da raça mediterrâneo e destinava-se a produção de leite e carne. No período de abril de 2009 a setembro de 2010 seis búfalos apresentaram diarreia crônica intermitente, perda de peso e apetite normal. Na neccropsia observaram-se vasos linfáticos proeminentes no mesentério e na mucosa do intestino. Os linfonodos mesentéricos estavam moderadamente aumentados, com áreas escuras entremeadas com focos multinodulares brancacentos, principalmente na região cortical. A mucosa do intestino se encontrava espessa e cerebriforme. Ao exame histológico, desde o duodeno até o intestino grosso havia infiltrado composto principalmente por macrófagos epitelióides e, em menor escala, linfócitos, plasmócitos e células gigantes tipo Langhans. Outros achados observados foram dilatação dos vasos linfáticos, linfangite granulomatosa e linfangiectasia. Nos linfonodos mesentéricos verificaram-se lesões histológicas semelhantes. A coloração de Ziehl-Neelsen e a imuno-histoquimica revelaram bacilos álcool-ácido resistentes no interior de macrófagos, de células gigantes de Langhans e livres na mucosa e submucosa do intestino e dos linfonodos mesentéricos. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano de amostras de fezes, raspado de mucosa intestinal e de linfonodos mesentéricos. A identificação do agente foi realizada pela técnica de PCR IS900. Foi realizado, também, levantamento sorológico de 136 búfalos da propriedade através da técnica de ELISA, na qual foram observados 15,45 % reagentes. O diagnóstico da paratuberculose foi baseado nos dados clínicopatológicos, sorologia, isolamento e identificação do agente. / It´s described the epidemiologic, clinical and pathologic aspects of paratuberculosis in a bubaline herd in Guaiba city, southern Brazil. The property had one hundred and ninety animals for milk and meet production. Six adults Mediterranean breed bubalines showed chronic intermittent diarrhea, lost of weight and normal appetite. At necropsy, the lymphatic vessels were prominent in the serosa, the mesenteric lymphnodes were slightly enlarged with dark areas interwoven by multinodular white spots mainly in the cortical region. The intestine mucosa were thickened and of corrugated aspect. The microscopic exam revealed severe diffuse granulomatous inflammation, marked dilatation of the lymphatic vessels, granulomatous lymphangitis and lymphangioectasia from the duodenum to the large bowl. The mesenteric lymphnodes showed similar histological lesions. Acid-fast bacilli in macrophages, in Langhans giant cells and freely in the mucosa and submucosa of the small intestine and lymphnodes were shown at Ziehl-Neelsen staining and immunohistochemistry. Mycobacterium avium subsp. paratuberculosis was isolated through bacterial cultivation of samples taken from feces, intestinal mucosa, mesenteric lymphnodes, and identified through IS900 PCR. In addition, a serologic survey was performed in the property by ELISA test. 15.45 percent of the animals reacted positive to the test. The diagnosis of paratuberculosis was based on clinical, pathological and epidemiological data, serology exam, bacterial isolation and identification of the agent.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-25T14:39:33Z No. of bitstreams: 1 Pedro Paulo Feitosa de Albuquerque.pdf: 705656 bytes, checksum: 3b387f372f7375364f614a52bba0c4df (MD5) / Made available in DSpace on 2016-07-25T14:39:33Z (GMT). No. of bitstreams: 1 Pedro Paulo Feitosa de Albuquerque.pdf: 705656 bytes, checksum: 3b387f372f7375364f614a52bba0c4df (MD5) Previous issue date: 2014-02-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The aim of the present study was to detect the IS900 region of MAP in bovine milk samples using the Real Time Polymerase Chain Reaction (qPCR) and conventional PCR, as well as to study the agreement between the tests used. In total, 121 bovine milk samples were collected from properties considered as a focus of MAP in the micro-region of Garanhuns, Pernambuco, Brazil. Of the 121 milk samples analyzed, it was possible to detect the DNA of MAP in 20 samples (16.52%) using conventional PCR and in 34 samples (28.09%) using qPCR. The DNA of the agent was detected on all of the six properties studied, ranging from 10.00% to 23.81% in the conventional PCR and from 10.00% to 36.84% in the qPCR. The agreement between qPCR and conventional PCR was moderate (Kappa =0.53; χ2 = 38.08; p<0.000). The sensitivity and specificity of conventional PCR in relation to qPCR was 50% and 96.6%, respectively. Based on these results, it was possible to conclude that the IS900 region of MAP is present in bovine milk in the micro-region of