No description available.

BACKGROUND:Bovine tuberculosis (TB) caused by Mycobacterium bovis is primarily a disease of ruminants, particularly cattle (Bos primigenius) and buffalo (Syncerus caffer), and is endemic in most developing countries. To date, studies done in Uganda have documented the prevalence of M. bovis in cattle, humans and wild life, in addition to non-tuberculous mycobacteria in pigs. Pigs are increasingly becoming an important component of the livestock sector and share the human ecosystem in rural Uganda. It is therefore of public health interest that they are not a source of human infections. As a follow up to previously published findings on mycobacteria in pigs, this study was aimed at investigating the occurrence and molecular characteristics of M. bovis detected in slaughter pigs in Mubende district, Uganda. One hundred fifty mesenteric lymph nodes with lesions suggestive of mycobacterial infections were collected from approximately one thousand slaughtered pigs in Mubende district over a period of five months. The isolation and identification of M. bovis was done using conventional mycobacteriological methods. Mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC) were identified to species level using deletion analysis. Molecular typing was done using Spoligotyping and MIRU-VNTR analysis. Molecular data were analysed and interpreted using MIRU-VNTR plus, SpolDB4.0 and the Mycobacterium bovis spoligo database.RESULTS:Of the examined animals, one boar and two sows from Madudu Sub County were infected with M. bovis which presented as lesions of a deep yellow colour and a grit-like texture in the mesenteric lymph nodes. This represents 2% (3/150) of the lymph nodes where lesions suggestive of mycobacterial infections were detected. Molecular analysis revealed that the isolates from the infected pigs showed identical MIRU-VNTR profile and spoligotype (SB1469).CONCLUSIONS:This is the first study documenting the occurrence of M. bovis in slaughter pigs in Uganda, revealing that one in fifty slaughter pigs with suspected lesions in mesenteric lymph nodes were infected. Molecular analysis revealed that the isolates were identical, showing a spoligotype previously reported from humans and cattle in the north eastern part of the Uganda cattle corridor. This finding is of public health importance, therefore there is a need for close cooperation between medical and veterinary professionals in designing and implementing control and prevention measures that safeguard the public from this potential source of zoonotic TB in Uganda.

Bovine Tuberculosis is a chronic debilitating disease of cattle and other animals with a worldwide distribution and transmitted mainly through the inhalation of aerosols. The aim of this study was to determine the prevalence of BTB in the cattle population of selected dip tanks in Swaziland. Furthermore, the zoonotic risk to farmers whose cattle are infected with BTB was assessed by means of a questionnaire survey. Abattoir surveillance identified 16 dip tanks of study where at least 10 % of the cattle were tested for BTB using the comparative intra-dermal skin. In five of these dip tanks, the same cattle were tested for BTB using the IFN-γ Test. Eight BTB skin test positive animals were slaughtered and a detailed post mortem examination was conducted and samples collected for the isolation of M. bovis. Concurrent with BTB testing, a questionnaire survey was conducted to determine risk factors for humans. The prevalence of BTB was found to be 6.75 % in the study population and 20 % of BTB positive animals were diagnosed by both the CIST and IFN-γ, indicating a correlation for the test positive animals in the two tests. M. bovis was isolated from seven of the eight animals slaughtered. Farmers’ knowledge of BTB as a cattle disease and serious zoonosis is insufficient and inadequate while consumption practices of products of bovine origin exposes them to the risk of infection by M. bovis. There is a need to investigate the extent of M. bovis infections in the human population. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-03-02T15:00:57Z No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2239638 bytes, checksum: a1bfd99261016639937e0a5a3ee8fb49 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T01:36:07Z (GMT) No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2239638 bytes, checksum: a1bfd99261016639937e0a5a3ee8fb49 (MD5) / Made available in DSpace on 2016-04-24T01:36:07Z (GMT). No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2239638 bytes, checksum: a1bfd99261016639937e0a5a3ee8fb49 (MD5) Previous issue date: 2013-04-10 / A tuberculose (TB) é uma doença de importância mundial. Os bacilos que causam a tuberculose entram no organismo principalmente pelas vias aéreas e a interação