Foi estabelecida uma parceria entre o Departamento de Medicina Veterinária Preventiva e Saúde Animal (VPS) da FMVZ-USP, a Coordenadoria de Defesa Agropecuária do Estado de São Paulo e o Serviço de Inspeção Federal (SIF) para organizar um sistema capaz de detectar focos de tuberculose bovina no Estado, com base nas rotinas de inspeção de carcaças em abatedouros, cujos objetivos foram: 1) conhecer a diversidade genética de isolados de Mycobacterium bovis em bovinos no Estado de São Paulo; 2) estudar a distribuição espacial desses focos; 3) estudar a tipologia das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) verificar se é possível, com a atual infra-estrutura existente em São Paulo, operar um sistema de vigilância para detecção de focos de tuberculose bovina. Assim, foi estruturado um sistema de coleta, envolvendo as redes SISP (Sistema de Inspeção do Estado de São Paulo) e SIF, que realizou as coletas de materiais e informações de maio de 2002 a janeiro de 2004. Todo o material seguiu para o VPS, onde foram processados. As propriedades caracterizadas como focos foram rastreadas e delas foi coletada outro conjunto de informações. Seguem os resultados alcançados: 1) foram identificados 33 diferentes espoligotipos dentre os 248 isolados de M. bovis de bovinos no Estado de São Paulo. Os isolados do espoligotipo SB0295 foram re-discriminados em 13 novos perfis genéticos de M. bovis pela técnica MIRU-VNTR; 2) dentre os dois espoligotipos mais prevalentes estudados (SB0295 e SB0121), apenas o SB0295 apresentou-se de forma agrupada nas análises espaciais; 3) foram geradas várias informações sobre a tipologia e o manejo das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) a atual infra-estrutura existente no Estado de São Paulo foi capaz de operar um sistema de detecção de focos de tuberculose bovina / A partnership between the Department of Preventive Veterinary Medicine and Animal Health (VPS) of the FMVZ-USP, the Coordination of Agriculture and Animal Defense of the State of São Paulo, and the Federal Inspection Service (SIF) was established to organize a work system for detection of bovine tuberculosis focus in the state, based on routine methods of carcass inspection in the abattoir, with the following objectives: 1) to determine the genetic diversity of the isolates of Mycobacterium bovis from bovines in the state of São Paulo; 2) to study the spatial distribution of the focuses; 3) to study the typology of the bovine breading units (farms), which were characterized as tuberculosis focus; 4) to verify the possibility of operating a surveillance system for detection of bovine tuberculosis focus based on the current network in the state of São Paulo. Thus, it was performed a system for data collection involving the current systems SISP (System of Inspection of the State of São Paulo) and SIF, who performed the collection of biological samples and information from May 2002 to January 2004. All samples were addressed to the VPS, where they were processed. Farms characterized as focus were traced to obtain new information. The results obtained in this study follow: 1) A total of 33 different spoligotypes were determined out of 248 bovine isolates of M. bovis in the state of São Paulo. The spoligotype SB0295 isolates were re-discriminated into 13 new M. bovis genetic profiles by the MIRU-VNTR technique; 2) From the two most prevalent spoligotypes analyzed in this study (SB0295 e SB0121), only SB0295 showed a cluster presentation by the spatial analyses; 3) Several information about typology and bovine breeding unit management were generated regarding the status of tuberculosis focus; 4) the current network in the state of São Paulo was capable of operating a system for detection of bovine tuberculosis focus

Realizou-se um inquérito epidemiológico para estimar a prevalência da tuberculose bovina no Estado de Rondônia. O Estado foi estratificado em três circuitos produtores, e em cada um dos circuitos escolheu-se aleatoriamente as propriedades a serem visitadas, totalizando 904 propriedades e 19.640.animais amostrados. Um número pré-estabelecido de animais foram aleatoriamente escolhidos e submetidos ao teste cervical comparativo, de modo que em propriedades com até 100 fêmeas com idade superior a 24 meses, foram testadas 20 fêmeas ou menos e, em propriedades com mais de 100 fêmeas com idade superior a 24 meses, foram testadas 40 fêmeas. Foram considerados focos as propriedades com um animal reagente entre os 20 amostrados e nas propriedades com 40 fêmeas amostradas que tiveram dois animais reagentes. As prevalências de focos e animais para o Estado foram, respectivamente, 2,3% [1,5; 3,5] e 0,1% [0,1; 0,2], e para os circuitos pecuários foram: Circuito 1 (Norte-Oeste-Sul), 1,7% [0,7; 4,0] e 0,1% [0; 0,4], Circuito 2 (Nordeste), 3,0% [1,6; 5,7] e 0,2% [0,1; 0,3] e Circuito 3 (Sudeste), 2,3% [1,1; 4,7] e 0,1%[0; 0,2]. Além dos testes tuberculínicos, aplicou-se um questionário para levantamento dos fatores de risco para tuberculose bovina. As variáveis foram submetidas à analise univariada e aquelas que apresentaram p < 0,20, foram submetidas à regressão logística. A aquisição de animais sem tuberculinização foi associada a condição de foco de tuberculose (OR = 7,1 [1,6;31,1], p=0,009). As baixas prevalências tanto de focos como de animais no Estado de Rondônia, sugerem a adoção de medidas de erradicação, previstas no PNCEBT. A obrigatoriedade de execução de testes tuberculínicos para trânsito interestadual independentemente da finalidade e a regulamentação de rastreamentos de novos focos a partir de amostras coletadas em frigoríficos tornam-se imprescindíveis para detecção de focos residuais e subsequente erradicação da tuberculose bovina. / An epidemiological survey was conducted to assess the prevalence of bovine tuberculosis in the State of Rondônia, Brazil. The State was divided into three epidemiological regions and, in each of them, a total of 904 farms were randomly chosen and 19,640 animals were sampled. A fixed number of animals were randomly chosen and submitted to the cervical comparative test, so that in farms with less than 100 bovine females over 24 months of age, 20 females, or less (if the herd was < 20), were tested. In farms with over 100 bovine females over 24 months of