Garanhuns, Pernambuco. It was also possible to conclude that the qPCR technique used in the present study was more sensitive than conventional PCR in terms of detecting these bacteria in milk samples. / Objetivou-se com este estudo detectar a região IS900 do MAP em amostras de leite bovino utilizando-se as técnicas de Reação em Cadeia da Polimerase em Tempo Real (qPCR) e a PCR convencional, e realizar um estudo de concordância entre os testes utilizados. Foram coletadas 121 amostras de leite bovino de propriedades consideradas foco para o MAP pertencentes à microrregião de Garanhuns, Pernambuco, Brasil. Das 121 amostras de leite analisadas foi possível detectar o DNA do MAP em 20 (16,52%) amostras na PCR convencional e em 34 (28,09%) na qPCR. O DNA do agente foi detectado em todas as seis propriedades estudadas, variando de 10,00% a 23,81% na PCR convencional e de 10,00% a 36,84% na qPCR. A concordância entre a qPCR e PCR foi moderada (Kappa =0,53; χ2 = 38,08; p<0,000) e a sensibilidade e especificidade da PCR convencional com relação a qPCR foi de 50% e 96,6%, respectivamente. Conclui-se que o DNA da região IS900 do MAP está presente no leite bovino na região estudada e que a qPCR é uma técnica sensível e rápida para detecção desse agente em amostras de leite.

Mycobacterium avium ssp. paratuberculosis (MAP) est l’agent pathogène responsable de la maladie de Johne qui est aussi connue sous le nom de paratuberculose. Cette maladie affecte principalement les ruminants, mais également d’autres animaux d’élevages et sauvages. Les mécanismes de pathogenèse de la paratuberculose sont toujours peu compris, mais il est connu que MAP est capable de s’évader du système immunitaire de son hôte pendant plusieurs années en utilisant les macrophages comme réservoir principal. L’effet de MAP dans l’infection des macrophages est souvent étudié en utilisant des vaches saines ou encore infectées de façon expérimentale, mais les études sur l’effet à long terme de la paratuberculose en étudiant les macrophages de vaches atteintes sont manquantes. Dans mon projet, le transcriptome de macrophages primaires dérivés des monocytes circulants infectés in vitro par MAP a été analysé par séquençage haut débit de l’ARN (RNA-seq). Des différences importantes dans les interactions hôtes-pathogènes ont été observées dans les signatures d’expression génique des vaches diagnostiquées comme négatives ou positives. Ces modifications mettent en lumière la mise en place de processus durables dans l’établissement de la paratuberculose. Les modifications clés observées lors de mon projet sont un débalancement immunométabolique, un changement dans l’homéostasie du cholestérol et des lipides intracellulaires, ainsi qu’un patron d’expression génique associé à l’établissement d’une tolérance mémoire chez les macrophages. En effet, les résultats obtenus lors de ce projet consolident l’hypothèse selon laquelle MAP pourrait induire un état de type tolérance dans les monocytes sanguins périphériques circulants lorsqu’ils se différencient en macrophages. De ce fait, MAP pourrait promouvoir davantage la persistance de la maladie en influençant le comportement des macrophages à long terme. Cette étude contribue à une meilleure compréhension du contrôle de MAP sur les cellules immunitaires et de ses mécanismes de survie.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-11-18T13:53:39Z No. of bitstreams: 1 texto completo.pdf: 1408584 bytes, checksum: 93c92bfb4c0654bc0272f8ef82b674ea (MD5) / Made available in DSpace on 2015-11-18T13:53:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1408584 bytes, checksum: 93c92bfb4c0654bc0272f8ef82b674ea (MD5) Previous issue date: 2015-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mycobacterium avium ssp. paratuberculosis (MAP) é o agente etiológico da doença de Johne ou paratuberculose, enterite granulomatosa crônica caracterizada por diarreia persistente e perda de peso progressiva que acomete ruminantes. Pode também ser isolado a partir de amostras intestinais de pacientes humanos, com doenças intestinais, principalmente portadores da doença de Crohn. Essa é uma doença de etiologia desconhecida, que se caracteriza por inflamação crônica, focal, assimétrica transmural e ocasionalmente granuloma- tosa, que pode acometer qualquer segmento do tubo digestivo. O isolamento de MAP e as semelhanças entre os processos clínicos e histopatológicos da paratuberculose e da doença de Crohn são algumas das razões pelas quais se investiga o potencial zoonótico da bactéria. As principais vias de eliminação do agente são através do leite e das fezes que contaminam os pastos e, direta ou indiretamente