inicial nos pulmões é com os macrófagos alveolares, que servem como células hospedeiras. A morte dos macrófagos infectados por apoptose constitui uma alternativa de defesa do hospedeiro capaz de remover o ambiente de suporte para o crescimento bacteriano. No entanto, cepas virulentas de micobactérias parecem ser capazes de modular este processo. Alguns estudos tem destacado a importância do fator de necrose tumoral alpha (TNF-α) na modulação da apoptose de macrófagos infectados. O TNF-a exerce suas atividades biológicas através de dois receptores de superfície celular, TNFR1 e TNFR2, cujos domínios extracelulares podem ser clivados por proteólise formando receptores solúveis (sTNFR-1 e sTNFR-2). A sinalização através do TNFR1 desencadeia a maioria das funções biológicas do TNF-a, resultando em sobrevivência ou morte celular, enquanto que o TNFR2 induz a sobrevivência da célula. O objetivo deste estudo foi avaliar a influência do TNF e seus receptores na modulação da apoptose de macrófagos alveolares durante a fase inicial de infecção por cepas atenuada e virulenta de Mycobacterium bovis, bem como, a possível interferência da micobactéria na sinalização TNF-TNFR. Para tal, camundongos C57BL/6 foram infectados, via intratraqueal, com as cepas atenuada (BCG Moreau) ou virulenta (ATCC19274) de M. bovis. Após 3 e 7 dias de infecção, os seguintes parâmetros foram avaliados: (1) expressão de receptores de TNF (TNFR1 e TNFR2) na superfície de macrófagos alveolares, (2) expressão gênica dos receptores TNFR1 e TNFR2, (3) níveis dos receptores solúveis sTNFR1 e sTNFR2 no lavado broncoalveolar e níveis de TNF-a no pulmão (4) freqüência da apoptose de macrófagos alveolares e (5) número de bacilos nos macrófagos alveolares. Além disso, o efeito do bloqueio da sinalização TNF-TNFR1 na modulação da apoptose de macrófagos foi avaliado em camundongos deficientes em TNFR1 infectados com M. bovis BCG. Um aumento significativo da apoptose e da expressão superficial de TNFR1 foram observadas em macrófagos alveolares 3 e 7 dias após a infecção com M. bovis atenuado, mas apenas no 7º dia de infecção com o M. bovis virulento. Baixa expressão superficial de TNFR1 e aumento dos níveis de sTNFR1 no 3º dia após a infecção pela cepa virulenta foram associados com reduzidas taxas de macrófagos apoptóticos. Além disso, uma redução significativa da apoptose de macrófagos alveolares foi observada em camundongos TNFR1-/- no 3º dia após a infecção com BCG. Estes resultados sugerem um papel potencial do TNFR1 na apoptose de macrófagos durante a infecção pela micobactéria. Neste contexto, a clivagem do TNFR1 parece contribuir para a modulação da apoptose de macrófagos. / Tuberculosis (TB) is a disease of worldwide importance. The tubercle bacilli enter the organism mainly via respiratory tract and initial interaction in the lungs is with the alveolar macrophages, which serve as host cells. Apoptosis of the infected macrophages constitutes a host defense alternative capable of removing the environment supporting bacterial growth. However, virulent strains of mycobacteria appear to be capable of modulate this process. Some studies have highlighted the importance of tumor necrosis factor-a (TNF-α) in the modulation of apoptosis of infected macrophages. TNF-a exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR-1 and sTNFR-2). The signaling through TNFR1 initiates the majority of the biological functions of TNF-a, leading to either survival or cell death, whereas TNFR2 induces survival. The aim of this study was to evaluate the influence of TNF-TNFR signaling in the modulation of apoptosis of alveolar macrophages during early stage of infection by virulent and attenuated strains of Mycobacterium bovis, as well as the possible interference of mycobacteria in this signaling. C57BL/6 mice were intratracheally infected with attenuated (BCG Moreau) or virulent (ATCC19274) strains of M. bovis. After 3 and 7 days of infection, the following parameters were assessed: (1) expression of TNF receptors (TNFR1 and TNFR2) on the surface of alveolar macrophages, (2) expression mRNA for TNF receptors, (3) levels of soluble receptors sTNFR1 e sTNFR2 in BAL and levels of TNF-a in lung (4) frequency of apoptosis alveolar macrophage and (5) number of bacilli in alveolar macrophages. Furthermore, the effect of abrogation of TNF-TNFR1 signaling in the modulation of macrophage apoptosis was assessed in TNFR1 deficient mice infected with M. bovis BCG. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated M. bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1-/- mice at day 3 after BCG infection. These results suggest a potential role of TNFR1 in macrophage apoptosis during infection by mycobacteria. In this context, the shedding of TNFR1 appears to contribute to the modulation of macrophage apoptosis.