age, 40 females were sampled. We have considered positive farms when at least one positive animal among the 20 sampled or two positive animals in the farm with 40 animals sampled were found. The prevalence of infected herds and positive animals in Rondonia State were, respectively, 2.3% [1.5; 3.5] and 0.1% [0.1; 0.2]. In each epidemiological region, the results were: Region 1 (North west - South) = 1.7% [0.7; 4.0] and 0.1% [0; 0.4]; Region 2 (Northeast) = 3.0% [1.6; 5.7] and in the Region 3 (Southeast) = 2.3%[1.1; 4.7] and 0.1% [0; 0.2]. In addition to the tuberculin tests a questionnaire was applied in each visited farm in order to determine the risk factors for bovine tuberculosis. The variables were submitted to univariate analysis and those with p < 0.20) were submitted to logistic regression. The acquisition of animals without tuberculin tests was associated to tuberculosis (OR = 7.1 [1.6; 31.1], p = 0.009). The low prevalence of bovine tuberculosis in Rondonia State suggests the adoption of eradication measures, provided by the National Program for the Eradication and Control of Bovine Brucellosis and Tuberculosis (PNCEBT). Compulsory enforcement tuberculin tests for interstate transit regardless of the purpose and regulation of new outbreaks tracing from abattoir samples makes it essential to detect residual foci and subsequent eradication of bovine tuberculosis.

Bovine Tuberculosis (bTB) is a disease of significant economic importance, being one of the most persistent animal health problems in the UK and the Republic of Ireland and increasingly constituting a public health concern especially for the developing world. Limitations of the currently available diagnostic and control methods, along with our incomplete understanding of bTB transmission, prevent successful eradication. This Thesis addresses the development of a complementary control strategy which will be based on animal genetics and will allow us to identify animals genetically predisposed to be more resistant to disease. Specifically, the aim of my PhD project is to investigate the genetic architecture of resistance to bTB and demonstrate the feasibility of whole genome prediction for the control of bTB in cattle. Genomic selection for disease resistance in livestock populations will assist with the reduction of the in herd-level incidence and the severity of potential outbreaks. The first objective was to explore the estimation of breeding values for bTB resistance in UK dairy cattle, and test these genomic predictions for situations when disease phenotypes are not available on selection candidates. Through using dense SNP chip data the results of Chapter 2 demonstrate that genomic selection for bTB resistance is feasible (h2 = 0.23(SE = 0.06)) and bTB resistance can be predicted using genetic markers with an estimate of prediction accuracy of r(g, ĝ) = 0.33 in this data. It was shown that genotypes help to predict disease state (AUC ≈ 0.58) and animals lacking bTB phenotypes can be selected based on their genotypes. In Chapter 3, a novel approach is presented to identify loci displaying heterozygote (dis)advantage associated with resistance to M. bovis, hypothesising underlying non-additive genetic variation, and these results are compared with those obtained from standard genome scans. A marker was identified suggesting an association between locus heterozygosity and increased susceptibility to bTB i.e. a heterozygote disadvantage, with the heterozygotes being significantly more in the cases than in the controls (x2 = 11.50, p < 0.001). Secondly, this thesis focused on conducting a meta-analysis on two dairy cattle populations with bTB phenotypes and SNP chip genotypes, identifying genomic regions underlying bTB resistance and testing genomic predictions by means of cross-validation. In Chapter 4, exploration of the genetic architecture of the trait revealed that bTB resistance is a moderately polygenic, complex trait with clusters of causal variants spread across a few major chromosomes collectively controlling the trait. A region was identified on chromosome 6, putatively associated with bTB resistance and this chromosome as a whole was shown to contribute a major proportion (hc 2= 0.051) of the observed variation in this dataset. Genomic prediction for bTB was shown to be feasible even when only distantly related populations are combined (r(g,ĝ)=0.33 (SE = 0.05)), with the chromosomal heritability results suggesting that the accuracy arises from the SNPs capturing linkage disequilibrium between markers and QTL, as well as additive relationships between animals (~80% of estimated genomic h2 is due to relatedness). To extend the analysis, in Chapter 5, high density genotypes were inferred by means of genotype imputation, anticipating that these analyses will allow the identification of genomic regions associated with bTB resistance more closely, and that would increase the prediction accuracy. Genotype imputation was successful, however, using all imputed genotypes added little information. The limiting factor was found to be the number of animals and the trait definitions rather than the density of genotypes. Thirdly, a quantitative genetic analysis of actual Single Intradermal Comparative Cervical Test (SICCT) values collected during bTB herd testing was conducted aiming to investigate if selection for bTB resistance is likely to have an impact on the SICCT diagnostic test. This analysis demonstrated that the SICCT has a negligibly low heritability (h2=0.0104 (SE = 0.0032)) and any effect on the responsiveness to the test is likely to be small. In conclusion, breeding for disease resistance in livestock is feasible and we can predict the risk of bTB in cattle using genomic information. Further, putative QTLs associated with bTB resistance were identified, and exploration of the genetic architecture of bTB resistance revealed a moderately polygenic trait. These results suggest that given that larger datasets with more phenotyped and genotyped animals will be available, we can breed for bTB resistance and implement the genomic selection technology in breeding programmes aiming to improve the disease status and overall health of the livestock population. Using the genomics this can be continued as the epidemic declines.