os cursos d’agua podendo dessa forma infectar humanos pela ingestão de água contendo o micro-organismo viável. MAP é uma bactéria resistente e responsável por grandes prejuízos econômicos e produtivos, sendo demonstrada sua sobrevivência no ambiente por longos períodos, além da resistência a pasteurização e à agentes de desinfecção aplicados ao tratamento da água para consumo humano. Diante disso, o estudo teve como objetivos: - identificar fatores de risco envolvidos com a presença de MAP em amostras de intestino humano, através da aplicação de questionário, em conjunto com da- dos prévios sobre a presença da bactéria em amostras de biópsias intestinais de pacientes portadores de Doença de Crohn, retocolite ulcerativa e portadores de doenças intestinais não inflamatórias, -verificar a presença do agente na água para consumo humano e animal através de PCR convencional e do cultivo microbiológico de amostras coletados em 10 propriedades de caprinocultura leiteira da Zona da Mata Mineira e realizar uma revisão bibliográfica dos estudos sobre a paratuberculose na América Latina. Quanto às amostras de água, MAP foi identificado viável em quatro (40%) das amostras de consumo animal, e identificado por PCR em três (30%) das de consumo humano além de uma quinta amostra de consumo animal (10%). Esse resultado demonstra o papel da água como reservatório do agente, mantendo o ciclo de infecção ativo e servindo de amostra confiável para o diagnóstico da presença do agente no rebanho já que, aparentemente não está condicionada a eliminação intermitente, como ocorre com as fezes desses animais. Nesse estudo também puderam ser identificados fatores de risco para a ocorrência do agente na água e em biópsias intestinais humanas, como o consumo de leite e derivados informais, assim como histórico familiar de agravos intestinais. Na América Latina MAP foi pesquisado em 10 países e identificado em nove, infectando, bovinos, caprinos e animais silvestres. Os resultados desse estudo contribuem para a identificação de fatores de risco envolvidos na transmissão de MAP para humanos, permitindo a sugestão de medidas que previnam ou reduzam a exposição ao agente. Esses fatores de risco identificados também demonstram a importância do leite na veiculação do agente por leite e produtos lácteos, com destaque para aqueles que não passaram pela pasteurização. Além disso, os estudos sobre água auxiliaram a elucidação do papel da ingestão da água na transmissão do agente, que não é pesquisado na rotina de tratamento, indicando exposição ao agente para humanos pode ser dar através do consumo de água contaminada por fezes de animais com paratuberculose. Esse estudo foi o primeiro sobre o agente na água no Brasil, e um dos poucos no mundo. / Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease or paratuberculosis, a chronic granulomatous enteritis characterized by persistent diarrhea and progressive weight loss that may affects ruminants. MAP is also be isolated from intestinal samples from human patients with intestinal diseases, particularly Crohn's disease patients. This is a disease of unknown etiology that is characterized by chronic inflammation, focal, transmural asymmetric and occasionally granulomatous lesions, which can affect any segment of the digestive tract. The isolation of MAP and the similarities between the clinical and pathologic processes of paratuberculosis and Crohn's disease are some of the reasons for investigating the zoonotic potential of bacteria. The main agent’s disposal routes are through feces and milk that contaminate pastures and directly or indirectly the watercourses and can thus infect humans by drinking water containing viable microorganism. MAP is a resistant bacteria and responsible for significant economic and productive loss, and demonstrated its survival in the environment for long periods, in addition to the resistance to pasteurization and disinfection agents applied to water treatment for human consumption. Thus, the study aimed to: - identify risk factors involved with the presence of MAP in human gut samples, through the questionnaire, together with previous data on the presence of bacteria in samples of intestinal biopsies of patients Crohn's disease, ulcerative colitis and patients with non- inflammatory intestinal diseases, -check the agent's presence in the water for human and animal consumption by conventional PCR and microbiological culture samples collected in 10 properties of dairy goat of the Zona da Mata Mineira and,- conduct a literature review of the studies on paratuberculosis in Latin America. As for the water samples, MAP was identified feasible in