Mycoplasma (M.) bovis est une bactérie pathogène des bovins, à l'origine de signes cliniques divers, comme des mammites, des arthrites, des otites et des bronchopneumonies, ces dernières étant majoritaires en France. Les mycoplasmoses à M. bovis ont un fort coût économique et leur contrôle impose une importante mobilisation sanitaire et un recours très fréquent à l'antibiothérapie. Peu de données étaient disponibles jusque récemment concernant le typage moléculaire et l'antibiosensibilité des souches françaises de M. bovis. Deux études antérieures à ce travail et réalisées au sein de l'UMR « Mycoplasmoses des ruminants » ont montré que les isolats cliniques de M. bovis collectés en France après 2000 appartiennent à un sous-type moléculaire majoritaire (ST2), très homogène et sont par ailleurs multirésistants à la plupart des familles antibiotiques à l'exception des fluoroquinolones. Ces résultats suggèrent la diffusion sur le territoire national d'un clone unique multirésistant. Le premier objectif de cette étude était de déterminer les mécanismes à la base de la perte de sensibilité aux antibiotiques des isolats français. Dans un deuxième temps, les liens entre les différents sous-types moléculaires, les profils d'antibiosensibilité, les maladies associées et le polymorphisme des gènes cibles des antibiotiques ont été investigués. Cette approche a été déployée pour trois familles d'antibiotiques utilisées en pratique vétérinaire: les macrolides, les tétracyclines et également les fluoroquinolones, quoique récemment classées comme molécules critiques. De façon générale, les mutations identifiées dans les cibles des antibiotiques expliquent à elles seules les phénotypes de résistance observés. Des mutations dans les ARNs ribosomaux, cibles des macrolides et des tétracyclines, ont été observées sur des isolats cliniques dès 1978 et sont devenues systématiques sur tous les isolats collectés après 2000 et appartenant au sous-type ST2 majoritaire. En ce qui concerne les fluoroquinolones, la faible augmentation des CMI (concentrations minimales inhibitrices) mesurée chez la plupart des isolats cliniques récents n'a pas été associée à des mutations des QRDR (« Quinolones Resistance-Determining Regions »). Par contre, des altérations cumulées de façon séquentielle dans ces QRDR, associées à une hausse des CMI, ont été mises en évidence lors d'expériences de sélection in vitro et majoritairement pour des souches appartenant à un sous-type récent minoritaire, ST3, apparemment plus variable et plus apte à fixer les mutations. En 2013, le premier isolat clinique présentant une CMI augmentée aux fluoroquinolones a été isolé: il appartient à ce sous-type ST3. L'ensemble des résultats obtenus montrent que les différents sous-types de M. bovis n'évoluent pas de la même façon vers la résistance. Ce constat ajouté à celui de la multirésistance des isolats récents (ST2 ou ST3) met en exergue l'intérêt de la surveillance (sous-typage et antibiosensibilité) et le suivi de l'évolution des isolats de M. bovis circulant en France. Ce suivi permettrait notamment d'anticiper une éventuelle émergence de la résistance aux fluoroquinolones / Mycoplasma (M.) bovis is a bacterial pathogen for cattle, responsible for various clinical signs, like mastitis, arthritis, otitis and respiratory diseases, the latter being the main syndrome present in France. Mycoplasmoses have a great economic impact and their control entails drastic sanitary measures and a frequent use of antibiotherapy. Few data was available until recently on the molecular subtyping and the antimicrobial susceptibility of the French strains of M. bovis. Two previous studies done in the UMR « Mycoplasmoses des ruminants » proved that clinical isolates collected in France after the year 2000 belonged to one major subtype (ST2), which is very homogeneous, and that they were multiresistant to the main antimicrobial families except fluoroquinolones. These results suggested the diffusion of one unique multiresistant clone on the