Rozprawy doktorskie: „Mycobacterium bovis”

1

Roring, Solvig Mary Margaret. "DNA fingerprinting of Mycobacterium bovis". Thesis, Queen's University Belfast, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287426.

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Mardare, Cornelia. "Interactions of Mycobacterium bovis with protozoa and the occurrence of Mycobacterium bovis in environmental protozoa". Thesis, University of Surrey, 2010. http://epubs.surrey.ac.uk/844633/.

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Bovine tuberculosis is in the UK a persistent disease, affecting cattle and badgers. The latter is suspected to be a reservoir for Mycobacterium bovis but the transmission between badgers and cattle remains unclear. Mycobacteria have been shown to survive ingestion by protozoa and some even multiplied inside amoebas. The aim of this study was to investigate some interactions of Acanthamoeba castellanii and Tetrahymena pyriformis with M bovis. Firstly, the long term survival of the bacilli in protozoa was monitored. Secondly, it was investigated whether bacilli internalized in protozoa cysts are protected from hypochlorous acid and desiccation. Thirdly, the identification of M bovis in environmental protozoa isolated from badger latrines was attempted. The long term incubation of M. bovis with A. castellanii showed that the amoebas had a negative effect on the survival of virulent M. bovis. M. bovis was not detectable after 6 months of coincubation but remained viable at high concentrations in the control experiments. This effect however, could not be seen in T. pyriformis. Cysts of A. castellanii did not protect M bovis from hypochlorous acid and desiccation. Results indicate that M. bovis was more susceptible to hypochlorous acid after the encystment in comparison with the controls. These findings suggest that A. castellanii contributes to the decrease of M. bovis and therefore, it can be suggested that protozoa might have a negative impact on the survival M. bovis in the environment. In one of the samples taken from Woodchester Park, acid fast rods could be identified. Acid fast microorganisms were also identified in trophozoites of protozoa. This indicates that trophozoites of enviromnental protozoa might be carrier of mycobacteria and possibly M. bovis. An infection with bacilli-containing trophozoites might therefore be a potential route of transmission between the enviromnent and animals.

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3

Sales, Mariana Lázaro. "Identificação de Mycobacterium bovis e Mycobacterium tuberculosis por PCR". Universidade Federal de Minas Gerais, 2012. http://hdl.handle.net/1843/SMOC-9L2PT7.

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Mycobacterium bovis and Mycobacterium tuberculosis are the causative agents of bovine and human tuberculosis, respectively. The standard diagnosis is based on the isolation and identification of bacterial colonies through biochemical methods. These methodologies in addition to being costly, require an environment with high level of biosecurity and a great time of cultivation and tests to obtain the results. The molecular tests based on the principles of real-time PCR using the fluorophore Eva Green were efficient in the identification of M. bovis and M. tuberculosis. In addition, analyses were performed with primers and molecular markers, described in the literature, able to differentiate the M. bovis of M. tuberculosis by PCR. The results showed that some of the molecular markers are found in both microorganisms. The work of this dissertation has enabled the development of real-time PCR techniques able to identify colonies of M. bovis and M. tuberculosis.
O Mycobacterium bovis e o Mycobacterium tuberculosis são os agentes causadores da tuberculose bovina e humana, respectivamente. O diagnóstico padrão se baseia no isolamento bacteriano e a identificação das colônias através de métodos bioquímicos. Estas metodologias além de serem onerosas, requerem um ambiente com alto nível de biossegurança e um grande tempo de cultivo e testes para obtenção dos resultados. Os testes moleculares baseados nos princípios da PCR em tempo real utilizando-se o fluoróforo Eva Green foram eficientes na identificação do M. bovis e M. tuberculosis. Além disso, foram realizadas análises com iniciadores e marcadores moleculares, descritos na literatura, capazes de diferenciar o M. bovis do M. tuberculosis por PCR. Os resultados mostraram que alguns dos marcadores moleculares são encontrados em ambos os micro-organismos. O trabalho dessa dissertação permitiu o desenvolvimento de técnicas de PCR em tempo real capazes de identificar colônias de M. bovis e M. tuberculosis, em substituição aos testes bioquímicos.

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4

Hamerman, Jessica Ann. "Macrophage activation during Mycobacterium bovis BCG infection /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8359.

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Michell, Stephen Lloyd. "Molecular characterisation of a novel lipoglycoprotein from Mycobacterium tuberculosis and Mycobacterium bovis". Thesis, Imperial College London, 1999. http://hdl.handle.net/10044/1/7477.

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In Great Britain a recent independent scientific review into bovine tuberculosis concluded that the best prospect for control of TB in the national herd is to develop a vaccine. The secreted antigens of the Mycobacterium tuberculosis complex have received much interest as possible vaccine candidates due to their ability to confer protection against tuberculosis in small animal models. The recent discovery that some of these mycobacterial antigens are glycosylated, a modification ubiquitous in eukaryotic proteins, may provide some novel insights into the properties of these antigens. The antigen MPB70 is the major secreted protein of Mycobacterium bovis, the causative agent of tuberculosis in cattle. It has previously been reported that this antigen, encoded for by the gene mpb70, is present in at least two forms, a 22 kDa unglycosylated form and a 25 kDa glycosylated form. A clone was isolated from an M. tuberculosis H37Rv mycobacterial shuttle cosmid library which expressed both the 22 and 25 kDa antigens. Genetic analysis of this cosmid revealed that the two antigens were encoded by separate genes. The gene encoding the 25 kDa antigen (MPT83), subsequently designated mpt83, is situated 2.4 kb upstream of mpt70 and transcribed in the same direction. Using a mycobacterial expression system and alkaline phosphate (PhoA) fusions, it was shown that MPT83 but not MPT70 is glycosylated by Mycobacterium smegmatis. Moreover, neither fusion protein was glycosylated in E. cW demonstrating that glycosylation of MPT83 was specific to the mycobacterial species. The use of site directed mutagenesis in conjunction with the PhoA reporter system identified both 0 and N-linked glycosylation sites within MPT83. Preliminary investigations into the role of glycosylation in the alteration of immune recognition showed a possible influence of cilycosylation on a T cell epitope of MPT83.

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Rennie, Bryan D. "Detection and identification of antigens from Mycobacterium bovis culture filtrate with immune sera from Mycobacterium bovis sensitized or infected cattle". Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28138.

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Bovine tuberculosis, caused by Mycobacterium bovis, infects approximately 50 million cattle worldwide and is diagnosed by the tuberculin skin test (TST). The purpose of this thesis was to characterise the culture filtrate proteins (CFP) of M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle. Sterile filtered PPD tuberculin (SF-PPD) resolved into approximately 200 discrete spots using two-dimensional PAGE. While 2D Western blot analysis of both M. bovis sensitized and M. bovis infected cattle sera demonstrated an antibody boost following comparative intradermal TSTs, M. bovis sensitized cattle responded with greater intensity to additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. In conclusion M. bovis sensitized cattle generated a more intense antibody response and recognized additional SF-PPD antigens as compared to M. bovis infected cattle within the first two months post infection/sensitization.

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Bouchez-Zacria, Malika. "Rôles de l’environnement et des contacts intra et interspécifiques dans la transmission de Mycobacterium bovis dans le système bovins-blaireaux en Pyrénées-Atlantiques – Landes". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS552/document.

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En France, pays officiellement indemne de tuberculose bovine depuis 2001, l’infection persiste dans certaines zones, dont les Pyrénées-Atlantiques-Landes, où les niveaux d’infection observés chez les bovins et les blaireaux font craindre un fonctionnement particulier de ce système multi-hôtes pour Mycobacterium bovis, agent de la tuberculose bovine. L’objectif de cette thèse a été de mieux comprendre les mécanismes de transmission de M. bovis au sein du système bovins-blaireaux dans cette zone. L’analyse statistique du risque d’infection localement concomitante chez les blaireaux et les bovins a montré l’importance de variables environnementales liées à la survie de la bactérie dans le sol, telles que le microrelief et le pourcentage de sable dans le sol de surface, ainsi que celle de variables associées aux mouvements des blaireaux. L’analyse du réseau de contacts entre élevages bovins, incluant des contacts directs liés au commerce ou au voisinage au pâturage et des contacts indirects induits par un voisinage avec des domaines vitaux de blaireaux, a montré l’aspect multifactoriel de la transmission de M. bovis entre élevages. Enfin, la modélisation dynamique de la transmission de M. bovis dans le système bovins-blaireaux a suggéré une interface asymétrique entre blaireaux et bovins dans notre zone d’étude, les terriers étant pour la plupart infectés suite à l’exposition des blaireaux à une pâture dont le sol était contaminé par un bovin infectieux, alors que les modes de contamination des élevages se partageaient entre l’exposition des bovins à un élevage infectieux voisin de pâture, et la contamination de ces pâtures par un blaireau infectieux. La validation du modèle dynamique proposé doit encore être poursuivie. L’ensemble de nos résultats permet cependant de conclure à l’importance de prendre en compte tous les mécanismes de propagation dans la lutte contre la tuberculose bovine dans la zone. Le modèle développé est destiné à devenir un outil permettant de préciser les rôles épidémiologiques des bovins et des blaireaux et de simuler différentes stratégies de contrôle, pouvant inclure la vaccination des blaireaux
France is officially free from bovine tuberculosis since 2001, but this infection is still persistent in some areas, including Pyrénées-Atlantiques - Landes. There, both cattle and badgers were found infected, suggesting that this multi-host system might have a particular functioning regarding Mycobacterium bovis (the main agent for bovine tuberculosis in France) transmission. The objective of the thesis was to better understand M. bovis transmission mechanisms throughout the badger-cattle system in this area. The statistical analysis of the local risk of the badger and cattle concomitant infection showed the importance of environmental variables such as microrelief and sand proportion of the topsoil as well as variables depicting badger movements. The analysis of the contact network between farms (that included direct contacts due to trade and to neighbouring pastures and indirect contacts due to the vicinity with badger home ranges) showed a multifactorial aspect of the transmission between farms in our study area. Finally, the dynamic modelling of M. bovis transmission throughout the badger-cattle system suggested an asymmetric interface where the infection of badgers due to soil contaminated by infected cattle was predominant, whereas, for the infection of cattle, the soil contaminated by infected badgers had a similar importance as the presence of infectious cattle on the neighbouring pastures. Our dynamic model validation should be continued. Nevertheless, our whole range of results underlined the importance of targeting all transmission mechanisms for infection control. The model we proposed is a tool designated to assess both cattle and badgers’ epidemiological roles as well as to simulate different control strategies, that could include badgers’ vaccination

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Whiteford, Danelle. "Stress survival in Mycobacterium tuberculosis and Mycobacterium bovis and the role of hup in Mycobacterium smegmatis". Pullman, Wash. : Washington State University, 2008. http://www.dissertations.wsu.edu/Dissertations/Fall2008/D_Whiteford_100908.pdf.

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9

Young, Jamie Stuart. "Molecular detection of Mycobacterium bovis in the environment". Thesis, University of Warwick, 2003. http://wrap.warwick.ac.uk/47689/.

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An investigation was carried out to determine the presence and persistence of Mycobacterium bovis in the environment. Soil samples were taken in April 2000 from a farm in Ireland which had undergone a bovine tuberculosis outbreak some four months prior. Total community DNA was extracted from these samples and PCR carried out targeted to two genes specific for the Mycobacterium tuberculosis complex; mpb64 and mpb70. These genes were detected in soil samples taken from entrances to two badger sets and in soil from two sites where the infected cattle grazed. Further analysis of DNA using oligonucleotide primers specific for the 16S rRNA genes of slow-growing mycobacteria was carried out. This revealed the presence of 16S rRNA genes relating to Mycobacterium bovis. RT-PCR was also carried out using these primers on total community RNA. Sequences relating to M. bovis were found, indicating that the DNA and RNA came from viable, intact cells in the soil, and that M. bovis persists in soil for up to four months after contamination of the soil. Sampling was repeated in November 2002 following a second TB outbreak in January 2001. DNA sequences for mpb64 and mpb7O were only found in the samples from the badger setts, as were 16S rRNA sequences. The survival of the vaccine strain M. bovis BCG was also determined, using soil microcosms experiments in defined environmental conditions. M. bovis BCG was found to survive for longest at temperatures of 37°C and at soil wetting levels of 30%. Culturable cells could not be detected after 60 days, however DNA and RNA relating to M. bovis BCG could be detected up to 18 months after initial inoculation. This suggests the cells persisted in a viable non-culturable state. Experiments to determine the rate of persistence of DNA in soil were carried out. DNA was found to persist for no longer than 10 days in soil. Diversity studies were carried out on the farm samples and on Warwick soil, to determine the diversity of the mycobacterial population. 16S rRNA analysis was carried out and showed the presence ofsequences relating to M. bovis, Al. hiberniae, M. avium, Al. fallax, and M. farcinogenes.

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Sales, Erica Bravo. "Genotipagem de Mycobacterium bovis pelo multispacer sequence typing". Universidade Federal de Minas Gerais, 2012. http://hdl.handle.net/1843/BUOS-8YGH2X.

