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 Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-03-14T18:51:10Z No. of bitstreams: 1 texto completo.pdf: 943385 bytes, checksum: 8adee72c3a2e9d49512f689910c76c69 (MD5) / Made available in DSpace on 2017-03-14T18:51:10Z (GMT). No. of bitstreams: 1 texto completo.pdf: 943385 bytes, checksum: 8adee72c3a2e9d49512f689910c76c69 (MD5) Previous issue date: 2016-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mastite causada por Escherichia coli tem a capacidade de estimular intensamente o sistema imunológico e desencadear rapida inflamação na glândula mamária. Em contraste, Mycobacterium avium subsp. paratuberculosis (MAP), agente etiológico da paratuberculose, caracterizada por enterite granulomatosa crônica, pode infectar a glândula mamária sem estimular intensamente a resposta inflamatória. A interação dessas duas bactérias na glândula mamária ainda é desconhecida. Em alguns casos, tanto a eliminação de MAP pelo leite como pelas fezes podem ocorrer de forma passiva, após a infecção ascendente da glândula mamária ou após ingestão de MAP, respectivamente. Estes animais, chamados passive-shedders, são importantes como uma fonte de infecção a animais suscetíveis no rebanho. O objetivo deste estudo foi investigar a relação entre MAP e E. coli em células de glândula mamária sob condições experimentais e verificar a presença de animais passive-shedders. A relação entre uma cepa K-10 de MAP e E. coli isolada de leite mastítico em linhagem de células epiteliais mamárias (MAC-T) foi avaliada. As células previamente infectadas com MAP diminuíram a invasão de E. coli durante 120 min de experimentação. Contudo, a eficiência da translocação de E. coli e a viabilidade das células MAC-T não foram comprometidas. Ao contrário, células previamente infectadas por E. coli aumentaram a capacidade de translocação baso-apical de MAP até os 30 min e diminuiu aos 120min pós-infecção. A quantificação de citocinas relevou que a expressão de IL-1β aos 120min foi significativa (P<0.05) para células infectadas por MAP + E. coli e E. coli apenas. As expressões de MAPKp 38 e IL-10 não foram significativas, independente do tempo pós-infecção. Para detectar a ocorrência de animais passive-shedders, 10 propriedades foram previamente investigadas para a presença de MAP. Treze cabras foram positivas por cultura de fezes e/ou PCR de leite. Dentre os animais positivos, quatro (4/13) foram adquiridas e avaliados por IS900-PCR, cultura de fezes, de leite e de tecido, e sorologia (ELISA). Todos os resultados foram negativos no período de um ano, demonstrando que os animais realizaram o fenômeno pass-through e a contaminação ascendente da glândula mamária, sem tornarem-se infectados. No geral, esses resultados indicam que a presença de MAP nas células mamárias pode dificultar a capacidade de invasão de E. coli, mas quando no interior da célula mamária, translocam-se mais eficientemente. No entanto, quando as células são previamente infectadas por E. coli, MAP é rapidamente atraído da região subepitelial para a superfície celular. A produção de IL-1β intensifica a atração de macrófagos para o sítio de infecção, onde MAP se beneficia, infectando-os. / Mastitis caused by Escherichia coli can intensely stimulate the immune system and rapidly trigger inflammation in the mammary gland. In contrast, Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, characterized by chronic granulomatous enteritis, can infect the mammary gland without intensely stimulating the inflammatory response. The interaction of these two species in the mammary gland is still unknown. In some cases, both the elimination of MAP by milk and faeces may occur passively, either through ascending infection of the mammary gland or through ingestion of MAP, respectively. These animals are called passive- shedders and are important as a source of infection to susceptible animals in the herd. The aim of this study was to investigate the relationship between MAP and E. coli in mammary gland cells under experimental conditions and verify the presence of passive- shedder animals. The relationship between a K-10 strain of MAP and E. coli isolated from mastitic milk in mammary epithelial cell lines was evaluated. Cells previously infected by MAP decreased E. coli invasiveness during 120min experimentation. However, the efficiency of E. coli translocation was not compromised, nor was the viability of the MAC-T cells. In contrast, cells previously infected by E. coli showed increased basal-apical translocation capacity of MAP up to 30 min and decreased at 120 min postinfection. Quantification of cytokines showed that IL-1β expression at 120 min was significantly increased in cells infected by MAP + E. coli and E. coli only. Expression of MAPKp 38 and IL-10 were not significant, regardless of time postinfection. To determine the occurrence of passive-shedders, 10 properties were previously investigated for MAP detection. Thirteen goats were positive by faeces culture and/or milk PCR. Among the positive animals, four (4/13) were evaluated by IS900-PCR, feces culture, milk and tissue culture and serology (ELISA). All the results were negative over a one-year period, demonstrating that the animals performed pass- through phenomenon and upward contamination of the mammary gland without becoming infected. Together, these results indicate that the presence of MAP in mammary cells may hamper capacity of E. coli invasion, but when within the mammary cell, the bacteria evade more efficiently. However, when the cells are pre-infected by E. coli, MAP is rapidly attracted from the subepithelial region to the cell surface. IL-1β production enhances the attraction of macrophages to the site of infection, where MAP benefits by infecting them.

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.

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.

The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.

To propose a solution for control of Mycobacterium avium subsp. paratuberculosis (MAP) infections in animals as well as in humans, and develop effective prevention, diagnostic and treatment strategies, it is essential to understand the molecular mechanisms of MAP pathogenesis. In the present review, we discuss the mechanisms utilised by MAP to overcome the host defense system to achieve the virulence status. Putative MAP virulence genes are mentioned and their probable roles in view of other mycobacteria are discussed. This review provides information on MAP strain diversity, putative MAP virulence factors and highlights the knowledge gaps regarding MAP virulence mechanisms that may be important in control and prevention of paratuberculosis.

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease mainly of domestic and wild ruminants. It occurs worldwide, causing significant economic losses through decreased productivity, low fertility, increased cull rates and mortality. It is listed by the OIE (World Organization for Animal Health) as a disease of concern to trade in animals. Prevalence of this disease can be studied by detecting anti-MAP antibodies by Enzyme linked immunosorbent Assay (ELISA). The aim of this study was to investigate the current prevalence of MAP infection in cattle in Khartoum State. The overall apparent prevalence of MAP infection was found to be 6.3% and 18.9% at animal and herd levels, respectively. All seropositive animals were cross-bred females of good body condition; most of them (>90%) were >3 years old and >50% were from medium-sized herds in Omdurman. No significant association (p > 0.05) was found between seropositivity and animal herd size. The prevalence of MAP infection in Khartoum State is still low to medium compared to other parts of the world, but it is comparable to those reported from other African countries. Further studies with the view of designing nationwide surveys in domestic ruminants and camels in other states of the country are needed for establishing control programmes.