national territory. The first aim of the present study was to decipher the molecular mechanisms underlying the loss of susceptibility to antimicrobials of the French strains. Secondly the links between the molecular subtypes, the antibiotics susceptibility profiles, the clinical origins and the polymorphisms of the target genes were assessed. This approach was used for 3 antimicrobial families currently used in veterinary medicine: macrolides, tetracyclines and fluoroquinolones, although recently classified as critical. Actually, the point mutations observed in the target genes of the antimicrobials accounted for the observed resistance phenotypes. Some mutations in the ribosomal RNAs, targets of the macrolides and the tetracyclines, were observed in clinical isolates as soon as 1978 and they were generalized in all isolates collected after 2000 and belonging to the major subtype ST2. Concerning the fluoroquinolones, the slight increase in MIC (Minimum Inhibitory Concentration) observed in most of the recent isolates was not associated with mutations in the QRDR (Quinolone Resistance-Determining Regions). However alterations that were associated with increased MICs were highlighted and proved to be sequentially cumulated during experiments of in vitro selection under antimicrobials pressure. This was mainly true for strains belonging to a recent and uncommon subtype, ST3, which is apparently more variable and more able to fix the mutations. In 2013 the first clinical strain showing an increased MIC to fluoroquinolones was isolated and proved to belong to ST3. The whole results of this study showed that the different subtypes did not evolve with the same speed towards resistance. This fact, associated with the multiresistant phenotype of the recent isolates (ST2 or ST3), highlights the urge to monitor (subtyping and antimicrobial susceptibility profiles) and to follow-up the evolution of the isolates of M. bovis circulating in France in order to anticipate a potential emergence of the resistance to fluoroquinolones

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T18:03:22Z No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:08:37Z (GMT) No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) / Made available in DSpace on 2016-10-22T13:08:37Z (GMT). No. of bitstreams: 1 michelefernandesrodrigues.pdf: 2462897 bytes, checksum: fecbbd695cdb846a7633c19e7fab1160 (MD5) Previous issue date: 2008-08-06 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Mycobacterium bovis é um membro do complexo Mycobacterium tuberculosis, um grupo de microorganismos com a capacidade de causar tuberculose em humanos. A característica marcante das micobactérias patogênicas é a sua habilidade para sobreviver e replicar dentro do fagossomo do macrófago. A apoptose dos macrófagos infectados pode ser uma alternativa de defesa do hospedeiro, capaz de eliminar o ambiente de suporte para o crescimento da micobactéria. No presente trabalho investigamos, in vivo, a influência da virulência e da carga bacteriana na ocorrência da apoptose de macrófagos durante a fase inicial da infecção pulmonar experimental por M. bovis. Camundongos BALB/c foram infectados intratraquealmente com alta ou baixa carga das cepas virulenta (ATCC 19274) e atenuada (BCG Moreau) de M. bovis. Após 3 e 7 dias de infecção, avaliamos a freqüência da apoptose dos macrófagos alveolares e sua correlação com o crescimento da micobactéria e com os níveis in situ das citocinas TNF-α, IL-10 e IL 12 e da proteína anti-apoptótica Bcl-2. Foi observado um aumento da apoptose de macrófagos após a infecção com ambas as cepas. Entretanto, a cepa virulenta induziu menos apoptose do que a cepa atenuada, sendo que, no 3o dia houve redução da apoptose na infecção com alta carga, enquanto que no 7o dia uma maior inibição ocorreu em baixa carga. Essa inibição da apoptose, promovida pela cepa virulenta, foi associada com o aumento da produção de IL-10, redução da produção de TNF-α e aumento da expressão de Bcl-2. Observamos ainda