four (40%) of the samples of animal feed, and identified by PCR in three (30%) of human consumption as well as a fifth sample of animal consumption (10%). This result demonstrates the role of water as an agent of the reservoir, keeping the active infection cycle and serving as a reliable sample for the diagnosis of the agent's presence in the herd since apparently is not subject to intermittent shedding, as with the feces of these animals. In this study could also be identified risk factors for the occurrence of the agent in in human intestinal biopsies, as the consumption of unpasteurized milk and informal derivatives as well as family history of bowel diseases. In Latin America MAP was investigated in 10 countries and identified in nine, infecting, cattle, goats and wild animals. The results of this study contribute to the identification of risk factors involved in the MAP transmission to humans, allowing the suggestion of measures to prevent or reduce exposure. These identified risk factors also demonstrate the importance of milk in placement agent for milk and milk products, especially those who have not gone through the pasteurization. Furthermore, studies on water helped to elucidate the role of water ingestion in the transmission of the agent, which is not searched in routine treatment, indicating exposure to the agent for humans can occur through the consumption of water contaminated by faeces of animals carrying paratuberculosis. This study was the first about the agent in water in Brazil, and one of the few in the world.

Mycobacterium avium ssp. paratuberculosis (MAP) est l'agent causal de la paratuberculose, maladie entérique, chronique et incurable des ruminants, avec un impact économique important. Une meilleure compréhension des facteurs de risque associés à l'introduction de la maladie dans un troupeau est essentielle pour sa prévention. L’amélioration des tests diagnostiques est aussi importante pour son contrôle. L’introduction des nouveaux animaux dans le troupeau et la présence et contact des différentes espèces sauvages et domestiques avec les vaches, semblent être des facteurs de risques d’introduction de la maladie. Nous avons réalisé une revue systématique dont l`objective était de recueillir l’information pertinente pour répondre à la question sur l’importance de ces facteurs et leur impact sur l’introduction de la maladie dans un troupeau. D`un autre côté, la détection de MAP dans les fèces par culture bactérienne demeure la méthode diagnostique de choix malgré les facteurs qui l`affectent. Une série de 3 étapes est requise afin de confirmer la présence du MAP : (1) culture (2) coloration, et (3) confirmation du MAP par PCR (si détecté à l´étape 2). Certains échantillons fécaux présentent une particularité en raison de leur forte charge de micro-organismes. Ces contaminants peuvent interférer avec la croissance et la détection de MAP. Une étude visant à : a) estimer l'impact des certain covariables sur les résultats de la culture de MAP parmi l`analyse rétrospective d`un banque des données et b) évaluer la possibilité d'optimiser le processus de diagnostic du MAP en effectuant l'analyse PCR sur les cultures déclarées comme contaminées a été réalisée. / Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis an enteric, chronic and incurable disease of ruminants, with a major economic impact. A better understanding of the risk factors associated with the introduction of the disease in a herd, is essential for its prevention. Improving diagnostic tests are also important for the control. The introduction of new animals into the herd and the presence and contact of wild and domestic species with cattle, appear to be a risk factor for disease introduction. We conducted a systematic review to obtain relevant information to answer the question about the importance and impact of these factors on the introduction of the disease into a herd. On the other hand, the detection of MAP in feces by bacterial culture remains the diagnostic method of choice despite of the factors that can affect it. A series of 3 steps are required to confirm the presence of MAP: (1) culture (2) acid fast stain (3) MAP confirmation by PCR (if detected in step 2). Some fecal samples exhibit a particularity due to their normal heavy load of micro-organisms. These contaminants can interfere with the growth and detection of MAP. A study to: a) assess the impact of some covariables on the culture results by a retrospective analysis of a databank and b) to evaluate the possibility to optimize MAP diagnostic process by performing a PCR analysis on cultures declared as contaminated was realized.

