Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2014. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2015-01-28T19:26:28Z No. of bitstreams: 1 2014_FlavioPereiraVeloso.pdf: 1224564 bytes, checksum: 6521faacfa36b45cd523140ab24b4634 (MD5) / Approved for entry into archive by Ruthléa Nascimento(ruthleanascimento@bce.unb.br) on 2015-02-13T14:05:29Z (GMT) No. of bitstreams: 1 2014_FlavioPereiraVeloso.pdf: 1224564 bytes, checksum: 6521faacfa36b45cd523140ab24b4634 (MD5) / Made available in DSpace on 2015-02-13T14:05:29Z (GMT). No. of bitstreams: 1 2014_FlavioPereiraVeloso.pdf: 1224564 bytes, checksum: 6521faacfa36b45cd523140ab24b4634 (MD5) / Com o intuito de subsidiar o planejamento estratégico do Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT), diversos Estados brasileiros vêm realizando estudos transversais por amostragem, sob a coordenação do Ministério da Agricultura, Pecuária e Abastecimento (MAPA) e com apoio científico da Universidade de São Paulo (USP) e da Universidade de Brasília (UnB). Em Santa Catarina, a Companhia Integrada de Desenvolvimento Agrícola de Santa Catarina (CIDASC) realizou estudo de prevalência e avaliação de fatores de risco em 2012. O Estado foi dividido em cinco regiões, de forma a considerar a heterogeneidade regional e de ecossistemas produtivos. Em cada região, foi feita uma amostragem independente, em duas etapas: (i) foram selecionadas, aleatoriamente, propriedades com atividade reprodutiva em cada região; e (ii) em cada rebanho uma amostra de fêmeas com idade igual ou superior a dois anos foi submetida à prova da tuberculinização comparada. Aplicou-se um questionário para coleta de dados relativos às características produtivas e práticas de manejo que pudessem estar associados à presença de animais reagentes no rebanho. Os resultados estimaram que a prevalência de propriedades positivas (focos) é de 0,50% [IC 95%: 0,074%; 0,93%] enquanto a prevalência de tuberculose em fêmeas adultas é de 0,06% [IC 95%: 0%; 0,12%]. Não houve diferença estatística nas prevalências de focos e de fêmeas positivas entre as cinco regiões. Foram desenvolvidos dois modelos de regressão logística que identificaram como fatores de risco o tamanho do rebanho (rebanhos com mais de 19 fêmeas – 3º quartil) e a finalidade da produção ser leiteira. Primeiramente construiu-se um modelo tradicional, sem considerar o peso amostral de cada propriedade testada para tuberculose bovina, o qual foi depois comparado com um modelo em que o delineamento amostral com estratificação regional foi considerado por via da inclusão dos pesos amostrais de cada propriedade. Este modelo, mais adequado para inferência populacional, revelou que os rebanhos com 19 ou mais fêmeas apresentaram um Odds Ratio (OR) de 7,68 [IC 95%: 1,22%; 48,39%] em relação aos rebanhos menores, enquanto as propriedades leiteiras apresentaram OR de 10,43 [IC 95%: 2,00%; 54,25%] em relação às demais. Os resultados sugerem que os rebanhos leiteiros, onde os animais são mantidos em confinamento parcial ou total, e os rebanhos maiores, que tendem a comprar animais com maior frequência, apresentam maior risco de tuberculose bovina. Considerando a prevalência baixa e a tipologia de propriedades mais afetadas, o sistema de vigilância da tuberculose bovina deve ser dirigido às bacias leiteiras do Estado, com destaque para a região Oeste, onde se concentram as principais indústrias e as propriedades leiteiras de produção mais intensiva. ___________________________________________________________________________________ ABSTRACT / Several Brazilian states have recently carried out major epidemiological studies to underpin the planning of the national programme for control and eradication of bovine brucellosis and tuberculosis. Such efforts are coordinated by the Ministry of Agriculture, Livestock and Food Supply and have the scientific support of the University of São Paulo and the University of Brasília. In Santa Catarina, the state animal health authority (CIDASC) implemented in 2012 a random sample survey with a view to estimating the prevalence and risk factors for bovine tuberculosis. The state was divided in five geographical regions and in each one of them was taken a simple random sample of bovine herds with adult females. In each selected herd a sample of adult females (> 2 years) was tested for bovine tuberculosis with the comparative cervical tuberculin test. In each selected herd was administered a questionnaire to collect data on animal production and management practices that could be associated to the presence of the disease at the herd-level. The results estimated the prevalence of herds with test-positive animals at 0.50% [IC 95%: 0.074%; 0.93%], whereas the prevalence for adult females was 0.06% [IC 95%: 0%; 0.12%]. There was no statistically significant difference between regions, both for herd and animal-level prevalence. With regard to the study of risk factors, two logistic regression models were constructed, which identified the larger herd size and the dairy production as the main factors associated to the chance of a herd being positive for bovine tuberculosis. After a preliminary univariable analysis, a standard model-based logistic regression was used to test for confounding and to assess the goodness-of-fit and the discriminatory performance of the predictive model. Then, the same variables were used to develop a design-based model, taking into account the sampling weights of each herd, considering the regions as a sampling strata, which is more appropriate for population inference. Both models gave results of the same magnitude, but the standard errors were wider in the design-based logistic regression, as expected: the Odds Ratio for herds with more than 18 adult females (3rd quartile of the herd-size) was 7.68 [IC 95%: 1.22%; 48.39%] and the dairy farms showed an Odds Ratio of 10.43 [IC 95%: 2.00%; 54.25%] compared to the beef or dual-purpose herds. These results confirm that dairy herds, where animals are kept in partial or full confinement, and larger herds, that tend to be more production intensive and buy more frequently replacement animals, have higher risk of having animals with bovine tuberculosis. Given the low prevalence found for the state and the type of higher risk herds, the surveillance system should be targeted at the main dairy basins, notably the western region, where most intensive dairy herds and industries are placed.