uma menor produção de IL-12 nos pontos de menor apoptose dos macrófagos o que reforça a hipótese de que a apoptose das células infectadas contribui para o desenvolvimento de imunidade específica contra o patógeno. Em conjunto esses dados sugerem que a cepa virulenta de M. bovis é capaz de modular a apoptose em um modo carga dependente de acordo com suas necessidades de crescimento intracelular e disseminação para células adjacentes. / Mycobacterium bovis is a member of the Mycobacterium tuberculosis complex, a group of organisms with the capacity to cause tuberculosis in humans. The hallmark of pathogenic mycobacteria is their ability to survive and replicate inside macrophage phagosome. The apoptosis of macrophages infected can be a host defense alternative capable of removing the environment supporting bacterial growth. In this work we investigate, in vivo, the influences of virulence and bacterial load on the apoptosis of macrophages during the initial phase of experimental pulmonary M. bovis infection. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) and attenuated (BCG Moreau) strains of M. bovis. The frequency of apoptosis and the growth of mycobacteria in macrophages from lung tissue, and the in situ levels of the cytokines TNF-α, IL-10 and IL-12 and of the anti apoptotic protein Bcl-2 were measured at day 3 and day 7 postinfection. An increase of macrophage apoptosis was observed after the infection with both strains, but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day of infection there was a reduction of apoptosis in the infection with high dose, whereas on the 7th day we found more inhibition in the lower dose. This inhibition of apoptosis was associated with the increased production of IL-10, reduced production of TNF-α and increases of Bcl-2. In addition, the production of IL-12 was reduced at the points of lowest apoptosis of macrophages, which suggests that the apoptosis of the infected cells contributes to the development of specific immune response against the pathogen. Our results indicate that the virulent strain of M. bovis is able to modulate apoptosis in a dose dependent fashion in accordance with its necessities for intracellular growth and dissemination to adjacent cells.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-11-01T14:25:20Z No. of bitstreams: 1 anamariadocarmo.pdf: 1166968 bytes, checksum: 902990ead7a531c64f6dd70ebcabe511 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-12-15T13:33:36Z (GMT) No. of bitstreams: 1 anamariadocarmo.pdf: 1166968 bytes, checksum: 902990ead7a531c64f6dd70ebcabe511 (MD5) / Made available in DSpace on 2016-12-15T13:33:36Z (GMT). No. of bitstreams: 1 anamariadocarmo.pdf: 1166968 bytes, checksum: 902990ead7a531c64f6dd70ebcabe511 (MD5) Previous issue date: 2008-02-28 / Sabe-se que existem inúmeros trabalhos envolvendo a modulação da resposta imune ao Mycobacterium. No entanto, o número de indivíduos apresentando tuberculose é cada vez maior. A resposta imune ao Mycobacterium é desencadeada principalmente por linfócitos Th1, com a produção de IFN-γ. As parasitoses intestinais também representam um importante problema médico-sanitário, tendo em vista o grande número de pessoas acometidas e as inúmeras alterações orgânicas que podem provocar no hospedeiro. Essas infecções helmínticas induzem preferencialmente uma resposta Th2 com a produção de IL-4, IL-5 e IL-13. Este trabalho avaliou a regulação da resposta imune no pulmão de camundongos co-infectados ou não por S. venezuelensis (SV) e/ou Mycobacterium bovis-BCG (MB), em dois pontos das duas infecções, denominados como ponto 1 (4° e 7° dia pós-imunização [dpi]) e ponto 2 (7° e 10° dpi) por MB e SV, respectivamente. Os animais foram infectados com 700 larvas de SV pela via subcutânea e, após 3 dias, com 1x106 UFC de MB cepa selvagem pela via intravenosa. Realizou-se a quantificação do número de ovos e vermes, a dosagem de citocinas (IFN-γ, IL-4 e IL-10) e quimiocinas (CCL2 e CCL5), o envolvimento de MPO e EPO, a detecção da infecção