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Mycobacterium bovis, which causes bovine tuberculosis, is a species belonging to Mycobacterium tuberculosis complex. It shows 99.9% of genetic homology with other species of the complex, making the diagnosis difficult. The bovine bacilli represent a serious problem due to the direct losses in cattle, caused by such infirmity and possible sanitary barriers to exportation. Molecular typing, provide an innovative way to identify bacterial strains, trough generate while genotypes by combining different polymorphic DNA markers, contributing to their epidemiological investigation. The techniques currently used for typing of M. bovis are Spoligotyping and VNTR. However these methods are not able to recognize all genetics analyzed strains diversity such as sequencing methods based in last ones allow analyzing molecular target sequence, thereby identifying all events present in the genetic marker used. This paper aims standardize Multispacer Sequence Typing (MST) for M.bovis genotyping. Seven intergenic spacer region were analyzed on 58 M.bovis samples, coming from six brazilian's states (MG, GO, SP, MT, PR, RS), isolated between 2006 and 2010, and on four M.boviss reference strains. Four types of genetic events were observed: nucleotide mutation (SNP), insertion, deletion and tandem repeat (TR), totaling from the combination of genotypes obtained a total of 28 events. Twenty-eight type sequences (TS) have been also produced, showing a discrimination index of 93%, including the standard strain AF2122/97 [Genbank BX248333.1] used for comparison in silico. These data were used to analyze the pattern of evolutionary lineage of M. boviss isolates and correlate them with phylogeographic lineages, based on the formation of clonal complexes, generated from eBURST. This is the first study aimed identify the variability of isolates of M. bovis using MST method. Results were quite satisfactory. Method allowed us to typing and differentiate M.bovis isolate from a sequencing of few regions, even in restricted locations and in short a time as established in Bueno Brandao city.The method has the sequencing advantage and the availability of sequences analyzed in public databases which can be used by professionals around the world as an analyzes tool.
O Mycobacterium bovis, causador da tuberculose bovina, está entre as espécies pertencentes ao Complexo Mycobacterium tuberculosis. A doença causada pelo bacilo bovino constitui um grave problema devido aos prejuízos diretos causados por esta enfermidade e possíveis barreiras sanitárias na exportação. O genoma do M. bovis é >99,95% geneticamente idêntico ao de M. tuberculosis, dificultando o diagnóstico. A tipificação molecular permite inovar a identificação de uma espécie e ao mesmo tempo gerar genótipos mediante a combinação de diferentes marcadores polimórficos, contribuindo para a sua investigação epidemiológica. As técnicas mais utilizadas atualmente para tipificar o patógeno M. bovis são o Spoligotyping e o VNTR, no entanto, esses métodos não são capazes de reconhecer toda a diversidade genética dos isolados analisados como é o caso de métodos baseados em sequenciamento, que permitem a análise de sequências alvos moleculares, identificando dessa forma todos os eventos genéticos presentes no marcador utilizado. O objetivo deste trabalho foi tipificar isolados de Mycobacterium bovis pela técnica Multispacer Sequence Typing (MST). Sete regiões espaçadoras intergênicas foram analisadas para um total de 58 isolados de M. bovis relativos aos anos de 2006 a 2010, procedentes de fazendas localizadas em seis estados brasileiros (MG, GO, SP, MT, PR. RS) e para quatro amostras padrão de M. bovis. Quatro tipos de eventos genéticos foram observados: mutação de nucleotídeo único (SNP), inserção, deleção e repetição em tandem (TR), totalizando a partir da combinação dos genótipos obtidos um total de 28 eventos. Os resultados, obtidos pela comparação in silico entre os fragmentos gerados pelo sequenciamento e a cepa padrão M. bovis AF2122/97 [Genbank BX248333.1] permitiu identificar 28 perfis genotípicos únicos, caracterizando 28 sequências tipo (ST) na amostragem analisada e apontando um índice de discriminação de 93%. Esses dados foram utilizados para analisar os padrões de descendência evolutiva dos isolados de M. bovis e correlacioná-los a linhagens filogeográficas com base na formação de complexos clonais gerados a partir do eBURST. Este foi o primeiro trabalho a identificar a variabilidade dos isolados de M. bovis pelo método MST. Os resultados obtidos foram bastante satisfatórios, dado que o método permitiu tipificar e diferenciar isolados de M. bovis a partir do sequenciamento de poucas regiões espaçadoras, mesmo em localizações restritas e em um período de tempo curto como foi verificado no município de Bueno Brandão. O método apresenta a vantagem do sequenciamento e a disponibilização de sequências analisadas em bancos de dados públicos, que podem ser utilizadas por profissionais em todo o mundo como ferramenta para análises futuras.

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Mpongoshe, Vuyiseka. "Gene expression changes in macrophages infected with pathogenic M. tuberculosis and non-pathogenic M. smegmatis and M. bovis BCG". Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86729.

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Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies.
AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ŉ poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ŉ bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ŉ nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ŉ filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.

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Tschiginewa, Valeria [Verfasser]. "Regulation der C-Quellenverwertung in Mycobacterium tuberculosis und Mycobacterium bovis BCG / Valeria Tschiginewa". Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2015. http://d-nb.info/1076828485/34.

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Himpens, Sabine. "Caractérisation du système à deux composants SenX3/RegX3 de Mycobacterium tuberculosis et Mycobacterium bovis BCG". Lille 1, 1999. https://pepite-depot.univ-lille.fr/LIBRE/Th_Num/1999/50376-1999-159.pdf.

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Les mécanismes contrôlant la virulence mycobactérienne sont encore peu connus. Pour étudier ces mécanismes, les systèmes de transduction sensorielle à deux composants constituent une cible privilégiée. Nos recherches ont permis de cloner des gènes codant des éléments de systèmes à deux composants chez Mycobacterium tuberculosis et Mycobacterium bovis BCG appelés senX3 et regX3. Ce système appartient à la famille des couples PhoB/PhoR et PhoP/PhoQ, impliqués dans la virulence chez diverses bactéries pathogènes. Nous avons produit ces deux protéines sous une forme recombinante chez Escherichia coli et les avons purifiées. Grâce à l'obtention d'anticorps dirigés contre ces protéines et à l'aide de fusions génétiques avec le gène rapporteur lac Z, nous avons montré que senX3 et regX3 sont exprimés chez le BCG et M. Tuberculosis. Nous avons caractérisé le mécanisme de phosphorylation du capteur senX3 et du régulateur regX3. Cette caractérisation a montré que ce système partage le mécanisme de base commun à tous les systèmes à deux composants. Nous avons pu montrer que le régulateur regX3 se lie à la séquence promotrice de son propre opéron, ce qui suggère que ce système est autorégulé, à l'instar de nombre d'autres systèmes à deux composants
De plus, nous n'avons pas observé d'effet majeur de la phosphorylation sur la liaison à l'ADN et la structure quaternaire de regX3. Ces observations suggèrent que la phosphorylation de regX3 pourrait exercer un effet d'activation en favorisant des interactions protéine-protéine avec l'ARN polymérase. En outre, une analyse plus détaillée du mécanisme de phosphorylation du capteur senX3 a été effectuée pour déterminer si comme les autres capteurs étudiés jusqu'à présent, les domaines cytoplasmiques de ces protéines se dimérisent et sont phosphorylés en trans. Cette étude a été effectuée en vue d'explorer la possibilité de produire ultérieurement une version mutante a effet dominant négatif in vivo. Par ailleurs, notre système de phosphorylation in vitro de senX3/regX3 pourrait permettre la recherche de molécules inhibitrices de systèmes à deux composants mycobactériens, qui pourraient correspondre à de nouvelles classes d'antibiotiques

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Baudoin, Norbert. "Tuberculose trachéale pseudo-tumorale à mycobacterium bovis : à propos d'un cas avec revue de la littérature". Montpellier 1, 1988. http://www.theses.fr/1988MON11273.

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Tadayon, K. "Molecular epidemiology of Mycobacterium bovis in cattle in Iran". Thesis, University of Aberdeen, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590987.

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Mycobacterium bovis causes bovine tuberculosis (BTB) that is an endemic disease of cattle in Iran. Despite benefiting from a successful test-and-slaughter programme, initiated in the late 1960s, which has reduced the prevalence to 0.05% (reactors in tuberculinated cattle) in 2006, there is little known about the epidemiology of M. bovis in Iran. The present study sought to improve this serious lack of information through combining traditional diagnostic methods (tuberculin tests in the field, mean inspections in abattoirs and microbiology in the laboratory) with modern molecular speciation and strain typing (Region of Deletion (RD) typing, spoligotyping and Variable Number Tandem Repeat (VNTR) typing). With information collected, a second objective of this thesis has been the analysis of historic field and recent laboratory observations to elaborate the origins of M. bovis in Iran and consequently developing a scenario for the history of BTB in this country. Molecular identification of 133 mycobacterial isolates collected from abattoir specimens confirmed M. bovis as the single principal cause of BTB in the Iranian environment. None of the studied isolates were M. bovis BCG and this categorically ruled out concerns over interpretation of the tuberculin test in cattle due to unauthorised vaccination of cattle with BCG vaccine in Iran. Spoligotyping revealed a genetically homogenous population of M. bovis. Only eight, highly similar, spoligotypes were identified with five of them not previously seen elsewhere. The ancestral, BCG-like spoligotype, SB0120, was the most prevalent (41%) along with two other similar patterns, SB1167 (39%) and SB1168 (17%) which were unique to Iran. VNTR typing recognised 23 types and again revealed a homogenous population with only a few predominant patterns. Generally speaking, the same genotypes of M. bovis were found to infect both European breeds and zebu bovids in Iran. The homogeneity of the M. bovis population, portrayed by this study, was unexpected. This lack of diversity is probably a reflection of the significant increase in population of BTB susceptible, Holstein-Friesian cattle in the last fifty years. The absence of adequate initial BTB control measures could have led to the rapid clonal expansion of a SB0120 strain which subsequently diversified into a few highly related strains. Whilst the origin of this strain might be from outwith Iran, the origins of the others are likely to have been within Iran and those Iranian spoligotypes that are also reported elsewhere in the world are most likely to be due to convergent evolution (homoplasy) rather than to common epidemiology.

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Carroll, Maria Veronica. "Interaction of innate immune proteins with mycobacterium bovis BCG". Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526549.

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Duarte, Elsa Maria Leclerc. "Tuberculose bovina: detecção molecular e genotipagem de Mycobacterium bovis". Doctoral thesis, Universidade de Évora, 2008. http://hdl.handle.net/10174/12147.

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Nesta dissertação, pretendeu-se avaliar e aplicar um conjunto de ferramentas moleculares a estirpes de Mycobacterium bovis e Mycobacterium caprae, com o objectivo de melhorar o diagnóstico da tuberculose bovina e de proceder à primeira genotipagem destas estirpes em Portugal, clarificando vias de transmissão e a eventual existência de reservatórios silvestres. A extracção de DNA por disrupção mecânica/ lise enzimática e o PCR-REA gyrB produziram resultados que permitiram aumentar a sensibilidade e especificidade do diagnóstico, e identificar um conjunto aleatório de isolados (N=293), de várias regiões do país, com vista à sua genotipagem. O spoligotyping permitiu atestar da elevada diversidade dos padrões Portugueses (Hunter-Gaston Index; h=0,9) e compará-los com outros países. A análise dos polimorfismos dos MIRU-VNTR permitiu alcançar uma superior discriminação (h=0,98), especialmente nos “spoligotypes” mais frequentes, permitindo comprovar, em consonância com dados das explorações, a transmissão entre efectivos bovinos e entre bovinos e espécies cinegéticas da mesma área. Pelos seus índices discriminatórios elevados, as duas técnicas de genotipagem demonstraram a sua utilidade futura para estudos de epidemiologia molecular, integrando estratégias mais eficazes de erradicação em Portugal. ### Summary - The evaluation of several molecular tools for Mycobacterium bovis and Mycobacterium caprae was undertaken in this thesis, aiming to improve bovina tuberculosis diagnosis, and to produce the first genotyping results of Portuguese strains, in order to clarify transmission routes and the possible existence of wildlife reservoirs. Results obtained by DNA extraction, with a combined mechanical disruption/ enzymatic Iysis, and PCR-REA gyrB analysis were proven to increase diagnosis sensitivity and specificity, and were applied to the identification of a random set of isolates (N=293), from several geographical regions, that were latter genotyped. Spoligotyping revealed the high diversity of Portuguese patterns (Hunter-Gaston lndex; h=0.9), and we were, able to compare them with other countries. MIRU-VNTR typing achieved a superior discrimination (h=0.98), specially within frequent spoligotypes, and was able to establish, in accordance with cattle herds data, inter-herd transmission as well as transmission between cattle and game animals, from the same area. By their high discriminatory power, these genotyping techniques are proven to be useful for future molecular epidemiological studies, adding increased effectiveness to eradication strategies in Portugal.

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Walrath, Jessica R. "The role of alpha 4 beta 1 integrin (VIa-4) in recruitment of mycobacterium tuberculosis-specific TH1-LIKE recall responses to the human lung". Cleveland, Ohio : Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1196182534.

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Rocha, Vivianne Cambuí Mesquita. "Discriminação de isolados de Mycobacterium bovis pelas técnicas de Spoligotyping, MIRU e ETR e suas aplicações epidemiológicas". Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-25022010-113545/.

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O Mycobacterium bovis é o agente causador da tuberculose em bovinos, zoonose que produz prejuízos para a produção de carne e leite em muitos países. Para apoiar estudos epidemiológicos no âmbito dos programas de controle, recentemente surgiram vários métodos de discriminação molecular de isolados de M. bovis. Os mais utilizados são o Spoligotyping, Mycobacterial Interspersed Repetitive Units (MIRU) e Exact Tandem Repeat (ETR), que apresentam diferentes poderes de discriminação. No presente estudo calculou-se a diversidade alélica para cada locus de MIRU e de ETR e o índice discriminatório de Hunter-Gaston (HGI) para o Spoligotyping, 10 MIRU e 3 ETR em 116 amostras de M. bovis isoladas de bovinos. Além disso, verificou-se se a capacidade de detectar agrupamentos espaciais de focos aumenta na medida em que é aumentado o poder discriminatório das análises moleculares. Comparou-se a utilização dessas três técnicas moleculares em análises espaciais para verificação de agrupamentos de focos da doença. A análise da diversidade alélica indicou que os MIRU 16, 26 e 27 e os ETR A, B e C foram os que apresentaram maior diversidade dentre os ensaiados. O HGI para cada uma das técnicas foi de: Spoligotyping = 0,738381; MIRU = 0,829835 e ETR = 0,825337. A associação desses métodos aumentou o poder discriminatório: Spoligotyping + MIRU = 0,930585; Spoligotyping + ETR = 0,931034; MIRU + ETR = 0,953373. O maior poder discriminatório foi alcançado quando as três técnicas foram associadas (HGI = 0,98051). Considerando as análises realizadas no presente estudo, o método inicial deveria ser o Spoligotyping, por diferenciar o M. bovis dos outros integrantes do complexo Mycobacterium tuberculosis. Como as associações do MIRU e do ETR com o Spoligotyping resultaram em HGI praticamente idênticos, depois do Spoligotyping, o método ETR parece ser a melhor escolha, pois é mais rápido e econômico do que o MIRU. Finalmente, o MIRU deve ser o último método a ser realizado. Assim, a escolha do método depende do poder discriminatório necessário para o objetivo em questão. Embora a capacidade de detectar agrupamentos espaciais de focos não tenha sido aumentada na medida em que cresceu o poder discriminatório das análises moleculares, a premissa continua válida.
Mycobacterium bovis is the causative organism of bovine tuberculosis, has zoonotic character and leads economic losses in livestock production in a lot of countries. To support the epidemiological researches on the disease control programs several molecular methods has been used to detect M. bovis strains. Among them, Spoligotyping, Mycobacterial Interspersed Repetitive Units (MIRU) and Exact Tandem Repeat (ETR) are the molecular analyses that present different powers of Mycobacterium bovis discrimination. In this study, allelic diversity of MIRU and ETR locus and Hunter-Gaston discriminatory index (HGI) were calculated for Spoligotyping, 10 MIRU and 3 ETR of 116 isolates of M. bovis from Brazilian bovines. The use of those three molecular techniques was compared in space analyses for verification of groupings of focuses of the disease. Besides, it was verified if the capacity to cluster of focuses increases in the measure in that the discriminatory power of the molecular analyses is increased. The analysis of the allelic diversity indicated that MIRU 16, 26, 27 and ETR A, B, C presented larger diversity among the rehearsed. The HGI for each individual technique was: Spoligotyping = 0.738381; ETR = 0.825337 and MIRU = 0.829835. The associations of these methods increased the discriminatory power: Spoligotyping + MIRU = 0.930585; Spoligotyping + ETR = 0.931034; MIRU + ETR = 0.953373. The greater discriminatory power was verified when the three techniques were associated (HGI = 0,98051). Considering the analyses performed, the initial method for differentiating M. bovis from the other species of the M. tuberculosis complex should be Spoligotyping. The association of MIRU and ETR with Spoligotyping resulted in HGI almost identical, after Spoligotyping, the ETR technique showed to be the best choice, due the shorter time to process and economic benefits when compared to MIRU. Finally, MIRU is the last method to be performed. Thus, the choice among the techniques depends on the discriminatory power necessary for the task. Although the capacity to detect clusters of focuses has not been increased in the measure in that it increased the discriminatory power of the molecular analyses, the premise continues valid.