La paratuberculosis (PTB) est une maladie endémique des ruminants causée par Mycobacterium avium subsp. paratuberculosis (Map). Les stratégies de maîtrise actuelles ne sont pas suffisamment efficaces. La réponse à l'exposition à Map varie entre les animaux avec une part de déterminisme génétique. Des marqueurs génétiques pourraient permettre une sélection. L'objectif était d'évaluer par modélisation l'efficacité potentielle attendue de stratégies de maîtrise utilisant la sélection génétique ou la réduction de l'exposition en élevage. Nous avons identifié quatre traits phénotypiques de résistance influençant principalement la propagation de Map à l'échelle du troupeau et montré la valeur ajoutée de leur amélioration simultanée. Nous avons évalué l'effet de l'environnement du troupeau et du système d’élevage sur la propagation et la maîtrise de Map. Nous avons montré une différence d’efficacité des stratégies de maîtrise les plus pertinentes entre deux systèmes d'élevage bovins laitiers contrastés d'Europe: l'ouest de la France et l'Irlande. Nous avons évalué l'efficacité que pourrait apporter la sélection génomique en évaluant le temps nécessaire pour atteindre des niveaux de variation des traits sélectionnés permettant un bon contrôle de l‘infection sous l’hypothèse que des marqueurs de sélection soient disponibles. Nous avons identifié 2 paramètres du modèle de sélection génomique influents sur l’efficacité de la sélection. Notre modèle permet d’intégrer de nouvelles connaissances biologiques sur le déterminisme génétique de la résistance à Map pour évaluer des stratégies de maîtrise complexes comprenant une composante de sélection génomique. / Paratuberculosis (PTB) is an endemic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Current control strategies are not effective enough. The response to Map exposure varies between animals with evidence of a partial genetic determinism. Genetic markers could allow selection. The objective was to assess the potential expected effectiveness of control strategies relying on genetic selection or reduction of exposure in herds, using a modelling approach. We identified four phenotypic traits of resistance mainly influencing the spread of Map at the herd scale and showed the added value of their simultaneous improvement. We evaluated the effect of the herd environment and management on the spread and control of Map. We showed a difference in effectiveness of the most relevant control strategies between two contrasting dairy cattle systems in Europe: western France and Ireland. We evaluated the effectiveness of genomic selection by assessing the time required to reach levels of variation in the selected traits allowing to achieve a good control of infection, assuming that associated genomic markers could be available. Effectiveness of selection was mainly influenced by 2 of the parameters of the developed genomic selection model. Our model allows to account for future knowledge about the genetic determinism of cattle resistance to Map in order to assess the effectiveness of complex control strategies including a genomic selection component.