pelo MB por PCR, a avaliação histopatológica e a expressão de moléculas coestimulat órias/imunomodulatórias (CD80, CD86, CD28, CTLA-4 e CD25) em células ou tecidos do pulmão dos animais infectados e/ou co-infectados. Os resultados mostraram que a presença do MB favoreceu para o aumento do número de ovos e vermes do SV observados nos animais nos dias 4° e 7° (ponto 1) e 7° e 10° (ponto 2) após a infecção por MB e SV, respectivamente, nos animais co-infectados (COIN). A reação de PCR foi efetiva em detectar a presença do MB no pulmão dos animais. Foi observado um aumento de IFN-γ e uma diminuição de IL-4 e EPO no pulmão dos animais do grupo COIN, além de aumento na expressão da molécula co-estimulatória CD80 no ponto 1 e uma diminuição no ponto 2. Houve uma alta produção de IL-10 no pulmão dos animais dos grupos MB e COIN, sendo que a histopatologia neste sítio mostrou formação de granulomas com grande influxo de neutrófilos, macrófagos e células epitelóides na periferia nos pulmões dos animais do grupo MB e um granuloma bem mais avançado, com centro necrótico nos animais do grupo COIN. Baseado nesses resultados, conclui-se que o MB modula a infecção pelo SV, fazendo com que os animais fiquem mais suscetíveis à infecção helmíntica. Por outro lado, o SV modula a infecção pelo MB, fazendo com haja uma modificação na formação de granuloma no pulmão dos animais do grupo COIN no ponto 1 da infecção pelo MB, que poderia ser justificada pela diminuição de IL-4 nos animais do grupo COIN. / A rising number of people have been contracting tuberculosis around the world even though a multitude of reports involving a modulation of the immune response to Mycobacterium have been published. The response to Mycobacterium is mainly mediated by Th1 lymphocytes through IFN-gamma production. Parasitic diseases account for a large proportion of human morbidity and mortality, considering the number of people affected by them and several pathologies associated to parasitic infection. Helminthic infections drive towards Th2 response which leads to IL-4, IL5 and IL-13 production. The present study evaluated the immune response of coinfected animals or not with Strongyloides venezuelensis (SV) and Mycobacterium bovis-BCG (MB) on pulmonary cells collected from BALB/c mice at time points 1 (4th and 7th days post-immunization [dpi] by MB and SV, respectively) and 2 (7th and 10th dpi by MB and SV, respectively). Animals were infected with 700 SV larvae subcutaneously, and 3 days after, 1x106 CFU of wild MB strain intravenously. The number of worms and eggs was counted as well as cytokine (IFN-gamma, IL-4 and IL-10) and chemokine (CCL2 and CCL5) assessments, and the MPO and EPO levels determination on pulmonary tissue from infected and/or coinfected animals. In addition, PCR for MB detection, the histopathology and the expression of costimulatory molecules such as CD80, CD86, CD28, CTLA-4 and CD25 on pulmonary tissue were also assessed. The results pointed that MB led to increase SV parasite burden in coinfected mice (COIN) at both time points analyzed. The PCR technique detected effectively MB. Moreover, elevated IFN-gamma and reduced IL-4 and EPO levels were detected on pulmonary tissue in the COIN group. In regard to CD80 molecule, there was an increased expression at time point 1 and diminished expression at time point 2. Also, higher amounts of IL-10 were found on pulmonary tissue in MB and COIN groups. The histopathological analysis revealed pulmonary granulomas with a number of neutrophils, macrophages and epithelial cells-like in the MB group as well as granulomas in an advanced stage with caseous necrosis in the COIN group. Based on these findings, it may be concluded that MB modulated the immune response to SV, leading coinfected animals to be more susceptible to helminthic infection. On the other hand, SV modulated the MB infection by modifying the characteristics of the pulmonary granulomas in the COIN group at time point 1 probably due the reduced IL-4 production in this group.