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Rocha, Vivianne Cambuí Figueiredo. "Avaliação do Spoligotyping, MIRU-VNTR e Multispacer Sequence Typing na discriminação de isolados autóctones de Mycobacterium bovis". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-22082014-160432/.

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A tuberculose continua sendo uma importante doença infecciosa, tanto nos humanos quanto nos animais, com índices de morbidade e mortalidade significativos e perdas econômicas em todo o mundo. A tuberculose bovina é uma doença infecciosa causada pela bactéria Mycobacterium bovis, que gera perdas na produção nos rebanhos infectados, sendo também considerada uma importante zoonose. Os métodos de diagnóstico direto têm fundamental importância para um sistema de vigilância para a tuberculose bovina e a agregação de métodos moleculares, notadamente aqueles que têm aplicação em epidemiologia, traz maior precisão diagnóstica para esses sistemas. Dentre as técnicas moleculares, destacam-se o TB Multiplex PCR, o RD Multiplex PCR e o Multispacer Sequence Typing, para a identificação dos isolados e o Variable Number Tandem Repeat (VNTR) e o Spoligotyping, como técnicas de fingerpint de M. bovis. Assim, o presente estudo teve como objetivo a identificação molecular de amostras oriundas de várias regiões do Brasil utilizando estes padrões de técnicas moleculares. Os espoligotipos identificados em maior abundância foram o SB0121, o qual apresentou-se amplamente distribuído entre as amostras, seguido pelo SB0295, SB1380, SB0140 e SB1050. Além disso, foram detectados quatro perfis nunca antes descritos na literatura, sendo que um deles foi o terceiro mais frequente entra as amostras pesquisadas. Os resultados observados neste trabalho demonstraram ainda que a tipagem pelo MIRU-VNTR revelou-se superior ao Spoligotyping para discriminar os isolados. Nesta perspectiva, acredita-se que as pesquisas moleculares voltadas a identificação de micobactérias, aliadas as técnicas epidemiológicas tradicionais, possam melhorar sensivelmente a performance dos sistemas de vigilância para tuberculose bovina no Brasil.
Tuberculosis remains a major infectious disease in both humans and animals, with morbidity and mortality and significant economic losses. Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis, with yield losses in infected herds and is also considered an important zoonosis. The methods of direct diagnosis are important for a surveillance system for bovine tuberculosis and aggregation of molecular methods brings greater diagnostic accuracy for these systems, especially those that have application in epidemiology. Among them, TB Multiplex PCR, RD Multiplex PCR, and Multispacer Sequence Typing for the identification of strains, and Variable Number Tandem Repeat (VNTR) and Spoligotyping, for fingerprint of M. bovis. Thus, the present study aimed to identify molecular samples from different regions of Brazil using these molecular techniques. The most abundant were the spoligotype SB0121, which has become widely distributed among the samples, followed by SB0295, SB1380, SB0140, and SB1050. In addition, four profiles never before described in the literature were detected, one of which was the third most frequent. The results of this study also showed that the MIRU-VNTR typing has proved superior to Spoligotyping to discriminate the isolates. In this perspective, it is believed that the research focused on molecular identification of mycobacteria, combined traditional epidemiological techniques, can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

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Zimpel, Cristina Kraemer. "Sequenciamento, anotação e análise do genoma completo de Mycobacterium bovis cepa SP38". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-25072017-120925/.

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A tuberculose é uma doença infectocontagiosa causada por bactérias do complexo Mycobacterium tuberculosis (MTBC) que afeta humanos e/ou animais. Membros desse complexo evoluíram clonalmente e possuem grande similaridade genômica, diferenciando-se por polimorfismos de nucleotídeo único (SNPs) e regiões de diferença (RDs). Dentre os patógenos da tuberculose em animais, Mycobacterium bovis, causador da tuberculose bovina, é o membro do MTBC de maior importância global. Desta maneira, o presente estudo tem por objetivo o sequenciamento, a anotação e a análise da estirpe brasileira SP38 de M. bovis, seguido da genômica comparativa desse com outros genomas de M. bovis depositados no GenBank. Mycobacterium bovis SP38 apresenta um genoma tradicional de micobactéria tuberculosa, sendo esse único, circular com 4.347.646 pb, alto conteúdo de GC (65,6%) e 4.216 genes, incluindo 154 pseudogenes, 3 genes de rRNA (RNA ribossomal), 45 de tRNA (RNA transportador), 2 de ncRNA (RNA não codificante), 1 tmRNA (RNA transferência-mensageiro) e 4.011 sequências de DNA codificante (CDSs) (NZ_CP015773.1). Dentre as CDSs, a maioria (2.805 - 69,93%) foi anotado com função e 1.206 (30,07%) como hipotéticos. Para a genômica comparativa, os 31 genomas completos ou em drafts de M. bovis depositados no GenBank, 32 genomas de Mycobacterium bovis BCG e 23 genomas de Mycobacterium tuberculosis foram selecionados. Análises in silico dos padrões de RD resultaram na exclusão de três genomas anotados equivocadamente como M. bovis virulentos. A análise de genes ortólogos sugere que M. bovis está sob processo de decay genômico. A quantificação de sítios polimórficos indica uma maior variabilidade genética em números totais (8.335 em M. tuberculosis, 3.448 em M. bovis virulentos, e 1.088 em M. bovis BCGs) e comparações par-a-par (p ≤0,05) de M. tuberculosis em relação a M. bovis virulentos e BCGs, indicando uma maior pressão evolutiva sob M. tuberculosis, contrastando com o fato de que M. bovis é capaz de infectar um maior número de espécies hospedeiras que M. tuberculosis. A maioria desses sítios polimórficos estão localizados em CDSs hipotéticos (31,7% - 51,3%), sendo associados a família gênica PE/PPE, e apresentam uma proporção de mutações não sinônimas crescentes pela ordem M. bovis BCG, M. bovis virulentos e M. tuberculosis (48,90%, 51,92% e 59,52%, respectivamente). Essa menor proporção de mutações não sinônimas e a categorização funcional dissimilar entre CDSs contendo sítios polimórficos, indica que M. bovis BCG está sujeito a diferentes pressões seletivas quando comparado a M. bovis virulentos e M. tuberculosis. Por fim, a análise filogenética baseada em sítios polimórficos indica agrupamentos filogenéticos de M. bovis suportados pela classificação dos Complexos Clonais (CCs) e não por hospedeiros de origem dos isolados, confirmando que sítios polimórficos podem ser utilizados para classificação filogenética de linhagens genéticas desta espécie bacteriana. Além do mais, 2/28 (7,14%) genomas de M. bovis não puderam ser classificados nos CCs atualmente descritos, sugerindo a existência de complexos ainda não determinados. Este estudo representa o primeiro genoma de uma estirpe nacional de M. bovis a ser completamente sequenciado e a primeira análise de genômica comparativa de genomas desta espécie bacteriana.
Tuberculosis is an infectious disease caused by bacteria of the Mycobacterium tuberculosisComplex (MTBC) that affects human beings and/or animals. Members of this complex clonally evolved and have high genomic similarity, differentiated by single nucleotide polymorphisms (SNPs) and regions of difference (RDs). Among the animal tuberculosis pathogens, Mycobacterium bovis, the causative agent of bovine tuberculosis, is the MTBC member of greatest global importance. Therefore, the aim of the present study is to sequence, assemble and annotate the genome of the Brazilian strain SP38 of M. bovis, followed by the comparative genomics with other M. bovis genomes available in GenBank. Mycobacterium bovis SP38 has a traditional mycobacteria genome. It has a single and circular chromosome with 4,347,646 bp, high GC content (65.6%), and 4,216 genes, including 154 pseudogenes, 3 rRNA genes (ribosomal RNA), 45 tRNA (transfer RNA), 2 ncRNA (non-coding RNA), 1 tmRNA (transfer-messenger RNA), and 4,011 coding DNA sequences (CDSs) (NZ_CP015773.1). The majority of CDSs (2,805 - 69,93%) was annotated with function and 1,206 (30,07%) are hypothetical. For the comparative genomics analyses, the 31 genomes (complete and drafts) of M. bovis available in GenBank, 32 Mycobacterium bovis BCG and, 23 of Mycobacterium tuberculosis were chosen. In silico analysis of the RDs patterns resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. Orthologous gene analysis suggests that strains of M. bovis are under genomic decay. The quantification of polymorphic sites indicates the greater variability in absolute numbers (8,335 in M. tuberculosis, 3,448 in virulent M. bovis, and 1,088 in M. bovis BCG) and in pairwise comparisons (p≤0,05) of M. tuberculosis compared to virulent M. bovis and M. bovis BCG, suggesting that M. tuberculosis is under high evolutionary pressure. This is in contrast to the fact that M. bovis is capable of infecting a higher number of host species than M. tuberculosis. Most of these polymorphic sites are located in hypothetical CDSs (31.7% - 52.3%), being associated with PE/PPE family, and demonstrating a nonsynonymous mutations proportion of the following increasing order: M. bovis BCG, virulent M. bovis and M. tuberculosis (48.90%, 51.92% and 59.52%, respectively). This lower proportion of nonsynonymous mutations and the dissimilar functional categorization of CDSs with polymorphic sites indicates that M. bovis BCG is subjected to different selective pressure when compared to virulent M. bovis and M. tuberculosis. Finally, the phylogenetic analysis based on polymorphic sites indicates that the phylogenetic grouping of M. bovis is supported by Clonal Complexes (CCs), and not by the host of M. bovis isolates, confirming that polymorphic sites can be used for phylogenetic classification of genetic lineages of this bacterial species. Furthermore, 2/28 (7.14%) genomes of M. bovis could not be classified in the currently described CCs, suggesting the existence of complexes yet to be determined. This study represents the first genome of a Brazilian strain of M. bovis to be completely sequenced and the first comparative genomic analysis of the genomes of this bacterial species.

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Ferreira, Emerson Ramalho. "A revacinação com a BCG modula a produção de citocinas frente a antígenos do Mycobacterium leprae, em contatos menores de 15 anos de pacientes com hanseníase". reponame:Repositório Institucional da UFC, 2010. http://www.repositorio.ufc.br/handle/riufc/4787.