Mycobacterium avium ssp. paratuberculosis (MAP) est l'agent causal de la paratuberculose, maladie entérique, chronique et incurable des ruminants, avec un impact économique important. Une meilleure compréhension des facteurs de risque associés à l'introduction de la maladie dans un troupeau est essentielle pour sa prévention. L’amélioration des tests diagnostiques est aussi importante pour son contrôle. L’introduction des nouveaux animaux dans le troupeau et la présence et contact des différentes espèces sauvages et domestiques avec les vaches, semblent être des facteurs de risques d’introduction de la maladie. Nous avons réalisé une revue systématique dont l`objective était de recueillir l’information pertinente pour répondre à la question sur l’importance de ces facteurs et leur impact sur l’introduction de la maladie dans un troupeau. D`un autre côté, la détection de MAP dans les fèces par culture bactérienne demeure la méthode diagnostique de choix malgré les facteurs qui l`affectent. Une série de 3 étapes est requise afin de confirmer la présence du MAP : (1) culture (2) coloration, et (3) confirmation du MAP par PCR (si détecté à l´étape 2). Certains échantillons fécaux présentent une particularité en raison de leur forte charge de micro-organismes. Ces contaminants peuvent interférer avec la croissance et la détection de MAP. Une étude visant à : a) estimer l'impact des certain covariables sur les résultats de la culture de MAP parmi l`analyse rétrospective d`un banque des données et b) évaluer la possibilité d'optimiser le processus de diagnostic du MAP en effectuant l'analyse PCR sur les cultures déclarées comme contaminées a été réalisée. / Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis an enteric, chronic and incurable disease of ruminants, with a major economic impact. A better understanding of the risk factors associated with the introduction of the disease in a herd, is essential for its prevention. Improving diagnostic tests are also important for the control. The introduction of new animals into the herd and the presence and contact of wild and domestic species with cattle, appear to be a risk factor for disease introduction. We conducted a systematic review to obtain relevant information to answer the question about the importance and impact of these factors on the introduction of the disease into a herd. On the other hand, the detection of MAP in feces by bacterial culture remains the diagnostic method of choice despite of the factors that can affect it. A series of 3 steps are required to confirm the presence of MAP: (1) culture (2) acid fast stain (3) MAP confirmation by PCR (if detected in step 2). Some fecal samples exhibit a particularity due to their normal heavy load of micro-organisms. These contaminants can interfere with the growth and detection of MAP. A study to: a) assess the impact of some covariables on the culture results by a retrospective analysis of a databank and b) to evaluate the possibility to optimize MAP diagnostic process by performing a PCR analysis on cultures declared as contaminated was realized.

Made available in DSpace on 2015-03-26T13:47:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1625922 bytes, checksum: 8e4e59f3e5a2481a04957bd2da9aa918 (MD5) Previous issue date: 2012-09-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease which causes chronic granulomatous enteritis preferably in domestic and wild ruminants. It is considered a disease of economic and social impact, because of losses in livestock and importance in human health, since there is evidence of possible correlation of MAP with Crohn's disease. However, much is still unknown about the developing the pathophysiology and immunopathogenesis this disease, in the sites of infection. Thus, this study has proposed verify the response of cytokines proinflammatory and anti-inflammatory by quantitative PCR and associate the expression of these cytokines with the lesions and the presence of the agent in organs of C57BL/6 mice infected intraperitoneal with strain MAP66115-98. It was applied 250&#956;l of inoculum at 3 x 108 UFC/ml in 20 mice, and it was applied saline phosphate buffers (PBS) in eight control species. The animals were sacrificed at 30, 60, 90 and 120 days post-inoculation. In each period were collected spleen, liver, intestine (ileum and colon) and Peyer's patches of five challenged mice and two controls. It was found in the majority an upregulation in the expression profiles of proinflammatory cytokines IL-2, IFN-, and TNF-&#945; &#1199;, and anti-inflammatory IL-4 and IL-10 analyzed in the organs compared to controls