L'une des particularités de Mycobacterium tuberculosis, agent pathogène de la tuberculose, est sa capacité à accumuler des lipides dans son cytoplasme, ce qui favorise son entrée en dormance. Le séquençage du génome de M. tuberculosis a permis d'identifier certains gènes codant pour des enzymes lipolytiques, parmi ceux-ci : le gène codant pour la protéine Rv3097c aussi appelée LipY (composée d'un domaine PE et d'un domaine lipase relié par un Linker). Dans la première partie de ce travail de thèse, nous avons procédé à la caractérisation biochimique de LipY, mais aussi de ses formes mutantes LipY(∆PE), LipY(∆149) et LipY(∆170), et à l'étude d'inhibition des membres de la famille Lip apparentés à la lipase hormono-sensible humaine (Lip-HSL). Nous avons pu déterminer les propriétés cinétiques de l'activité lipase de LipY et de ses différentes formes mutantes.Dans la seconde partie, nous nous sommes intéressés à la compréhension du rôle des différents domaines de LipY, et du linker dans l'hydrolyse des lipides au cours de l'infection en utilisant des macrophages spumeux (macrophage riche en lipides) infectés au préalable par des souches de M. bovis BCG recombinantes. Ces résultats et les hypothèses posées durant ce travail de thèse, pourraient être appuyés par l'obtention de la structure tridimensionnelle de LipY. Pour cela, nous avons initié et procédé à la cristallogenèse de LipY. La poursuite des études d'optimisation des cristaux obtenus pourrait permettre d'aller plus en profondeur dans l'élucidation du rôle et du mécanisme d'action de LipY. / Mycobacterium tuberculosis is a pathogenic agent, responsible of the tuberculosis, which can store lipids into the cytoplasm. This accumulation allows the bacteria to enter in the dormancy phase. The sequencing of M. tuberculosis genome, allows to identify some genes coding for lipolytic enzymes, among which a gene coding for Rv3097c protein, also called LipY. (possessing PE domain linkto a lipase domain). During my PhD thesis, we first biochemically characterized LipY and its mutant forms LipY(ΔPE); LipY(Δ149) and Lip(YΔ170) and studied the inhibition of Lip family members related to the human hormone-sensible lipase (Lip-HSL). We determined the kinetic properties for the lipase activity of LipY and its mutants. In the second part, based on these previous results, we studied the role of these different domains and the linker on the hydrolysis of lipids during the infection phase, in infected foamy macrophages (lipids rich). For these studies, we used foamy macrophages infected by recombinants strains of M. bovis BCG (LipY(wt) and its mutants.These results and hypothesis can be confirmed and supported by resolving the tridimensional structure of LipY. Crystallogenesis tests allowed us to have some crystals of LipY(wt), which after optimization would allow us to have a better understanding of the role and action mechanism of LipY.