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FERREIRA, Emerson Ramalho. A revacinação com a BCG modula a produção de citocinas frente a antígenos do Mycobacterium leprae, em contatos menores de 15 anos de pacientes com hanseníase. 2010. 104 f. Dissertação (Mestrado em Microbiologia Médica) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2010.
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Studies in different populations have shown that vaccination with BCG provides partial protection against leprosy. However, need of boost BCG vaccination and your impact in response immune against antigens of Mycobacterium leprae is poorly understood, mainly in children who are contacts of these patients with leprosy, and constitute population of risk for illness. This study, we investigated the paper a second dose of BCG given to contacts of leprosy patients under the age of fifteen years in modulate the response imune. Blood samples of twenty five children, who received a first dose of BCG at birth, were collected immediately before and two months after BCG revaccination. Your peripheral blood mononuclear cells (PBMC) stimulated by proteins and peptides of M. leprae, were measured IFN-γ and IL-10 by an ELISA assay. Before revaccination the serum anti-PGL1 IgG and IgM isotypes were measured by ELISA assays performed with native PGL1- coated microplates, and PPD-skin reaction was measured 2 to 3 months after revaccination. The study was approved by the local Ethic Committee, number 006-08, May 7th, 2008. BCG revaccination can induces IFN-gamma and/or IL-10 production related to antigen and age, but did not correlated with PPD-skin reaction or anti-PGL1 levels. The PBMC’s production of IFN-gamma against MLT (total M. leprae antigen) after BCG was higher on children under 4 years old (N= 8, 4203,0 ± 539,2 pg/mL before BCG x 9141,0 ± 860,9 pg/mL after BCG, p=0,015, Mann-Whitney’s test). Children between 5 and 9 years old showed an increase either of IFN-gama levels (N=11, 4912 ± 1065 pg/mL x 9249,0 ± 1171 pg/mL, p=0,024, Mann-Whitney’s test) or IL-10 levels (N=11, 210,6 ± 39,4 pg/mL x 680,3 ± 76,74 pg/mL, p=0, 0,001, Mann-Whitney’s test). However children between 10 and 15 years old failed to increase the cytokines levels after BCG booster. Contacts of multibacillary patients produced higher IFN-gamma levels (N= 13, 4536,0 ± 543,1 pg/mL x 9263,0 ± 989,0 pg/mL, p=0,0012, Mann-Whitney’s test) and IL-10 levels (N= 13, 235,9 ± 46,5 pg/mL x 743,5 ± 87,8, p=0,0012, Mann-Whitney’s test) against MLT after BCG, but only IFN-gamma levels (N=12, 4975,0 ± 995,8 pg/mL x 8126,0 ± 788,6, p=0,0342, Mann-Whitney’s test) increased after BCG on contacts of paucibacillary patients. The production of IFN-gamma and IL-10 against two synthetic peptides (p38 and p67), before and after BCG vaccine, were similar as seen with MLT antigen, but the absence of cytokine production against p69 help to identify this peptide as a effective diagnostic marker.
Estudos em diferentes populações têm mostrado que a vacinação com a BCG concede proteção parcial contra a hanseníase. Entretanto, a necessidade da revacinação e seu impacto na resposta imune contra antígenos do Mycobacterium leprae é pouco compreendida, principalmente nas crianças que são contatos destes pacientes com hanseníase, e constituem uma população de risco para o adoecimento. Neste estudo, investigamos o papel da revacinação com a BCG na modulação da resposta imune em contatos menores de quinze anos de pacientes com hanseníase. Amostras de sangue periférico de vinte e cinco crianças, vacinadas com a BCG ao nascer, foram coletas antes e dois meses após a revacinação com a BCG. Suas células mononucleares (PBMC) foram estimuladas com proteínas e peptídeos do M. leprae, dosando-se as quantidades de IFN-γ e IL-10 por ELISA. Anteriormente a revacinação, foram coletas amostras para dosagem de IgM e IgG anti-PGL-1 e realizado o teste tuberculínico (PPD) para avaliação da eficácia vacinal após a revacinação. Identificamos o potencial papel da BCG em estimular a produção de IFN-γ e/ou IL-10 dependendo do antígeno e da idade do contato, sem relação com o PPD e sorologia anti-PGL-1. Estes resultados sugerem que a BCG modula a resposta imune dos contatos frente aos antígenos do M. leprae, com o frequente aumento nos níveis de IFN-γ, frente ao MLT, nas crianças com idade entre 1 a 4 anos (4.203 + 539,2 pg/mL pré-BCG x 9.141 + 860,9 pg/mL pós-BCG, p=0,001). Já os contatos entre 5 a 9 anos mostram aumento na produção de IL-10 (210 +39,4 pg/mL pré-BCG x 680 + 76,74 pg/mL pós-BCG, p=0,001) e IFN-γ (4.912 + 1.065 pg/mL x 9.249 + 1.171 pg/mL, p=0,024). Estes achados são acompanhados com o aumento dos casos de hanseníase em crianças entre 5 a 9 anos na comunidade. Entretanto, nos contatos entre 10 a 15 anos a revacinação falha em induzir o aumento de IFN-γ e IL-10. Os contatos de MB produziram, simultaneamente, altos níveis de IFN-γ e IL-10 em resposta ao MLT, enquanto que os contatos de PB somente produziram altos níveis de IFN-γ após a revacinação. A produção de IFN-γ e IL-10 frente aos peptídeos sintéticos p38 e p69 indica que estes antígenos podem ser possíveis marcadores de infecção. Assim, a BCG pode ser protetora, sendo eficiente em induzir o aumento da resposta por IFN-γ nestes contatos, frente aos antígenos do M. leprae.

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23

Tuffal, Gilles. "Analyse structurale et fonctionnelle des lipopolyméthylglucoses de Mycobacterium bovis BCG". Toulouse 3, 1995. http://www.theses.fr/1995TOU30281.

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La paroi des mycobacteries joue un role majeur dans la survie intramacrophagique des mycobacteries pathogenes telles que mycobacterium tuberculosis, l'agent etiologique de la tuberculose. Elle contient une forte proportion d'acides mycoliques, supposes former une barriere physique relativement impermeable aux molecules hydrophiles et en particulier aux antibiotiques polaires. Bien que les etapes clef de la biosynthese des acides mycoliques soient connues, aucun schema de biosynthese ne specifie l'origine des acides gras precurseurs. Ces derniers pourraient provenir du cytoplasme ou leur biosynthese est catalysee par l'acide gras synthetase de type i (ags i), une enzyme dont l'activite est modulee par des polysaccharides partiellement methyles: les lipopolymethylglucoses (lpmg) et les polymethylmannoses (pmm). L'analyse structurale des lpmg, realisee auparavant chez m. Smegmatis, a ete reprise sur la base de resultats de spectrometrie de masse indiquant que les pmg (la structure polysaccharidique des lpmg) de m. Bovis bcg forment un melange. Celui-ci a ete resolu en utilisant la chromatographie liquide d'echange d'anions a haut ph, chaque pmg purifie etant analyse par spectrometrie de masse. Cette strategie analytique nous a permis de caracteriser une dizaine de pmg isoles chez m. Bovis bcg ainsi que chez d'autres mycobacteries, se differenciant par leur degres de polymerisation et leur taux de methylation. Elle a aussi revele des differences de distribution des pmg entre souches a croissance rapide et lente. De plus, l'analyse par resonance magnetique nucleaire bidimensionnelle des pmg a permis de redefinir leur structure generale et surtout d'elucider la structure d'un nouveau compose present chez m. Bovis bcg, contenant un residu de 2-n-acetamido-2,6-didesoxy-glucopyranose. Dans le but de mieux cerner les proprietes des lpmg, les complexes stoechiometriques lpmg/palmityl-coa ont ete analyses en utilisant l'electrophorese capillaire couplee a la spectrometrie de masse

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24

Venisse, Anne. "Caractéristiques structurales et fonctionnelles du lipoarabinomannane de Mycobacterium bovis BCG". Toulouse 3, 1994. http://www.theses.fr/1994TOU30025.

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Le lipoarabinomannane (lam) est un antigene majeur de la paroi des mycobacteries et les fonctions biologiques attribuees au lam de mycobacterium tuberculosis le designent comme un immunomodulateur important de la reponse immunitaire de l'hote, voire comme un des facteurs de virulence de ce pathogene. L'objet de nos travaux a ete d'etablir la structure du lam de m. Bovis bcg, souche a la virulence attenuee utilisee comme vaccin antituberculeux, et d'etudier ses proprietes fonctionnelles comparativement a celles du lam d'une souche virulente de m. Tuberculosis. Les analyses par rmn bidimensionnelle homo- et heteronucleaire et la caracterisation, par hplc et fab-ms, d'oligosaccharides obtenus apres hydrolyse partielle du lam, ont permis de montrer que ces deux lams possedent la meme structure globale, avec des unites oligomannosyles presentes aux extremites des chaines arabinanes, unites decrites comme caracteristiques des souches virulentes. De plus, la masse moleculaire du lam a ete determinee pour la premiere fois avec precision par spectrometrie de masse grace a l'ionisation par desorption par laser assistee d'une matrice et est de l'ordre de 17,3 kda pour les deux molecules. Cette etude ainsi que l'analyse structurale plus approfondie du domaine mannane de la molecule montrent une heterogeneite importante de la molecule fonction du nombre d'unites osidiques et de phosphates. Apres mise au point du marquage du lam de m. Bovis bcg avec de la biotine, son interaction avec les cellules phagocytaires murines a ete mise en evidence par cytofluorimetrie. Cette liaison semble faire intervenir les residus mannosyles de la molecule qui pourrait constituer un mediateur de l'adhesion de la mycobacterie sur leurs cellules hotes, favorisant ainsi l'invasion de ces cellules. L'ensemble des resultats suggerent que le lam pourrait constituer un facteur de virulence intervenant de facon precoce dans l'infection

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25

PHILIPP, WOLGANG. "Organisation genomique de mycobacterium tuberculosis et de m. Bovis bcg". Paris 7, 1995. http://www.theses.fr/1995PA077246.

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Mycobacterium tuberculosis, l'agent causant la tuberculose humaine est la premiere source de mortalite due a un agent infectieux. Depuis une decennie, le developpement et l'application des outils de la biologie moleculaire ont bien fait progresser la recherche sur cet organisme qui etait auparavant difficilement abordable. Dans ce memoire nous decrivons la construction des cartes genomiques d'une souche virulente de m. Tuberculosis, la souche de reference h37rv, ainsi que pour la souche vaccinale m. Bovis bcg pasteur. Une carte integree du genome de m. Tuberculosis h37rv a ete realisee a l'aide de l'electrophorese en champs pulses et de l'agencement de cosmides. Le chromosome de m. Tuberculosis d'une taille de 4,4 mb est legerement (50-100 kb) plus grand que celui de bcg mais possede 16 copies de l'element d'insertion is6110 alors qu'un seul exemplaire se trouve chez le bcg. Plus de 180 marqueurs genetiques ont ete localises sur le chromosome de m. Tuberculosis. La comparaison affinee de ces chromosomes mycobacteriens a montre une forte conservation mais a neanmoins conduit a l'identification de plusieurs regions restructurees ou ayant subi des deletions. L'etude comparative des genomes de m. Tuberculosis et de m. Leprae a revele un assemblage de facon mosaique avec certaines parties extremement bien conservees et d'autres organisees differemment. Pour le moment, huit regions conservees d'une taille entre 30 et 200 kb ont pu etre identifiees. Les outils mis au point lors de cette etude serviront de base au sequencage total du genome de m. Tuberculosis et aideront a identifier des parties genomiques impliquees dans le pouvoir pathogene mycobacterien

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26

Abbas, Rabiya. "Genetic basis of survival of Mycobacterium bovis inside Acanthamoeba castellanii". Thesis, University of Surrey, 2011. http://epubs.surrey.ac.uk/843112/.

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Tuberculosis remains a major threat to human health accounting for 2 million annual deaths worldwide. M. bovis causes TB in cattle which is a serious issue in the UK. Mycobacteria are widely distributed in the environments that are also colonized by free living amoebae. In the present study, free-living amoeba (A. castellanii) has been used to study the genetic factors required for the intracellular survival of M. bovis. Role of region of difference 1 (RD1), isocitrate lyase (Rv0467), ClgR (Rv2745) and the VapC (Rv2548) toxin-antitoxin system was examined for survival in amoebae. While the role of RD1 in mycobacterial survival in amoebae could not be observed, isocitrate lyase and a transcriptional regulator (ClgR) might play some part in survival of M. bovis in A. castellanii. It was found that although the mycobacteria were able to remain inside the amoebae vacuoles, they were unable to control the pH as the vacuoles remained acidic. This is very interesting as it is in contrast to macrophages where the mycobacteria are controlling and keeping the phagosomal pH only weakly acidic. A library of ~2500 M. bovis mutants was also created and TraSH mutagenesis was performed to provide a systematic assessment of the importance of mycobacterial genes for intra-amoebic survival. The results indicate that perhaps Rv3087 plays some role in the ability of M. bovis to grow inside amoebae. Rv3087 is considered to be essential for growth in murine infection model and induced when tubercle bacilli are exposed to acidic conditions in vitro and in macrophages. However, these are preliminary findings that need to be confirmed by further research.

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Lima, Cristiane. "Análise funcional do regulador transcricional Rv3405c em Mycobacterium bovis BCG". reponame:Repositório Institucional da FIOCRUZ, 2013. https://www.arca.fiocruz.br/handle/icict/8057.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, Brasil
Em 2011 foram notificados quase 9 milhões de novos casos de tuberculose (TB), culminando em aproximadamente 2 milhões de mortes. A vacina atual contra tuberculose (BCG) apresenta eficácia variável na proteção de adultos contra a tuberculose pulmonar (0% - 80%), com isso destacamos a necessidade de maiores investigações sobre a vacina. A cepa da vacina utilizada no Brasil é a BCG Moreau. Este trabalho incide sobre a caracterização de Rv3405c, um regulador transcricional (repressor) presente em todas as cepas de BCG, porém truncado em BCG Moreau devido à perda da região genômica RD16. Analisamos por RT-PCR a expressão de genes adjacentes a rv3405c em BCG Pasteur e Moreau, comprovando que sua perda funcional afeta a expressão de rv3406, que codifica uma proteína envolvida no metabolismo do enxofre. O aumento da expressão de rv3406 em BCG Moreau pode representar um ganho funcional, devido à perda desse repressor transcricional (Rv3405) A fim de caracterizar melhor a sua função, rv3405c de BCG Pasteur e rv3406 de BCG Moreau foram clonados e expressos em E. coli. O soro policlonal produzido em camundongos imunizados contra a proteína purificada rRv3406 foi utilizado para analisar sua expressão sob diferentes condições de crescimento. Rv3406 é encontrada na fração intracelular e na superfície de BCG Moreau, não tendo sido detectado em BCG Pasteur. Rv3405c recombinante purificada foi usada no ensaio de retardo em gel (EMSA), confirmando a sua capacidade para se ligar a um motivo de DNA conservado, identificado na região intergênica entre rv3405c-rv3406, sugerindo seu papel na regulação destes genes e justificando a expressão constitutiva de rv3406 em BCG Moreau. Além disso, a adição de bile e seus componentes provoca o deslocamento da proteína desfazendo sua interação com DNA. A caracterização de rv3405c e suas funções é importante para uma análise de genômica funcional comparativa entre BCG Moreau e Pasteur. Este trabalho contribui para uma melhor compreensão da vacina brasileira contra a tuberculose
Tuberculosis (TB) was responsible for almost 9 million new cases in 2011, culminating in approximately 2 million deaths. The current approved TB vaccine (BCG) presents variable efficacy in adult protection against pulmonary TB (0%-80%), highlighting the need for further investigations on this vaccine. The vaccine strain used in Brazil is BCG Moreau. This work focuses on the characterization of Rv3405c, a transcriptional regulator (repressor) present in all BCG strains but truncated in BCG Moreau due to the loss of genomic region RD16. We have analysed the expression of adjacent genes in both BCG Moreau and Pasteur by RT-PCR finding that loss of Rv3405c impacts the expression of rv3406, encoding a protein involved in sulfur metabolism. Its increased expression in BCG Moreau may represent a functional gain through the loss of a transcription repressor. In order to better characterize their function, rv3405c from BCG Pasteur and rv3406 from BCG Moreau were cloned and expressed in E. coli. The polyclonal sera produced in mice immunized with purified rRv3406 were used to follow the expression of this protein under different growth conditions. Rv3406 is found in the intracellular fraction and the surface of BCG Moreau, and was not detected in BCG Pasteur. Purified recombinant Rv3405c was used in electrophoretic mobility shift assays (EMSA), confirming its ability to bind a conserved DNA motif identified in the intergenic region between rv3405c-rv3406, suggesting its role in regulating these genes and justifying the constitutive expression of rv3406 in BCG Moreau. Furthermore, the addition of bile and its components is able to displace Rv3405c from DNA. The characterization of rv3405c and its functions is important for the comparative functional genomics analysis of BCG Moreau and Pasteur. This work contributes to a better understanding of the Brazilian vaccine against TB.