during period of the experiment. It was observed also negative regulations in the fourth week in spleen and colon, twelfth in the spleen, ileum and Peyer's patch and the sixteenth week in the liver, colon and ileum. Statistical differences were only observed at the eighth week after the challenge for IFN-&#1199; in liver, in spleen for expression of IL-2 and IFN-&#1199; and in the ileum for IL-4 and TNF-&#945;. In the fourth week, it was possible to identify increased levels of mRNA transcripts of the cytokines studied. In the liver, ileum and colon IFN-&#1199; showed the highest level of mRNA transcripts compared to the negative control, however at higher levels spleen were observed for IL- 10. Only eight weeks it was possible to observe a statistically significant difference for the cytokine IFN-&#1199; in the liver and spleen samples for TNF-&#945; and IL-4 in the ileum and IL-2 in spleen. The highest levels of mRNA transcript were observed in the liver for IFN- &#1199;, spleen for IL-2, and in the ileum Peyer's patch for IL-4 and colon for IL-10. In the twelfth week, the major mRNA transcript levels were observed in liver for IL-10 in spleen and ileum for IL-2 in colon for TNF-&#945; and Peyer's patch for IL-4. In the sixteenth week, the major mRNA transcript levels were observed in liver and colon for IL-10 in spleen and ileum for IFN-&#1199; and Peyer's patch for TNF-&#945;. The animals showed increased expression of mRNA transcripts of cytokines studied organs after challenge with MAP, although not verified gross and microscopic changes (hematoxylin and eosin staining). However it was possible to observe the presence of alcohol-acid resistant rods in two samples of Peyer's patches (Ziehl-Neelsen) and nested-PCR technique was possible to observe the presence of MAP in every organ analyzed. Thus, these results contribute to a better understanding of the expression of proinflammatory and anti-inflammatory in local infection at different post-inoculation in both organs taken as site specific as MAP intestine (colon and ileum) and Peyer's patch and organs extraintestinal, and thus find a better understanding of the immunopathogenesis of infection in the local. / Mycobacterium avium subespécie paratuberculosis (MAP) é o agente etiológico da paratuberculose, uma enfermidade que causa enterite granulomatosa crônica preferencialmente em ruminantes domésticos e silvestres. É considerada uma doença de impacto econômico e social, devido às perdas no rebanho e na saúde humana, uma vez que se têm indícios da possível relação de MAP com a doença de Crohn. Contudo, muito ainda se desconhece sobre o desenvolvimento da patofisiologia e imunopatogênese desta doença, nos locais da infecção. Desse modo, este estudo propôs verificar a resposta de citocinas pró e anti-inflamatórias por meio da técnica de PCR quantitativo e associar a expressão destas citocinas com as lesões e a presença do agente em órgãos de camundongos C57BL/6 infectados por via intraperitoneal com a cepa MAP66115-98. Em 20 camundongos foram aplicados 250&#956;l de inóculo a 3 x 10 8 UFC/ml e, em oito controles foram aplicados PBS. Os animais foram sacrificados aos 30, 60, 90 e 120 dias pós-inoculação. Em cada período, foram coletado baço, fígado, intestino (cólon e íleo) e placas de Peyer de cinco camundongos desafiados e de dois controles. Verificou-se na maioria, uma regulação positiva nos perfis de expressões das citocinas pró-inflamatórias IL-2, IFN-&#1199; e TNF-&#945;, e anti-inflamatórias IL-4 e IL-10 nos órgãos analisados em relação aos controles durante o período do experimento. Observaram-se também regulações negativas na quarta semana no baço e colón, na décima segunda no baço, íleo e placa de Peyer e na décima sexta semana no fígado, colón e íleo. Diferenças estatísticas foram observadas somente na oitava semana após o desafio no fígado para IFN-&#1199;, no baço para expressão da IL-2 e IFN-&#1199; e no íleo para IL-4 e TNF-&#945;. Na quarta semana, foi possível identificar aumento dos níveis de transcritos de RNAm das citocinas estudadas. No fígado, íleo e colón o IFN-&#1199; apresentou maior nível de transcritos de RNAm em relação ao controle negativo, no entanto no baço os maiores níveis foram observados para a IL-10. Somente na oitava semana foi possível observar diferença estatística significativa para a citocina IFN-&#1199;, em amostras do fígado e baço, para o TNF-&#945; e IL-4 no íleo e para a IL-2 no baço. Os maiores níveis de transcrito de RNAm foram observados no fígado, para o IFN-&#1199; , no baço para a IL-2 , no íleo e placa de Peyer para a IL-4 e no colón para a IL-10. Na