Cette thèse a pour but de contribuer à une meilleure compréhension de la tuberculose (TB) et desmycobactéries non-tuberculeuses dans la Caraïbe. La tuberculose a hanté l’humanité depuisplusieurs millénaires et reste de nos jours une des maladies infectieuses faisant le plus de victimeschaque année (1,5 millions de décès en 2013). La connaissance de l’épidémiologie de la tuberculoseest essentielle afin de concevoir des programmes de lutte anti-TB adaptés aux spécificitésrégionales et donc plus efficaces. Dans cette optique, la première partie de ce travail fourni un suivià long-terme de la résistance aux antituberculeux observée en Guadeloupe, Martinique et Guyanefrançaise ainsi qu’un aperçu de la diversité génétique et de la résistance aux antituberculeux dansla Caraïbe. Les données montrent une baisse graduelle de la fréquence des infections causées pardes souches résistantes parmi les nouveaux cas de TB dans les départements français d’Amérique.En ce qui concerne la Caraïbe, des différences marquées ont été observées entre les différentsterritoires, ce qui semble refléter le passé historique de cette région.La deuxième partie est consacrée à la phylogénie et l’évolution de M. tuberculosis, étudié à l’aide dedivers marqueurs génétiques comme spoligotypes, LSP, SNP et MIRU-VNTR. Les profils MIRUVNTR(format 12-loci) ont été étudiés afin de déterminer leur utilité comme marqueurphylogénétique. Il a été montré que ce marqueur est adapté pour retracer la phylogénie ducomplexe M. tuberculosis et que la précision du classement basé sur les MIRUs est supérieure àcelle du classement basé sur les spoligotypes. De plus, la technique MIRU-VNTR permetégalement d’observer la diversification évolutive d’une souche de M. tuberculosis au cours del’infection ou alors d’identifier des patients infectés par plusieurs souches de M. tuberculosis enmême temps. Les deux phénomènes ont été observés au cours de ce travail de thèse et les casconcernés sont décrits dans ce deuxième chapitre.Enfin, un premier aperçu de la diversité des mycobactéries non-tuberculeuses isolées desprélèvements cliniques en Guadeloupe, Martinique et Guyane est présenté dans la troisième partiede ce travail. Des différences marquées dans la fréquence d’isolement de certaines espèces ont puêtre observées entre les trois départements français d’Amérique. M. intracellulare par exemple étaitsignificativement plus abondant en Guadeloupe. Cependant l’existence d’une niche écologiquespécifique à cette île n’a pas pu être mise en évidence. La problématique de l’identification desmycobactéries non-tuberculeuses est abordée également à travers une étude rétrospective del’utilisation de hsp65-PRA pour l’identification des mycobactéries dans un laboratoire de routinemais aussi sous forme d’un travail prospectif visant à la mise en place d’un protocoled’identification de mycobactéries non-tuberculeuses avec MALDI-TOF MS. / This thesis aims at providing a better understanding of tuberculosis (TB) and non-tuberculousmycobacteria (NTM) in the Caribbean. TB is an ancient scourge of humanity and remains one ofthe deadliest infectious diseases today having claimed around 1.5 million lives in 2013.Understanding the epidemiology of TB is essential for optimizing regional TB control programs. Inthis context, the first part of this work provides long-term data on drug-resistance in Guadeloupe,Martinique and French Guiana as well as an insight in the genetic diversity and drug-resistance ofM. tuberculosis in twelve Caribbean territories. Encouragingly, the results show a gradual decreaseof drug-resistant TB in newly infected patients in Guadeloupe, Martinique and French Guiana. Onthe Caribbean level, distinct differences were observed from one territory to the next and thecurrent epidemiological landscape seems to reflect the historical past of the region.The second part addresses the phylogeny and evolution of M. tuberculosis using various geneticmarkers such as spoligotyping, large sequence polymorphism (LSP), single nucleotidepolymorphism (SNP), and MIRU-VNTRs. The suitability of 12-loci MIRU-VNTR profiles for use inphylogenetic studies was evaluated and it was found that this marker is not only able to resolvethe evolutionary relationships within the M. tuberculosis complex but also allows to achieve ahigher phylogenetic precision than spoligotyping. MIRU-VNTR also permits the identification ofon-going evolution in TB patients (in-patient microevolution) as well as mixed strain infections.Both phenomena were observed in our setting and the respective cases are described herein.Finally, a first insight in the diversity of NTM isolated from clinical specimen in Guadeloupe,Martinique and French Guiana is provided. The isolation frequency of some NTM species variedconsiderably between the three departments, the most striking example being the relativeabundance of M. intracellulare in Guadeloupe. However, no evidence of a privilegedenvironmental niche/infection source on this island could be found. Last but not least, the subjectof NTM identification is addressed in the form of a retrospective evaluation of hsp65-PRA basedidentification in a routine laboratory and in the form of a prospective study towards theimplementation of a MALDI-TOF MS based identification of NTM at the Pasteur Institute ofGuadeloupe.