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Gallant, James Luke. "The effect of glutamate homeostasis on the survival of M. bovis BCG". Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/97831.

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Thesis (MScMedSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of the tuberculosis disease, is estimated to infect a third of the world’s population and is therefore, arguably, the most successful human pathogen in recorded history. Immense efforts to understand the genetic factors and biochemical processes underlying the complex interactions between M. tuberculosis and its host cells have delivered staggering insights into the profound proficiency by which this bacterium establishes and maintains an infection. It is now clear that M. tuberculosis can interfere with the immune responses initiated by host cells in such a manner as to subvert the various bactericidal conditions established by these cells and thus eliminate the tubercle bacilli that infect them. Specific characteristics of M. tuberculosis which provide it with this ability include a nearly impenetrable cell wall, secretion systems which secrete special factors which directly interact with host immune factors. This enables M. tuberculosis to modulate the activities of the host environment and unique metabolic adaptations of M. tuberculosis allows the organism to survive in the hypoxic, oxidative, nitrosative, acidic and nutrient poor environment of immune cell phagosomes and to persist for decades in a quiescent state in otherwise healthy people. New observations into the pathways which constitute energy, carbon and central nitrogen metabolism, among others, in M. tuberculosis, suggest that a carefully orchestrated homeostasis is maintained by the organism which may modulate the concentrations and ameliorate the effect of molecules that are important to defensive strategies employed by host cells. Here we discuss various recent studies as well as new information provided by this study, focusing on central metabolism and its regulation in M. tuberculosis. We aim to highlight the importance of nitrogen metabolism in the subversive response employed by M. tuberculosis to survive, colonise and persist in the host. We argue that the homeostatic regulation of nitrogen metabolism in M. tuberculosis presents a profound vulnerability in the pathogen which should be exploited with compounds that inhibit the activities of various effector proteins found in this pathway and that are unique to the organism. Such compounds may provide valuable novel chemotherapies to treat tuberculosis patients and may alleviate the burden of multiple drug resistance which plagues tuberculosis treatments. Specifically, in this study we investigate the role of M. bovis BCG glutamate dehydrogenase (GDH) and glutamate synthase (GltS) by subjecting knockout mutants of the aforementioned gene products to various cellular stress conditions. Furthermore, we investigated how the genomes of each M. bovis BCG strain was affected post deletion of the aforementioned protein products. The role of GDH was also tested in an murine macrophage model of infection to elucidate potential importance to colonisation and infection. This study provides novel results indicating an importance of GDH toward the resistance of nitrosative stress as well as a requirement for optimal persistence in RAW 264.7 macrophages. In addition, it was found that GltS is dispensable for resistance against nitrosative stress.
AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat die aansteeklike siekte tuberkulose veroorsaak, infekteer ongeveer ‘n dêrde van die wêreld populasie en is daarom, waarskynlik, een van die mees suksesvolle menslike patogene in geskiedenis. In die afgelope jare is daar noemenswaardige poging aangewend om genetiese faktore sowel as biochemiese prosesse te verstaan wat die komplekse interaksies tussen M. tuberculosis en sy gasheer selle verduidelik. Dit is nou voor die hand liggend dat M. tuberculosis kan inmeng met die reaksies van die immuun sisteem, om dus die bakteriosidiese omgewing wat geskep word deur die selle van hierde sisteem te vermy. Daar is spesifieke kenmerke van M. tuberculosis wat toelaat dat die bacilli so ‘n omgewing kan weerstaan. Hierdie kenmerke is, onder andere, ‘n byna ondeurdringbare selwand en uitskeiding sisteme wat spesiale faktore vrystel. Hierdie faktore het die vermoë om direk met die gasheer immuun sisteem ‘n interaksie te hê wat dus die immuun sisteem moduleer. Verder, is M. tuberculosis se metabolisme aan gepas om die organisme te help teen die lae suurstof, hoë oksidatiewe en stikstof stress, lae pH en lae voedingswaarde omgewing te oorleef. M. tuberculosis het ook die vermoë om vir ‘n onbeperkte tyd in ‘n statiese toestand te oorleef, in gashere wat toon as gesond. Nuwe waarnemings in die energie, koolstof en sentrale stikstof metaboliese paaie stel voor dat ‘n homeostase gehandhaaf word deur M. tuberculosis, wat die konsentrasies van verskeie molekules moduleer of die effek van molekules wat deur die gasheer vrygestel word as ‘n verdedigings meganisme versag. In hierdie dokument bespreek ons verskeie studies, asook nuwe inligting voortgebring deur hierdie studie, wat fokus op sentrale metabolisme en sy regulering in M .tuberculosis. Ons raak aan die vermoë van M. tuberculosis om intrasellulêr te oorleef, koloniseer en voort te bestaan in ‘n gasheer. Ons vemoed dat die homeostatiese regulering van stikstof metabolisme in M. tuberculosis n diepgaande kwesbaarheid in die patogeen skep wat die potentiaal het om uit gebuit te word. Molekules kan gesintiseer word wat die aktiwiteite van verskeie ensieme in hierdie padweg inhibeer en sodoende die organisme hinder. Sulke molekules mag dalk as waardevolle en oorspronklike medisynes ontwikkel word om tuberkulose patiënte meer suksesvol te behandel asook om die las van middelweerstandige bakterieë te verlig. Met betrokke tot hierdie spesifieke studie, het ons die rol van glutamaat dehidrogenase (GDH) en glutamaat sintase (GltS) van M. bovis BCG bestudeer deur om die uitslaan mutante van die genoemde geen produkte aan verskeie sellulêre stress toestande bloot te stel. Die effek van die verlore gdh en gltBD gene op die evolusie van die genome van elke M. bovis BCG uitslaan mutant ras ten opsigte van die wilde tipe was ook bestudeer. Die rol van GDH was getoets in ‘n muis makrofaag model van infeksie om te bepaal of GDH n funksie het in koloniseering en infeksie van M. bovis BCG. Hierdie studie het nuwe bevindinge voort gebring wat die belangrikheid van GDH in die weerstand teen stikstof oksied stress. Daar is verder bevind dat GDH n vereiste toon vir die suksessvolle oorlewing van M. bovis BCG in RAW 264.7 macrofage

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Blankenheim, Thalita Masoti [UNESP]. "Resposta à tuberculinização em bovinos sensibilizados com inóculos inativados de Mycobacterium avium e de Mycobacterium bovis". Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/141991.

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A tuberculose causada pelo Mycobacterium bovis é uma importante doença dos bovinos e constitui um grande problema de saúde animal, podendo também atingir humanos. Para o diagnóstico da infecção, e para desencadear as medidas sanitárias decorrentes desse diagnóstico, o Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose (PNCEBT) estabelece a utilização de testes intradérmicos de tuberculinização. O objetivo deste estudo foi avaliar as respostas à tuberculina (PPD) aviária e à tuberculina bovina apresentadas por animais sensibilizados com inóculos inativados de M. bovis e de M. avium, e comparar os resultados do teste da prega caudal (TPC), do teste cervical simples (TCS) e do teste cervical comparativo (TCC) para diagnóstico da tuberculose bovina nos animais sensibilizados e em animais não sensibilizados. Os resultados mostraram que: a repetição dos testes não influiu na proporção de resultados positivos; houve animais sensibilizados com M. bovis que apresentaram reação até 500 dias após a sensibilização; em animais sensibilizados com M. avium, a especificidade do TCC foi superior à do TCS e à do TPC, e o TCC mostrou-se efetivo para discriminar reações induzidas pelo inóculo desse microrganismo; em animais sensibilizados com M. bovis, o TCC apresentou menor sensibilidade do que os outros dois testes; o ponto de corte do TCS e do TCC com melhor combinação de sensibilidade e especificidade foi inferior ao ponto adotado pelo PNCEBT para diagnóstico em animais naturalmente infectados; o TCS apresentou boa concordância com os outros dois testes, mas a concordância entre o TCC e o TPC foi apenas regular; as frequências das repostas às tuberculinas não apresentaram distribuição gaussiana, a não ser em alguns períodos pós-sensibilização ou então após transformação radicial ou logarítmica dos dados.
Tuberculosis caused by Mycobacterium bovis is an important disease in cattle e a great problem for animal health that can reach humans. For the diagnosis of the infection and the consequent sanitary measures, the National Program for Control and Eradication of Brucellosis and Tuberculosis (PNCEBT) establish the use of intradermal tuberculin tests. The aim of this study was to analyze the response to the avian and bovine tuberculin (PPD) developed by cattle sensitized with inactivated inoculum of M. avium and M. bovis. Another aim was to compare the results of the caudal fold test (CFT), the comparative cervical test (CCT), and the simple cervical test (SCT) for tuberculosis diagnosis in the sensitize animals and in animals that have not been sensitized. Repetition of the tests did not influence the proportion of positive results. There were animals sensitized with M. bovis showing reaction up to more than 500 days post sensitization. In animals sensitized with M. avium, the specificity of the CCT was higher than that of CFT and SCT, and CCT was able to discriminate the unspecific reaction induced by M. avium inoculum. In animals sensitized with M. bovis, CCT had lower sensitivity than the other two tests. The SCT and CCT cut-off with the best combination of sensitivity and specificity was lower than that adopted by the PNCEBT for the tuberculosis diagnosis in naturally infected animals. SCT hat good agreement with the other two tests, but the agreement between CFT and CCT was only moderate. The frequency of the responses to tuberculin did not show Gaussian distribution, but in some post sensitization periods or after square root or logarithmic transformation of the data.

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Blankenheim, Thalita Masoti. "Resposta à tuberculinização em bovinos sensibilizados com inóculos inativados de Mycobacterium avium e de Mycobacterium bovis /". Jaboticabal, 2016. http://hdl.handle.net/11449/141991.

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Orientador: Luis Antonio Mathias
Banca: Anna Monteiro Correia Lima
Banca: Adolorata Aparecida Bianco Carvalho
Banca: Raphaella Barbosa Meirelles Bartoli
Banca: Samir Issa Samara
Resumo: A tuberculose causada pelo Mycobacterium bovis é uma importante doença dos bovinos e constitui um grande problema de saúde animal, podendo também atingir humanos. Para o diagnóstico da infecção, e para desencadear as medidas sanitárias decorrentes desse diagnóstico, o Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose (PNCEBT) estabelece a utilização de testes intradérmicos de tuberculinização. O objetivo deste estudo foi avaliar as respostas à tuberculina (PPD) aviária e à tuberculina bovina apresentadas por animais sensibilizados com inóculos inativados de M. bovis e de M. avium, e comparar os resultados do teste da prega caudal (TPC), do teste cervical simples (TCS) e do teste cervical comparativo (TCC) para diagnóstico da tuberculose bovina nos animais sensibilizados e em animais não sensibilizados. Os resultados mostraram que: a repetição dos testes não influiu na proporção de resultados positivos; houve animais sensibilizados com M. bovis que apresentaram reação até 500 dias após a sensibilização; em animais sensibilizados com M. avium, a especificidade do TCC foi superior à do TCS e à do TPC, e o TCC mostrou-se efetivo para discriminar reações induzidas pelo inóculo desse microrganismo; em animais sensibilizados com M. bovis, o TCC apresentou menor sensibilidade do que os outros dois testes; o ponto de corte do TCS e do TCC com melhor combinação de sensibilidade e especificidade foi inferior ao ponto adotado pelo PNCEBT para diagnóstico em animais n... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Tuberculosis caused by Mycobacterium bovis is an important disease in cattle e a great problem for animal health that can reach humans. For the diagnosis of the infection and the consequent sanitary measures, the National Program for Control and Eradication of Brucellosis and Tuberculosis (PNCEBT) establish the use of intradermal tuberculin tests. The aim of this study was to analyze the response to the avian and bovine tuberculin (PPD) developed by cattle sensitized with inactivated inoculum of M. avium and M. bovis. Another aim was to compare the results of the caudal fold test (CFT), the comparative cervical test (CCT), and the simple cervical test (SCT) for tuberculosis diagnosis in the sensitize animals and in animals that have not been sensitized. Repetition of the tests did not influence the proportion of positive results. There were animals sensitized with M. bovis showing reaction up to more than 500 days post sensitization. In animals sensitized with M. avium, the specificity of the CCT was higher than that of CFT and SCT, and CCT was able to discriminate the unspecific reaction induced by M. avium inoculum. In animals sensitized with M. bovis, CCT had lower sensitivity than the other two tests. The SCT and CCT cut-off with the best combination of sensitivity and specificity was lower than that adopted by the PNCEBT for the tuberculosis diagnosis in naturally infected animals. SCT hat good agreement with the other two tests, but the agreement between CFT and CCT was... (Complete abstract click electronic access below)
Doutor

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Sigola, Lynnette Brenda. "The role of innate immunity in the host response to Mycobacterium bovis". Thesis, London School of Hygiene and Tropical Medicine (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265950.

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Kanno, Alex Issamu. "Expressão de antígenos de Schistosoma mansoni em BCG recombinante". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-14052014-095103/.

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O BCG é um bacilo atenuado de Mycobacterium bovis atualmente investigado na expressão de antígenos heterólogos. Genes oriundos de vírus, bactérias e parasitas são clonados em vetores de expressão micobacterianos e a partir deles, são geradas cepas de BCG recombinante, rBCG. Duas cepas micobacterianas, BCG e M. smegmatis, demonstraram a expressão do antígeno SmStoLP-2 e animais imunizados com este BCG não demonstra diferença na proteção contra a infecção por cercárias, apesar da resposta imune diferenciada. A otimização do códon de SmTSP-2 e SmVAL-5 permitiu sua expressão em M. smegmatis recombinante, mas não em BCG. M. smegmatis transformado com uma biblioteca de plasmídeos contendo o promotor L5p mutagenizado à montante de egfp demonstra acentuada diferença na fluorescência. 3 destes plasmídeos definidos como \'\'forte\'\' e \'\'fraco\'\', com base em sua capacidade de produzir GFP foram utilizados para expressar em BCG o antígeno Sm29 em maior e menor nível.
BCG is an attenuated bacillus derived from Mycobacterium bovis currently being investigated to express foreign antigens. Genes from viruses, bacteria and parasites are cloned to mycobacterial vectors and with them, recombinant BCG strains are created. Recombinant BCG and M. smegmatis strains where capable to express SmStoLP-2 and mice immunized with these rBCGs do not show better protection against infection with cercariae, although the distinct immune response observed. The use of codon optimization made possible the expression of SmTSP-2 and SmVAL-5 in M. smegmatis but not BCG. M. smegmatis transformed with a library of plasmids containing the L5p upstream egfp gene show a wide range in fluorescence. 3 of these plasmids strong and weak defined as their ability to produce GFP were used to express in BCG the antigen Sm29 at different levels by each promoter.

