Estudio de la patogénesis causada por mycobacterium bovis en cerdos domésticos y desarrollo de estrategias para el control de la tuberculosis en suinos
Tuberculosis (TB) is a chronic infectious disease, produced by members of the genus Mycobacterium. Mycobacterium bovis (M. bovis) is the main cause of bovine tuberculosis\n(TBb), a disease that significantly affects cattle, but which can also affect other farm animals\nsuch as goats, deer and pigs,...
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| Formato: | Tesis doctoral acceptedVersion |
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Facultad de Ciencias Veterinarias
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_6338 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_6338.dir/6338.PDF |
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| Sumario: | Tuberculosis (TB) is a chronic infectious disease, produced by members of the genus Mycobacterium. Mycobacterium bovis (M. bovis) is the main cause of bovine tuberculosis\n(TBb), a disease that significantly affects cattle, but which can also affect other farm animals\nsuch as goats, deer and pigs, as well as wild species, being a problem for public health as it is a zoonotic disease. In pigs, the main route of infection is the oral route, and occurs when\nthey are in contact with sick animals or when they are fed with milk or dairy unpasteurized\nproducts, as well as contaminated food waste. The disease is characterized by the formation\nof nodular granulomas, with a higher frequency of lesions in the mandibular or\nretropharyngeal lymph nodes, compatible with a predominantly oral route of infection. However, there are also more generalized lesions, including the locations in thorax and\nabdomen, suggesting that both respiratory and foodborne transmission may occur. Although TB in pigs is not eradicated, it remains under control in developed countries. However, in countries where extensive production systems predominate and TBb is\nendemic such as Argentina, M. bovis has a big impact on production and public health. On the other hand, in Argentina, small and medium-scale production systems represent more\nthan 95% of the total pig production, being the priority of this area to place the consumption of pork meat in the market. Therefore, improving food safety conditions and reducing\nenvironmental contamination in these systems will have an economic impact, as well as on animal and public health conditions. At the same time, improving the health status of these\nestablishments will help them to develop their maximum productive potential, and thus to\nposition them in a more competitive way and increase commercialization within and outside\nthe country. In our country, data on swine TB is scarce, and the prevalence of the disease in pigs is\nestimated by the presence of TB like-lesions at inspection in slaughterhouse. Between 1969 and 2005, the incidence of swine TB decreased from 8,4 to 0,7% and reached 0,3% in\naccordance with the prevalence of TB in cattle. This is consistent with a reduction in transmission to pigs and other species due to the progress of TB eradication program\nlivestock. Currently, there are no effective ante mortem diagnostic tools to detect infected animals. In Argentina, there is a swine TB eradication and control program, based on a\ndouble comparative tuberculin skin test with PPDb and PPDa, as in cattle. Particularly in\npigs, this technique is difficult to perform and often provides false positive results. Thus, the\ninformation regarding the TB health status of the farms is uncertain. On the other hand, pigs constitute a source of infection for other animals in the farm as well as for humans, being\nable to act as a reservoir or as a ?spill-over? host suggesting an important role in the epidemiology of TB and acting as a reservoir where new variants of M. bovis may occur.\nThe aim of the present work was to develop strategies for the detection and control of swine TB. Also, to understand the virulence of M. bovis strains, through the study of the pathogenesis and immune response in different experimental models, together with a\ngenomic approach, in order to contribute to TBb eradication programs.\nTo this aim, we first develop a diagnostic serological test, to detect the disease in pigs?\npopulations. For this end we developed and validated an in house-ELISA, which proved to be highly repeatable and reproducible, comparable with a commercial ELISA test, and with a sensitivity of 69% and a specificity of 100%, allowing to detect sick animals within a given\npopulation, without the risk of eliminating healthy animals. Additionally, M. bovis strains from TB-like lesions from slaughtered pigs were isolated and\ngenotyped. We focused our attention on strain MB894 that presented a spoligotype\nassociated with human infections. This strain was selected for further characterization in\nanimal models.\nWe first evaluated the pathogenesis and immune response in pigs experimentally infected\nwith MB894 strain. Two groups of 10 animals each were infected by the oral route in previously sensitized animals or not, with a heat- inactivated M. bovis suspension (HIMB).\nThe results obtained allowed us to confirm the virulence of the strain, since all the animals presented TB-like lesions. The study of these lesions, their distribution and histopathology, as well as the excretion of mycobacteria, allowed us to better understand the pathogenesis\nof M. bovis infection in pigs. In addition, we were able to evaluate the humoral immune response triggered after infection, detecting seropositive animals after 70 days post infection. We observed that previous sensitization with HIMB slightly increased the intensity of the response, given by a higher lesion score, a more fibrotic component and an increase\nin the humoral immune response. Although the differences were not statistically significant\nfor the lesion score between the groups, we observed that the oral route of infection, combined with a previous oral sensitization, could modify the course of the infection towards\nthe development of lesions with a higher fibrotic component, which could suggest resistance\nto infection under the right conditions. Subsequently, we studied the virulence of the strain MB894 in the pulmonary TB model in\nBALB/c mice in comparison with an already characterized M. bovis 04-303 strain, MB303.\nMice were infected by the intratracheal route, and the virulence of the strains was evaluated by the study of macroscopic lesions, histopathology, colony-forming units and cytokines\nexpression. The results obtained showed a degree of virulence comparable to the hypervirulent strain MB303 isolated from wild boar, evidencing the high virulence of the M.\nbovis strain isolated from a pig TB-like lesion.\nTo understand the highly virulence phenotype, we performed the whole genome-sequencing\nof MB894 using the M. bovis AF2122/97 strain as reference. A comparative genomic study\nwas performed between MB894 and MB303 strains, and SNPs/ INDELs were found in 10 genes that could have a high effect at the protein level and could be relevant for the virulence\nof the bacteria. We also looked for previously characterized genes involved in lipid\nmetabolism reported in the bibliography, probably related with virulence. Finally, the pangenome of MB894 was performed together with the hypervirulent strain MB303 and an attenuated strain M. bovis 534 (MB534), and MB894 exclusive genes were found that could\nbe associated with differences in virulence |
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