La tuberculose bovine est une maladie zoonotique se transmettant principalement par inhalation. C’est une maladie qui peut avoir des répercussions économiques (ex : saisie d’un animal infecté à l’abattoir) et sur les échanges commerciaux, car les pays peuvent bannir l’importation des animaux provenant des pays touchés par cette maladie. Au Canada, le contrôle de cette maladie a commencé en 1923 chez les bovins et a ensuite été appliqué chez les cervidés depuis 1989. Ce programme est composé par la surveillance, la réponse aux éclosions, ainsi que le contrôle des mouvements des cervidés. La capacité de détecter cette maladie lors des activités de surveillance est influencée par la performance des méthodes diagnostiques utilisées à chaque étape de la surveillance. À l’abattoir, la surveillance est effectuée par l’inspection visuelle post-mortem et par différentes procédures diagnostiques réalisées sur des prélèvements de tissus anormaux (histopathologie, réaction de polymérisation en chaîne (PCR) ou culture bactérienne). Chez le cervidé vivant, la surveillance est plutôt réalisée par des tests cutanés ou sérologiques. L’objectif principal de ce projet était de fournir, à travers une revue systématique de littérature, les estimés des valeurs des principaux paramètres pouvant influencer la probabilité de détection de la tuberculose bovine dans un contexte de surveillance de cette maladie à l’abattoir chez les cervidés d’élevage et chez les cervidés vivants. Les études qui ont estimé la probabilité de présence des lésions macroscopiques ont trouvé qu’elle variait de 20% à 100% alors que la probabilité de détection des lésions tuberculeuses était supérieure ou égale à 90%. L’estimé de la sensibilité des tests diagnostiques pouvant être utilisés pour la détection de la tuberculose bovine à l’abattoir (histopathologie, PCR et culture) variait de 63.6% à 98.6%. Chez les animaux vivants, les tests sérologiques et sanguins avaient une sensibilité qui variait de faible à élevée (1.4% à 100.0%) alors que les tests cutanés avaient une sensibilité qui variait de 65.0% à 100.0%. Comme le stade de la maladie (temps depuis l’infection) influence la sensibilité de la majorité des tests diagnostiques et que ce facteur n’a généralement pas été rapporté par les études, la performance de ces tests peut difficilement être comparée. La prévalence de la tuberculose estimée lors des études transversales ou de contrôle de la maladie était faible (prévalence au niveau animal au sein d’un troupeau). Une étude effectuée au Canada sur les facteurs de risque de la tuberculose bovine chez les cervidés d’élevage a constaté que l’augmentation de la taille du troupeau était associée à une augmentation du risque de positivité (du troupeau) à la tuberculose bovine. Ces informations sur la performance et les limites des méthodes diagnostiques sont essentielles pour orienter le choix du test diagnostique et pour permettre l’évaluation de la sensibilité des activités de surveillance / Bovine tuberculosis is a zoonotic disease mostly transmitted by inhalation of contaminated material. This disease leads to the economic losses (e.g. condemnation of meat from infected animals at the abattoir) and impede trade since countries can stop importing live cervids from tuberculosis endemic countries. In Canada, the control of bovine tuberculosis started in 1923 in cattle and then was implemented in farmed cervids in 1989. This program is composed of surveillance, response to the outbreaks and movement control of cervids. The capacity of detecting this disease during the surveillance activities depends on the performance of diagnostic methods used at each step of the component of the surveillance system. At the abattoir, the inspection is carried out by visual inspection of organs and carcass and diagnostic tests carried on abnormal tissues by using histopathology, PCR or culture. In live cervids, the detection is carried out through skin or serological testing. The main objective of this project was to provide, through a systematic literature review, the estimates of main parameters that can influence the probability of detecting bovine tuberculosis in the context of surveillance activities in farmed cervids at the abattoir and in live cervids. The probability of presence of macroscopic lesions estimated by studies ranged from 20% to 100% while the estimate of probability of detection of lesions was greater than or equal to 90%. The estimate of sensitivity of diagnostic tests used during bovine tuberculosis surveillance at the abattoir (histopathology, PCR and culture) ranged from 63.6% to 98.6%. In live cervids, studies that investigated the performance of serological tests reported an estimate of sensitivity ranging from low to high (1.4% to 100.0%) while skin tests were reported to have a sensitivity ranging from 65.0% à 100.0%. Given that the stage of the disease affects the performance of most diagnostic test while that variable was not reported by studies, it was difficult to compare the performance of the various tests. Bovine tuberculosis prevalence estimated by studies using a cross-sectional or disease control design reported a low prevalence (prevalence at the animal level within a herd). One study carried out in Canada evaluated the risk factors for bovine tuberculosis infection and found that the increase of herd size was associated with an increase of risk of herd positivity to bovine tuberculosis. This information on the performance and limits of bovine tuberculosis diagnostic tests is critical for orienting the choice of appropriate diagnostic method and for a valid evaluation of the surveillance activities

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.