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Dib, Cristina Corsi. "Utilização de uma técnica rápida para o isolamento de Mycobacterium bovis a partir de amostras de leite experimentalmente inoculadas". Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-13082007-103556/.

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A técnica de camada delgada em placas com meio de Middlebrook 7H11 foi comparada com a técnica padrão de cultura em meio de Stonebrink, a fim de se avaliar a sensibilidade e o tempo de detecção de micobactérias em amostras de leite, experimentalmente inoculadas com Mycobacterium bovis (estirpe AN5), em uma diluição 10-2, e submetidas a duas diferentes técnicas de processamento, utilizando-se da gordura (técnica 1)e do sedimento (técnica 2), descontaminadas pelo método de Petroff modificado (adicionado de Tween 80) e confrontadas com a técnica do leite total submetida ao método de Petroff e semeadas nos dois meios. Os resultados destas técnicas (1 e 2) foram comparados entre si pelo teste não paramétrico de Wilcoxon, e entre os resultados obtidos na técnica de leite total submetida ao método tradicional de Petroff, por meio do teste não paramétrico de Mann-Whitney. Posteriormente, amostras de leite nas diluições 10-3, 10-4 e 10-5 foram então submetidas às mesmas técnicas de processamento e descontaminação para averiguação de sensibilidade. Os resultados obtidos demonstraram que: 1) a técnica de cultivo de micobactérias em amostras de leite no meio de Middlebrook 7H11 se mostrou viável frente à tradicional; 2) a técnica de camada delgada permitiu a visualização precoce das micobactérias quando comparadas ao meio de Stonebrink; 3) as técnica 1 e 2 forneceram maior recuperação de micobactérias e maior proporção de cultivos positivos nos dois empregados; 4) a técnica de camada delgada em meio de Middlebrook 7H11 modificado pode ser usada como uma técnica complementar aos métodos tradicionais de diagnóstico da tuberculose bovina em amostras de leite para fins de vigilância epidemiológica.
The modified thin layer Middlebrook 7H11 cultivation technique was compared to the tradicional culture technique using the Stonebrink in order to evaluate the sensibility and the time for the detection of positive cultures of mycobacteria in milk samples, experimentally inoculated by Mycobacterium bovis (strain AN5), 10-2 dilution, were submitted by two types of procedures named technique 1 (milk fat) and 2 (milk sediment), descontamined by modified Petroff method, added with Tween 80, and compared with total milk samples submitted to tradicional Petroff method, in the same media types. The results of the two tecniques were compared within each other by the non-parametric Wilcoxon test, and within the results of standard milk submitted by the tradicional Petroff method, by the non-parametric Mann-Whitney. Milk samples were diluted to 10-3, 10-4 and 10-5 and submitted by the tecniques 1 and 2 descontaminated by the modified Petroff method, and total milk by tradicional Petroff method to averiguate the sensibility. The results found in this experiment demonstrated that 1) the modified thin layer technique of isolation of mycobacteria in milk samples was practicable against the tradicional one; 2) the time needed for the detection of mycobacteria colonies was slightly less with the thin layer technique than the tradicional culture method; 3) techniques 1 and 2 gave more number of colonies and more proportion of positive culture than the tradicional one in all media used; 4) this technique should be used as a complementary method to the tradicional one in the diagnostic of bovine tuberculosis from milk samples in orther to improve epidemiologic vigilance.

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Morato, Flávia. "Avaliação da atividade micobactericida de desinfetantes químicos utilizando a técnica de cultivo em camada de ágar Middlebrook 7H11". Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-15082007-093534/.

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Avaliou-se a atividade micobactericida de cinco desinfetantes químicos frente a uma estirpe de Mycobacterium bovis isolada de caprinos, tipificada por PCR (polymerase chain reaction) e com 32 dias de cultivo no meio de Stonebrink. O teste de desinfetantes foi realizado utilizando-se a técnica de cultivo em camada delgada de ágar Middlebrook 7H11 modificado e foi comparado ao teste em tubos com meio de Stonebrink, tradicionalmente utilizado no laboratório de zoonoses bacterianas da FMVZ/USP. Os cinco desinfetantes ensaiados foram: \"A\" : grupo controle; \"B\" - hipoclorito de sódio (2,5 % de cloro ativo); \"C\"- glutaraldeído (2 %); \"D\" - ácido peracético 0,25 % e peróxido de hidrogênio 5 %; \"E\" - iodóforo (2,6% de iodo) e \"F\"- compostos fenólicos (orto-fenilfeno 12,243 g; orto-benzil paraclorofenol 11,080 g; para-terceário amilfeno 4,1222 g.). A diluição destes produtos foi feita conforme recomendação do fabricante. Os meios de cultura adotados para o procedimento de isolamento e preparo da suspensão bacteriana foram o meio de Stonebrink e o meio de Middlebrook 7H11 modificado. Os testes foram realizados na presença e ausência de matéria orgânica e à temperatura ambiente (21 ± 2°C) e à temperatura de 4 °C. Os resultados obtidos nas contagens de colônias foram transformados em percentual de redução para análise estatítica e demostraram que: a técnica de cultivo de micobactérias em camada delgada no meio de Middlebrook 7H11 permitiu uma visualização precoce das micobactérias e se mostrou viável para realização de testes de desinfetantes; os cinco tipos de desinfetantes analisados apresentaram atividade micobactericida e o melhor desempenho foi obtido pelo ácido peracético seguido pelo hipoclorito de sódio. A atividade micobactericida dos iodóforos foi instisfatória na presença de matéria orgânica.
The mycobactericidal activity of five chemical disinfectants was evaluated against a strain of Mycobacterium bovis isolated from a goat, typified by PCR (polymerase chain reaction) and with 32 days of growth in the Stonebrink medium. The disinfectants were tested using the modified thin layer Middlebrook 7H11 cultivation technique and it was compared to the test made in tubes with Stonebrink medium, which is tradicionally used at the Bacterian Zoonosis Laboratory of the Veterinary Medicice Faculty of the University of São Paulo. The five disinfectants were: \"A\" was the control group; \"B\"- sodium hypochlorite (2,5% of active chlorine); \"C\"- glutaraldehyde (2 %); \"D\"- peracetic acid (0,25 %) and hydrogen peroxide (5 %); \"E\" - iodine compounds (2,6%) e \"F\"- fenolic compounds (orto-fenilfeno 12,243 g; orto-benzil paraclorofenol 11,080 g; para-terceario amilfeno 4,122 g.). The products were diluted according to label instructions. The culture media used for the isolation procedure and preparation of the bacterian suspension were the Stonebrink and modified Middlebrook 7H11 medium. The assays were performed either in the presence or absence of organic matter, at temperatures of 4 °C and 21 ± 2°C The colony counting results were transformed into reduction percentages for the statistical analysis and concluded in: the modified thin layer Middlebrook 7H11 cultivation technique permitted an earlier visualization of the colonies and was practible for the realization of the disinfectants tests; the five disinfectants showed mycobactericidal activity and the peracetic acid had the best performance followed by the sodium hypochlorite. The mycobactericidal activity of the iodine compound was unsatisfactory when in the presence of organic matter.

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Ambrosio, Simone Rodrigues. "Métodos bacteriológicos aplicados à tuberculose bovina: comparação de três métodos de descontaminação e de três protocolos para criopreservação de isolados". Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-31102006-162337/.

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Dada a importância do Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT), a necessidade de uma eficiente caracterização bacteriológica dos focos como ponto fundamental do sistema de vigilância e as dificuldades encontradas pelos laboratórios quanto aos métodos de isolamento de Mycobacterium bovis fizeram crescer o interesse do meio científico por estudos, sobretudo moleculares, de isolados M. bovis. Para a realização dessas técnicas moleculares, é necessária abundância de massa bacilar, obtida através da manutenção dos isolados em laboratório e repiques em meios de cultura. Entretanto o crescimento fastidioso do M. bovis em meios de cultura traz grandes dificuldades para essas operações. Assim sendo, o presente estudo teve por objetivos: 1º) Comparar três métodos de descontaminação para homogeneizados de órgãos, etapa que precede a semeadura em meios de cultura, onde 60 amostras de tecidos com lesões granulomatosas, provenientes de abatedouros bovinos do Estado de São Paulo, foram colhidas e imersas em solução saturada de Borato de Sódio e transportadas para o Laboratório de Zoonoses Bacterianas do VPS-FMVZ-USP, onde foram processadas até 60 dias após a colheita. Essas amostras foram submetidas a três métodos de descontaminação: Básico (NaOH 4%), Ácido (H2SO4 12%) e 1- Hexadecylpyridinium chloride a 1,5% (HPC) e o quarto método foi representado pela simples diluição com solução salina (controle). Os resultados foram submetidos à comparação de proporções, pelo teste de χ², na qual verificou-se que o método HPC foi o que apresentou menor proporção de contaminação (3%) e maior proporção de sucesso para isolamento de BAAR (40%). 2º) Comparar três diferentes meios criopreservates para M. bovis, foram utilizados 16 isolados identificados pela técnica de spoligotyping. Cada um desses isolados foi solubilizado em três meios (solução salina, 7H9 original e 7H9 modificado), e armazenado em três diferentes temperaturas (-20ºC, -80ºC e -196ºC), sendo descongelado em três diferentes tempos (45, 90 e 120 dias de congelamento). Antes do congelamento e após o descongelamento foram feitos cultivos quantitativos em meios de Stonebrink Leslie. Os porcentuais de redução de Unidades Formadoras de Colônias (UFC) nas diferentes condições foram calculados e comparados entre si através de métodos paramétricos e não-paramétricos. Os resultados obtidos foram: na análise da variável tempo, em 90 dias de congelamento foi observada uma maior proporção de perda de M. bovis, quando comparado ao tempo 120 dias (p=0,0002); na análise da variável temperatura, foi observada uma diferença estatística significativa entre as proporções de perda média nas temperaturas de -20ºC e -80ºC (p<0,05); na análise da variável meio, foi observada uma diferença significativa (p=0,044) entre os meios A e C, para 45 dias de congelamento e -20ºC de temperatura de criopreservação. Embora as medianas dos porcentuais de perdas de UFC terem sido sempre inferiores a 4,2%, permitiram sugerir que o melhor protocolo de criopreservação de isolados de M. bovis é solubilizá-los em 7H9 modificado e mantê-los à temperatura de -20ºC
In the context of the National Program of Control and Eradication of Brucellosis and Tuberculosis (PNCEBT), the necessity of an efficient bacteriologic characterization of the infected herds as a cornerstone of the monitoring system and the difficulties faced by the laboratories regarding the methods for Mycobacterium bovis isolation led to a growing interest for scientific studies, especially molecular, of M. bovis isolates. To use these molecular techniques it is necessary to have an abundant bacillary mass, obtained through the maintenance of isolates in laboratory and replication in culture media. However the fastidious growth of M. bovis in culture media brings out great difficulties for these activities. Thus, the present study has the following objectives: First, to compare three decontamination methods for organ homogenates, phase that precedes the sowing in culture media, 60 samples of tissues with granulomatosis injuries, proceeding from bovine slaughterhouses in the State of São Paulo, were obtained, immersed in sodium borato saturated solution and transported to the Laboratório de Zoonoses Bacterianas of the VPS-FMVZ-USP, where they were processed up to 60 days after the sampling. These samples were submitted to three methods of decontamination: Basic NaOH 4%, Acid (H2SO4 12%) and 1- Hexadecylpyridinium chloride (HPC) 1.5% and a simple dilution with saline solution (control method). The results were analysed by means of the &chi test to compare proportions, and it was verified that HPC method presented the smallest proportion of contamination (3%) and the greatest proportion of success for M. bovis isolation (40%). Second, to compare three different cryopreservation media for M. bovis, 16 isolates identified by the technique of spoligotyping were used. Each one of these isolates was solubilized in three media (original saline solution, 7H9 and 7H9 modified), and stored in three different temperatures (-20ºC, -80ºC and -196ordm;C), and defrosted in three different time periods (45, 90 and 120 days of freezing). Before the freezing and after the unfreezing, quantitative cultivations in Stonebrink Leslie media were carried out. The proportions of Colony-Forming Units (CFU) loss in the different conditions were calculated and compared with one another through parametric and non-parametric methods. The results obtained were: in the analysis of the variable time, at 90 days of freezing a bigger proportion of CFU loss was observed when compared to 120 days (p=0,0002); in the analysis of the variable temperature, a statistically significant difference was observed between the average proportions of CFU loss for the temperatures of -20ºC and -80ºC (p<0,05); in the analysis of the variable media, a significant difference was observed (p=0,044) between the media A and C, for 45 days of freezing and -20ºC of cryopreservation temperature. Althougth the medium ones of the proportion of losses of CFU to always have been inferior 4,2%, had allowed to suggest that the best protocol for cryopreservation of M. bovis isolates is to solubilize them in 7H9 modified medium and to keep them at a temperature of -20ºC

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Agunso, Camila Noia. "Enriquecimento seletivo para pesquisa de Mycobacterium bovis em leite e queijo". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-22042014-162454/.