décima segunda semana, os maiores níveis de transcrito de RNAm foram observados no fígado para a IL-10, no baço e íleo para a IL-2, no colón para o TNF-&#945; e na placa de Peyer para a IL-4. Na décima sexta semana, os maiores níveis de transcrito de RNAm foram observados no fígado e colón para a IL-10, no baço e íleo para o IFN-&#1199; e na placa de Peyer para o TNF-&#945;. Os animais apresentaram uma maior expressão dos transcritos de RNAm das citocinas estudadas nos órgãos após o desafio com MAP, embora não tenham sido verificadas alterações macro e microscópica (coloração de Hematoxilina e Eosina). No entanto foi possível observar presença de bastonetes álcool ácido resistentes em duas amostras de placas de Peyer, (coloração de Ziehl-Neelsen) e pela técnica de nested-PCR foi possível observar a presença de MAP em todos os órgão analisados. Assim, estes resultados contribuem para a melhor compreensão da expressão de citocinas pró e anti-inflamatória nos locais da infecção em diferentes tempos pós-inoculação tanto em órgãos tidos como sitio específicos de MAP como intestino (colón e íleo) e placa de Peyer e órgãos extra-intestinais, e com isso buscar o melhor entendimento da imunopatogênese nos locais da infecção.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-02-03T21:22:32Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-04T14:47:40Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) / Made available in DSpace on 2014-02-04T14:47:40Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) Previous issue date: 2005 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / 514 amostras de sangue bovino foram analisadas para detecção de anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis, utilizando um kit comercial do teste ELISA indireto. Os animais eram todos mestiços, machos e fêmeas, classificados em dois grupos, de acordo com a idade, menores ou iguais a 36 meses e maiores de 36 meses, provenientes de 23 municípios do Estado do Pará. Além deste teste, também foram colhidas 100 amostras de fragmentos de intestino delgado e de linfonodo mesentérico para análise bacteriológicaatravés da coloração de Ziehl-Neelsen, a fim de identificar o Mycobacterium avium (subsp.) paratuberculosis. As amostras foram colhidas aleatoriamente em matadouro da região metropolitana de Belém. No teste ELISA indireto, das 514 amostras de sangue bovino 182 (35,4%) foram reagentes para anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis e 332 (64,6%) foram não-reagentes. Em todas as mesorregiões encontraramse animais soropositivos. As fêmeas com idade superior a 36 meses mostraram maior respostas ao teste , 92,06% de animais soropositivos. Entre os machos, a maior prevalência (76,79%), foi obtida nos mais novos, com idade abaixo de 36 meses. Das 100 lâminas coradas através do método de Ziehl-Neelsen, nenhum Mycobacterium sp. foi identificado. Conclui-se assim, que o elevado número de animais com anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis indica a presença da doença em todas as mesorregiões estudadas, devendo-se atentar aos riscos desta enfermidade, através de pesquisas mais abrangentes sobre a Paratuberculose, com aplicação de métodos diagnósticos diretos e indiretos. / 514 blood samples from beef cattle were examined of to find anti- M. avium (subsp.) paratuberculosis antibodies, using commercial ELISA test kit. The animals from samples were all compous from cross between Indicus x Taurus race, between male and female, with two age class, over 36 months old and below 36 months old, from of the 23 municipality of Para State. Also were collected 100 samples of small intestine and mesenteric lymph nodes of bacteriological test through Ziehl-Neelsen method to detect the M. avium (subsp.) paratuberculosis. Were at random collected in slaughterhouse of Belem. In ELISA test, through these 514 samples, 182 (35,4%) were seropositive and 332 (64,6%) samples were seronegative. All the middle region were found soropositives animals. The female with over 36 months old presented larger response test, of the 92,06% seropositives animals. Among male the prevalency larger (76,79%,) were obtained between the most new, with below 36 months old. Of 100 samples stained through Ziehl-Neelsen method, not were found either Mycobacterium spp.. The conclusion, is the biggest number of seropositive animals of anti- M. avium (subsp.) paratuberculosis sign of disease is presented in all the regions studied, watching the risks of this disease with the elaborate more covering stud ies about the paratuberculosis with the appling of directs and indirects diagnosis methods.