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O Mycobacterium bovis causa a tuberculose bovina, doença zoonótica que propaga, provavelmente com baixa prevalência, em todo o território nacional e pode ser transmitida pelo leite. A adoção sistemática da pasteurização do leite contribuiu para a redução dos casos humanos da doença, mas em alguns países, como o Brasil, é comum o consumo de leite cru e de seus derivados, o que pode contribuir para ocorrência de casos humanos. A participação desse agente nos atuais índices de tuberculose humana, cujo principal agente é o M. tuberculosis, é pouco investigada, mas acredita-se que seja maior do que parece. As razões que contribuem para isso incluem o fato do tratamento humano ser o mesmo independentemente do agente,e as dificuldades e alto custo de distinguir as espécies. O método de diagnóstico \"gold standard\" em laboratório para Mycobacterium bovis em amostras clínicas é o isolamento do agente em meios como Stonebrink-Leslie ou similares. A riqueza em nutrientes dos meios, mais o lento metabolismo deste agente e o alto grau de contaminantes das amostras torna imprescindível o uso de descontaminantes que, via de regra, são tóxicos para o agente, dificultando o isolamento em amostras com níveis baixos do patógeno; cenário provável no caso do leite devido à mistura com o leite de animais sadios. Mas, a despeito de constituir-se uma importante zoonose transmitida pelo leite, não existe uma metodologia oficial para detecção desse agente em alimentos lácteos. Esses fatos justificam um estudo para avaliar o desempenho de meios líquidos, já utilizados em caros sistemas automatizados para detecção de micobactérias, como alternativa para um enriquecimento seletivo do M. bovis em amostras lácteas. Assim, amostras de leite integral esterilizado e de queijo tipo parmesão foram contaminadas com 10 a 100 UFC/ml ou g de M. bovis AN5 e enriquecidas em dois meios líquidos seletivos (MGIT e MGIT modificado), foram analisados nos dias 0, 7, 14, 21, 24, 28 e 32, mantidas em estufa a 37°C. Nessas datas, uma alíquota foi semeada em meio Stonebrink-Leslie e incubada a 37°C por 60 dias. No leite, houve crescimento exacerbado do M. bovis principalmente no MGIT modificado. No queijo, não foi possível isolar M. bovis de nenhuma amostra em MGIT modificado, devido à contaminação excessiva que deteriorou o meio de cultura. O MGIT modificado foi mais eficaz como enriquecimento para o Mycobacterium bovis, mas foi menos seletivo que o MGIT. Estudos futuros devem concentrar a investigação da curva de crescimento do agente na primeira semana de enriquecimento.
Mycobacterium bovis causes bovine tuberculosis, zoonotic disease raging, probably with low prevalence, throughout the national territory and can be transmitted through the milk. The systematic adoption of milk pasteurization helped reduce human cases of the disease, but in some countries, like Brazil, is the common consumption of raw milk and its derivatives, which may contribute to the occurrence of human cases. The participation of this agent in the current rates of human tuberculosis, the principal agent is M. tuberculosis, it is not investigated, but it is believed to be larger than it looks, the reasons contributing to this include the fact that human treatment is similar regardless of the agent and the difficulty / cost to distinguish between species. The diagnostic method \"gold standard\" for Mycobacterium bovis in clinical samples is the isolation of the agent means, Leslie as Stonebrink or the like. The species richness in nutrient media, over the slow metabolism this agent and the high degree of contamination of the samples makes indispensable using decontaminant that, as a rule, are toxic to the agent, making isolation in samples with low levels of the pathogen; scenario likely due to the milk mixture with the milk of healthy animals. But, despite constitute an important zoonosis transmitted by milk, there is no official method for detection of this agent in dairy foods. These facts justify a study to evaluate the performance of liquid media, as used in expensive automated systems for detection of mycobacteria, as alternative to a selective enrichment of M. bovis in milk samples. Thus, samples of sterilized milk and Parmesan cheese type were infected with 10 to 100 CFU / ml or g of M bovis AN5 and enriched in two selective liquid media (MGIT and modified MGIT) were analyzed on days 0, 7, 14, 21, 24, 28 and 32, maintaining at 37 ° C. On that date, an aliquot was plated on Stonebrink half- Leslie and incubated at 37 ° C for 60 days. In milk, there was overgrowth of M. bovis mainly in MGIT modified. Cheese, it was not possible to isolate M. bovis no sample in MGIT modified due to excessive contamination it deteriorated the culture medium. The modified MGIT was more effective as enrichment for Mycobacterium bovis, but was less selective than the MGIT. Future studies should focus on investigating the growth curve of the agent in the first week of enrichment.

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Patiño, Suaza Ana Eugenia. "Comparación de métodos para el diágnostico de Mycobacterium bovis en Chile". Tesis, Universidad de Chile, 2015. http://repositorio.uchile.cl/handle/2250/145135.

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Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias mención Medicina Preventiva Animal.
Con el objetivo de implementar métodos que disminuyan los tiempos de respuesta diagnóstica y por ende proporcionen rapidez en la emisión del resultado de tuberculosis bovina, se evaluaron y compararon los resultados obtenidos por los métodos BACTEC MGIT 960, cultivo bacteriológico en medio Stonebrink (ST), reacción en cadena de la polimerasa en tiempo real (RT-PCR), e Histopatología. Todo el trabajo se realizó en el Laboratorio de Micobacterias del Subdepartamento Laboratorios y Estación Cuarentenaria Pecuaria del Servicio Agrícola y Ganadero (SAG), siguiendo los protocolos establecidos por el laboratorio. Se utilizaron 100 muestras de tejido obtenidas de nódulos linfáticos y/u órganos con lesiones del tipo granulomatosas (LTG), provenientes de bovinos de plantas faenadoras o por inspección de seguimiento. Los resultados mostraron que el cultivo en el sistema BACTEC MGIT 960 puede disminuir el tiempo de detección de Mycobacterium bovis y favorecer la rapidez en la emisión del resultado en el diagnóstico de la Tuberculosis Bovina (TBB). En combinación las cuatro pruebas detectaron 89 muestras positivas y todas tuvieron concordancia positiva entre sí, que varió entre débil a moderada, según los índices de Kappa, con significancia estadística (p < 0,05). En cuanto a la eficiencia, cada método sobresalió por un parámetro diferente, siendo RT-PCR menos compleja y más rápida, los cultivos los más económicos comercialmente y de mayor certidumbre, aunque la Histopatología resultó con el mejor nivel de eficiencia combinado. Se propone implementar un esquema que incluya los cuatro métodos (histopatológico, molecular, un cultivo en medio sólido y un cultivo en medio líquido), que permita detectar el número máximo de resultados positivos, en el menor tiempo posible y con alta certidumbre.
In order to implement methods to shorten the time of growth and thus provide speed in issuing the results in the diagnosis of bovine tuberculosis were evaluated and compared the results obtained by the methods BACTEC MGIT 960, bacteriological culture amid Stonebrink (ST ), polymerase chain reaction in real time (RT-PCR), and histopathology. All work was performed in the Laboratory of mycobacteria at Subdepartment of Laboratories and Quarantine Station of the Agricultural and Livestock Service (SAG), following the protocols established by the Laboratory. 100 tissue samples obtained from bovine lymph nodes and / or organs with granulomatous lesions (LTG) were used. The results showed that cultivation in the MGIT 960 system can shorten the growth of Mycobacterium bovis and the time required for diagnosis of bovine tuberculosis. In combination, the four tests detected 89 positive samples and all had positive agreement among themselves, with significant (p<0,05) Kappa indexes which ranged from weak to middle values. Regarding efficiency, each method excelled some parameter, with RT-PCR being less complex and faster, bacteriological culture were the cheapest, although histopathology had the highest combined efficiency. These results support a diagnostic scheme that includes the four methods (histopathological, molecular, solid medium and a liquid medium) for achievement the maximum number of isolates, in the shortest time and with highest certainty.

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38

Whelan, Adam Owen. "Development and optimisation of reagents for the discrimination of Mycobacterium Bovis infected from M. Bovis BCG infected cattle". Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270776.

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Barbier, Elodie. "Prévalence de Mycobacterium bovis dans les agroécosystèmes : analyse de réservoirs environnementaux potentiels (sol, eau douce, faune du sol et faune aquatique) et traçage de la circulation de cette bactérie entre les différents compartiments". Thesis, Dijon, 2016. http://www.theses.fr/2016DIJOS026/document.

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La tuberculose bovine est une maladie infectieuse contagieuse causée par Mycobacterium bovis. Cette maladie touche les bovins et de nombreuses espèces de mammifères domestiques et sauvages, ainsi que l’homme. La circulation de la bactérie dans des systèmes multi-hôtes variés favorise l’entretien de la maladie et la contamination des bovins vivant à proximité des animaux sauvages infectés. En marge de la transmission directe de M. bovis par voie respiratoire, la transmission indirecte aux bovins, liée à l’inhalation ou à l’ingestion de matrices environnementales contaminées par un animal infecté excréteur, est suspectée dans plusieurs régions du monde. L’existence de réservoirs environnementaux où le bacille M. bovis est capable de persister, pourrait donc être un facteur important de la réémergence puis du maintien de la maladie dans les systèmes multi-hôtes.En Côte d’Or, département fortement touché par la tuberculose bovine depuis 2004, la transmission indirecte de la bactérie entre la faune sauvage infectée et les bovins est suspectée dans plusieurs élevages. Pour évaluer la présence et la survie de cette bactérie dans l’environnement, nous avons analysé un grand nombre d’échantillons prélevés dans des zones partagées par les bovins et/ou la faune sauvage infectés dans le but de déterminer la distribution environnementale de M. bovis. Pour ce faire, nous avons développé ou modifié des systèmes de détection moléculaire adaptés aux matrices environnementales complexes. Nous avons également évalué l’impact de la température et des propriétés physico-chimiques de deux sols sur la survie de M. bovis, ainsi que le rôle de la mésofaune du sol (lombrics en particulier) dans la dissémination de la bactérie à partir de matière organique contaminée. L’étude environnementale a mis plus particulièrement en évidence la contamination de deux biotopes: les zones humides des pâtures et les sols de terriers de blaireaux. De plus, les études expérimentales ont montré que M. bovis pouvait survivre plusieurs mois dans le sol à 4°C et que les lombrics pouvaient disséminer la bactérie dans le sol, voire jouer un rôle potentiel de vecteur pour les animaux qui les consomment. Ces résultats apportent de nouvelles connaissances sur la persistance et la circulation de M. bovis dans l’environnement en Côte d’Or et permettront de proposer des améliorations aux mesures de biosécurité déjà existantes dans les élevages bovins
Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis. This disease affects cattle, and many species of domestic and wild mammals, and humans. The circulation of the bacteria in various multi-host systems promotes the maintenance of the disease and the contamination of cattle in the vicinity. Beside direct transmission of the bacteria through the respiratory route, indirect transmission, through inhalation or ingestion of environmental matrices contaminated by an infected animal excretory, is suspected in several countries. Environmental contamination with M. bovis appears to be a crucial factor in the persistence of the infection in multi-host systems.In Côte d'Or, a French department affected by bovine tuberculosis since 2004, the indirect transmission of the bacteria from infected wildlife to cattle is suspected in several cases. To assess this type of transmission of the bacillus, we evaluated the environmental contamination with M. bovis on a large number of samples taken in areas shared by cattle and / or wildlife infected. For this purpose, we developed or modified molecular detection systems adapted for environmental complex matrices. We also assessed the impact of physicochemical properties of both soil and temperature on survival of M. bovis and the role of earthworms in the spread of the bacteria from contaminated organic material. The environmental study showed the contamination of two media in particular: wetlands pastures and soil badger setts. Moreover, experimental studies have shown that M. bovis can survive in soil for several months at 4 ° C and the worms could spread the bacteria in the soil, or even play a potential role for vector animals that consume them. These results will propose improvements to existing biosecurity measures on cattle farms and provide new knowledge about the persistence and circulation of M. bovis in the environment in Côte d'Or

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Morar, Darshana. "Development of ELISAs for the detection of interferon-gamma in rhinoceroses and elephants as diagnostic tools for Mycobacterium bovis and Mycobacterium tuberculosis infections". Thesis, Pretoria : [s.n.], 2009. http://upetd.up.ac.za/thesis/available/etd-12032009-193314/.

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Rodrigues, Lívia de Andrade. "Inativação do Mycobacterium bovis (espoligotipo BR024) em creme de leite submetido à alguns parâmetros comerciais de pasteurização". Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-10022011-080624/.

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A resistência térmica dos microrganismos sofre influência, entre outros fatores, das características do agente e das características do substrato, como o teor de gordura. Um dos objetivos da pasteurização do creme é a eliminação dos patógenos eventualmente presentes no leite. Entretanto, não há padrão de tempo e temperatura de pasteurização para este produto na legislação. O Mycobacterium bovis é considerado o patógeno não formador de esporo de maior resistência térmica que pode normalmente ser transmitido pelo leite. Assim, este trabalho se propõe a avaliar a inativação de Mycobacterium bovis (espoligotipo BR024) em creme de leite fresco submetido a alguns parâmetros comerciais de pasteurização. Creme de leite foi contaminado e pasteurizado em Banho-Maria a 75°C, 80°C, 85°C e 90°C, por 5 e 15 segundos. O agente foi quantificado por semeadura em duplicata das diversas diluições em meio Stonebrink, após incubação a 36°C/45 dias. A redução na população variou de 3,9 log UFC/mL até a 6,8 log UFC/mL o que mostra que, nas condições do estudo, todos os binômios estudados mostraram-se capazes de reduzir a carga contaminante para níveis tão baixos ou menores que 0,1 log UFC/mL, considerando a máxima contaminação inicial natural do leite por M. bovis (4 log UFC/mL), segundo Ball (1943)
The thermal resistance of microorganisms is influenced by the agent in question, the initial microbial load and the characteristics of the substrate that can exert a protective effect on the cell, for example, the fat content. The pasteurization of whipping cream aims to eliminate pathogen microorganisms that may occasionally be present in milk. However, there is no standard in the law of time and temperature for this product, making it necessary more detailed study to consider the specific feature of thermal resistance of microorganisms at different temperatures for pasteurization, especially considering the high fat product. Mycobacterium spp is considered the pathogen spore-non-forming of higher heat resistance that can be transmitted by milk, among species, M.bovis is the most pathogenic. Therefore, this study aims to evaluate the inactivation of Mycobacterium bovis (spoligotype BR024) in fresh whipping cream subjected to some parameters of commercial pasteurization. Whipping cream was contaminated and pasteurized in a water bath at 75°C, 80°C, 85°C and 90°C for 5 and 15 seconds. The agent was measured in duplicate in the middle Stonebrink after incubation at 36°C/45 days. The reduction in the population ranged from 3.9 log CFU / mL up to 6.8 log CFU/ mL which shows that under the conditions of the study, all binomials studied were able to reduce the contaminant load to such low levels or lower than 0.1 log CFU / ml, the maximum initial natural contamination of milk by M. bovis (4 log CFU / mL), according to Ball (1943)

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Amaral, Eduardo Pinheiro. "Avaliação da virulência micobacteriana e modulação da resposta imune durante a infecção por isolados clínicos de Mycobacterium bovis e Mycobacterium tuberculosis". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-26102011-153630/.

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A tuberculose é considerada um problema emergente de saúde pública. Este estudo tem como objetivo avaliar a associação da patogenicidade/virulência e propriedades imunomoduladoras de isolados clínicos de Mbv (cepas B2 e MP287/03) e Mtb (cepa Beijing 1471), e da cepa de Mtb H37Rv, como referência de virulência. Os isolados, MP287/03 e Beijing 1471, apresentaram maior virulência em relação às demais cepas, levando os camundongos à morte ainda na fase aguda de infecção. Foi verificada baixa produção de mediadores pró-inflamatórios nos animais infectados com o isolado MP287/03, enquanto nos infectados com o isolado Beijing 1471 os níveis destes mediadores foram exacerbados. O desbalanço na produção destes mediadores pode ter contribuído para morte precoce dos animais. Baseado nesse estudo, nós podemos concluir que as propriedades que conferem hipervirulência aos isolados clínicos de Mbv e Mtb estão principalmente relacionadas à alta capacidade de crescimento intracelular das bactérias, que parece ser pouco alterada pela presença de citocinas pró-inflamatórias. Sendo assim, as infecções por isolados hipervirulentos podem acarretar consequências semelhantes, mesmo quando associadas a diferentes padrões de modulação da resposta imune.
Tuberculosis is an emergent problem of public health. This study aimed to evaluate the association between pathogenicity/virulence and immunemodulatory ability of Mbv (B2 and MP287/03) and Mtb (Beijing 1471) clinical isolates, using H37Rv strain as reference of virulence. The virulence was assessed in C57BL/6 mice infected with a low dose of bacilli (~100 bacteria) via intratracheal route. MP287/03 and Beijing 1471 isolates showed higher virulence than all others strains, leading to mice death during the acute phase. It was verified low production of pro-inflammatory mediators in mice infected by MP287/03 bacteria, whereas in mice infected by Beijing 1471 bacteria were observed exacerbated levels of pro-inflammatory mediators. The disbalance of these mediators may have contributed to the early mouse death. Based on this study, we concluded that the properties that confer hypervirulence to Mbv and Mtb clinical isolates are primarily related to the high intracellular growth capacity of the bacteria, which seems to be marginally affected by the presence of pro-inflammatory cytokines. Therefore, the infection by hypervirulent isolates can lead to similar outcomes, even when associated to different patterns of modulation of the immune response.

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Pothi, Radhika. "Global analysis of gene regulation in Mycobacterium bovis (BCG) and Streptomyces coelicolor". Thesis, University of Surrey, 2016. http://epubs.surrey.ac.uk/811050/.

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Streptomyces coelicolor and Mycobacterium bovis are high G+C gram-positive members of the phylum Actinobacteria. M. bovis is a member of the M. tuberculosis species complex and M. bovis, BCG is the attenuated vaccine strain used as a model organism as it can be manipulated in containment Category 2 laboratories. When cells are exposed to different environments/stresses they need to adapt their physiology and biochemistry for their effective survival. A key mechanism of adaptation is the ability to quickly alter the molecular composition of the cell through the regulation of gene expression both at transcriptional and translational level. While transcriptional regulation of gene expression has been studied extensively, little information regarding translational regulation in bacteria is available. To begin to study translational regulation in Streptomyces and Mycobacterium, a genome-wide approach was adapted to study the extent of translational regulation when cells are exposed to heat shock. As an initial attempt the protocol to isolate polysomes from both S. coelicolor and M. bovis (BCG) using classical sucrose density gradient under normal and heat shock conditions was optimized. Using DNA microarrays and RNA sequencing, the relative distribution of mRNA in the polysome fractions were analysed and compared it to the total transcriptome of the cell. Major heat shock responsive genes such as dnaK, grpE, groEL, groES, lon, hspR and dnaJ were significantly differentially expressed or were found to be translationally/transcriptionally potentiated (significantly differentially expressed genes in both transcription and translation) during heat shock in S. coelicolor, and in M. bovis (BCG) a smaller number of genesincluding SerX coding for tRNA biosynthesis and an ArsR repressor anti-toxin coding gene were shown to be translationally regulated. The study was further extended in S. coelicolor to investigate the use of a method based on the purification of affinity tagged ribosomes as a way to isolate actively translated mRNA. Selected ribosomal proteins were successfully tagged using strep-tag and the presence of tagged ribosomal proteins in cell lysates was checked using blotting techniques.

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44

Wooff, Emine Esen. "The application of functional genomics to understand the virulence of Mycobacterium bovis". Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407184.

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45

Taylor, Stephanie Jemma. "The role of protozoa and nematodes in the survival of Mycobacterium bovis". Thesis, University of Surrey, 2003. http://epubs.surrey.ac.uk/802/.

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46

Benton, Clare Helen. "Spatio-temporal distribution and persistence of Mycobacterium bovis in a badger population". Thesis, University of Exeter, 2017. http://hdl.handle.net/10871/28495.

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Studying the dynamics of pathogen transmission within wildlife populations presents an array of challenges. Where populations are socially structured, this can influence parasite transmission, impacting on the effectiveness of disease management strategies. In this thesis, I focus on a well-studied social mammal, the European badger (Meles meles) which is a key wildlife reservoir of a disease of economic importance; bovine TB (caused by infection with Mycobacterium bovis). The social structuring, characteristic of high density badger populations, is of well-established importance in the transmission of bovine TB and has resulted in unexpected management outcomes. However, little is known about the role of kin structure or host genotype on transmission dynamics. In this thesis, I combine traditional spatial epidemiology and ecological analysis of a well-studied badger population with more novel genetic and genomic approaches. Firstly, I investigate the role of kin structure within badger social groups in determining early life infection risk (Chapter 3). Using host genotype data, I demonstrate that cubs who are related to infected adults experience enhanced infection risks. I then explore the role of badger genotype on outcomes of M. bovis exposure and demonstrate that inbred badgers are more likely to show evidence of progressive infection (Chapter 4). Where the social structure of badgers is stable and unmanaged, this is predicted to result in a stable spatial distribution of M. bovis infection. Motivated by an observation of change in the spatial distribution of M. bovis infection in the study population, in the absence of management, I characterise the attrition of a spatially stable infection distribution (Chapter 5). To explore the drivers of this, I detect changes in the genetic population structure (Chapter 6) and present evidence that the population has experienced a period of demographic flux. Finally, I use a novel dataset generated by whole genome sequencing of M. bovis isolates and present evidence of spatial spread of M. bovis infection across the study population (Chapter 7). To conclude, I discuss how my findings demonstrate how genetic and genomic approaches can complement traditional wildlife epidemiology approaches, how they contribute to our understanding of heterogeneity in transmission dynamics and discuss their implications for wildlife disease management.

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Rodrigues, Gabriel Santos Cruz. "Papel da obesidade na imunomodulação da infecção experimental por Mycobacterium bovis BCG". Universidade Federal de Juiz de Fora, 2014. https://repositorio.ufjf.br/jspui/handle/ufjf/730.

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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A obesidade é um grande problema de saúde mundial e com os hábitos e estilos de vidas atuais a sua importância social só tende a crescer. Hoje associa-se a obesidade com o mau prognóstico de diversas doenças, como câncer, diabetes e doenças auto-imune brandas, além disso, ela também está associada a uma resposta imune deficitária frente a infecções por diversos patógenos, como alguns vírus e bactérias. A tuberculose é uma doença infecto-contagiosa, mais comumente pulmonar, causada pelo Mycobacterium tuberculosis (ou bacilo de Koch) e outros membros do complexo Mycobacterium tuberculosis. Esta doença causa cerca de 1,4 milhões de morte ao ano, sendo constatados cerca de 8 milhões de novos casos ao ano , estando apenas atrás da AIDS na taxa de mortalidade por doenças infecciosas e seu sucesso se deve em parte à capacidade de viver de meses à décadas no hospedeiro, em estado assintomático. Neste trabalho, avaliamos a relação entre a obesidade e o desenvolvimento da tuberculose, através do modelo de infecção experimental por Mycobacterium bovis BCG em camundongos C57/BL6 controles e tratados com dieta hiperglicídica. Os resultados demonstraram que os animais obesos apresentaram um menor recrutamento leucocitário, dentre estes, menor influxo de neutrófilos e eosinófilos. Além disso, animais obesos apresentaram menor formação de corpúsculos lipídicos, menor produção de PGE2, maior produção de mediadores anti-inflamatórios, como IL-10 e adiponectina, e menor produção de mediadores pró-inflamatórios, como leptina, em comparação aos animais controles. De maneira geral, nossos resultados apontam que a obesidade induz um perfil mais anti-inflamatório, inibindo a expressão de fatores de favorecimento do patógeno, como a formação de corpúsculos lipídicos, PGE2 e eosinofilia, sugerindo que a obesidade, nos estágios iniciais de infecção, pode modular negativamente a resposta inflamatória induzida pela infecção micobacteriana.
Obesity is a huge world health problem and with the habits and lifestyles of these days, it’s important only tend to grown. Today obesity is associated with a bad prognosis of a large number of diseases , like cancer, diabetes and light self-immune diseases, and besides that, its also associated with an uncompleted response to several pathogens, like some virus and bacteria. The tuberculosis is an infect-contagious disease, commonly pulmonary, caused by Mycobacterium tuberculosis (or, Koch bacillus), and other members of the Mycobacterium tuberculosis complex. This illness cause about 1,4 million deaths per year, being registered 8 million new cases per year, being only behind AIDS on the mortality ratio by infectious diseases in the world and ur success can be related to your capacity to live from months to decades on the host , in a asymptomatic state. In this work, we evaluated the relationship between the obesity and the development of tuberculosis, by using the experimental infection model by Mycobacterium bovis BCG. C57/BL6 mice, male, with about 4 to 6 weeks of life, had being fed with standard ration (control group) or hiperglicidic ration (obese group), for 0, 2 or 3 months. The body weight was monitored during this period, and the fat gain and glucose tolerance was measured at the end, so the state of obesity could be characterized. At the end of these periods, this animals received a intraperitoneal inoculation of 100 mL of saline solution (infection control) or 100 mL of BCG solution (infected group) and characteristic processes of this infection were observed, like leukocitary recruitment , lipid bodies formation on these cells, the production of pro and anti-inflammatory cytokines, eicosanoids (PGE2) and adipokines. Was observed then, that the obese animals, presented a lower leukocitary recruitment , among then, less influx of neutrophils and eosinophils, lower formation of lipid bodies and production of PGE2 , higher production of anti-inflammatory mediators (IL-10 and adipokine) and lower production of pro-inflammatory mediators (leptin) . Overall , our data points to a more anti-inflammatory profile on the obese animals , occurring in a minor expression of factors that are important to the pathogen survival , like the formation of lipid bodies and eosinophilia , indicating that, the obesity, on the early stages of the infection, may be representing a disadvantaging factor to the disease.

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48

Beckley, Nicholas. "Epidemiological dynamics of Mycobacterium bovis and population suppression in badgers (Meles meles)". Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/49412.

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Bovine TB in cattle is a major economic problem in the UK, costing the government approximately £100m a year. Badgers are a wildlife host of the infection that causes bovine TB, and there is strong evidence that they transmit the infection to cattle. Understanding the ecology and epidemiology of infected badger populations is therefore crucial for implementing disease management strategies relating to badgers. Genetic and phenotypic data of badgers captured during a large-scale field trial of repeated, widespread badger culls were used to assess the importance of parental roles on the impacts of badger culling. Further ecological and epidemiological dynamics were investigated through developing a stochastic simulation model of an infected badger population. An estimated 72.8% of badgers were captured during initial culls of the trial, and an estimated 57.8% during follow-up culls, based on badger parentage assignments. Further analyses of these parentage assignments revealed evidence of a genetic predisposition in infection susceptibility from parents to cubs, but no evidence of a significant infection transmission route from mothers to their young, dependent cubs. There was also no evidence that badger welfare was compromised during the trial through not capturing dependent cubs of culled mothers. Analysis of a simulation model found that moderate levels of disease-induced mortality in an infected badger population could significantly reduce the size of badger social groups with a higher prevalence of infection. The impact of these findings relating to potential disease management strategies is discussed, together with suggested directions for future research.

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Brown, Julian A. "Transmission of bovine tuberculosis (Mycobacterium bovis) from badgers (Meles meles) to cattle". Thesis, University of Bristol, 1993. http://hdl.handle.net/1983/d277aaf1-a1b1-4142-b0e5-ffa1f3d12bb7.

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Delmas, Carole. "Analyse structurale comparative des lipoarabinomannanes de quatre souches de Mycobacterium bovis BCG". Toulouse 3, 1997. http://www.theses.fr/1997TOU30134.

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Depuis les annees 20, mycobacterium bovis bcg (bacille calmette-guerin) est le vaccin vivant utilise pour la protection antituberculeuse. Pourtant, l'existence d'une variabilite, de 0 a 80%, de la protection antituberculeuse induite par ce vaccin demontre clairement une derive des souches de bcg actuellement utilisees. Un composant majeur des parois mycobacteriennes, le lipoarabinomannane ou lam est suppose, aujourd'hui, etre un facteur determinant dans l'infection et la survie intramacrophagique des mycobacteries, etapes necessaire a la mise en place d'une immunite protectrice par le bcg. L'objectif de ce travail a donc consiste en une analyse comparative de quatre souches de bcg (pasteur, copenhague, glaxo et tokyo), chacune dotee d'un pouvoir vaccinant variable. Cette etude est centree sur l'analyse structurale des lams produits par ces souches vaccinales. Dans un premier temps, nous avons tente d'ameliorer la strategie de purification du lam, travail qui nous a conduits a la caracterisation de deux sous-populations de lams par souche. Tous ces composes se sont averes appartenir a la famille des lams mannosyles ou manlams. Dans un second temps, une etude rmn bidimensionnelle homo- (cosy, hohaha) et heteronucleaire (hmqc, hmbc) nous a permis, pour la premiere fois, de localiser, avec precision, des residus de succinates sur la molecule. Une approche semi-quantitative par l'analyse des spectres 1d (#1h) a egalement ete menees sur ces manlams. Enfin, afin d'evaluer le degre de mannosylation des differents manlams. , nous nous sommes interesses a la mise au point d'une strategie utilisant la chromatographie echangeuse d'anions a haut ph (hpaec) et la fab -ms. Les resultats peu probants obtenus nous ont conduit a nous orienter vers l'electrophorese capillaire qui, dans un premier temps, nous a permis d'acceder a l'aspect qualitatif de la mannosylation des lams. Nous nous sommes ensuite focalises sur l'elaboration d'une strategie en vue d'aborder, a l'aide de cette technique, l'aspect quantitatif de cette analyse.

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