Técnicas de genotipificação do Mycobacterium bovis baseadas na amplificação do DNA via PCR como MIRU-VNTR constituem importantes ferramentas na investigação epidemiológica da tuberculose bovina (TB) em áreas de baixa prevalência, como no Estado de Mato Grosso. Isto posto e diante de limitadas informações a respeito dos genótipos grassantes no Estado, foi realizado monitoramento nas linhas de inspeção sanitária pós-abate em 35 matadouros-frigoríficos sob inspeção oficial para detectar lesões granulomatosas sugestivas de TB e proceder a coleta e análise de amostras. Foram processadas e analisadas 167 amostras mediante isolamento bacteriano e técnicas moleculares. Através do método de genotipagem MIRU-VNTR, 49 isolados de M. bovis foram analisados aplicando-se 24 pares de primers diferentes. Desses, 20 marcadores de VNTR mostraram polimorfismo genético. O locus QUB26 apresentou alto poder discriminatório (h=0,66) enquanto ETRC (0,54), Mtub21 (0,5), QUB11b (0,33) e Mtub34 (0,31) mostraram moderada diversidade alélica. Os outros 15 loci (Mtub 04, 29 e 39; MIRU 04, 16, 23, 24, 26, 27, 31, 39 e 40; ETR A; ETR B; QUB 4156) revelaram baixo poder discriminatório (h=0,02 - 0,27). Quatro loci (MIRU 2, 10 e 20; Mtub30) não apresentaram diversidade alélica. Foram observados 28 perfis genéticos (V1-V28) nos isolados de M. bovis obtidos em 35 propriedades rurais. Dentre essas, 30 (85,71%) apresentaram somente 1 tipo de perfil genético. Apenas 19 amostras (38,77%) apresentaram perfil MIRU-VNTR único revelando a existência de isolados com o mesmo perfil MIRU-VNTR entre diferentes regiões geográficas no Estado. O isolamento, seguido da identificação molecular e discriminação genotípica por MIRU-VNTR em isolados de M. bovis constituíram fontes de relevantes informações auxiliares no desenvolvimento de estratégias de erradicação da TB na região de estudo. / Mycobacterium bovis genotyping techniques based on PCR amplification as MIRU-VNTR are important tools in the epidemiological investigation of bovine tuberculosis in areas of low prevalence, such as in the state of Mato Grosso. Based on limited information about the genotypes in state, post-slaughter sanitary inspection lines were monitored in 35 slaughterhouses under official inspection to detect granulomatous lesions suggestive of TB and to collect and analyze samples. 167 Samples were processed and analyzed by bacterial isolation and molecular techniques. Through the MIRU-VNTR genotyping method, 49 M. bovis isolates were analyzed by applying 24 different primer pairs. Of these, 20 VNTR markers showed genetic polymorphism. The QUB26 locus presented high discriminatory power (h = 0.66) while ETRC (0.54), Mtub21 (0.5), QUB11b (0.33) and Mtub34 (0.31) showed moderate allelic diversity. The other 15 loci (Mtub 04, 29 e 39; MIRU 04, 16, 23, 24, 26, 27, 31, 39 e 40; ETR A; ETR B; QUB 4156) showed low discriminatory power (h = 0.02 - 0.27). Four loci (MIRU2, MIRU10, MIRU20 and Mtub30) did not present allelic diversity. Twenty-eight genotypes (V1-V28) were observed in the M. bovis isolates obtained from 35 rural properties. Among these, 30 (85.71%) presented only 1 genotype type. Only 19 samples (38.77%) presented a single MIRU-VNTR profile revealing the existence of isolates with the same MIRU-VNTR profile among different geographic regions in the State. Isolation, followed by molecular identification and genotypic discrimination by MIRU-VNTR in M. bovis isolates were sources of relevant auxiliary information in the development of strategies to eradicate TB in the study region.

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose e tuberculose bovinas na região de Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim e São João da Boa Vista. Foram aleatoriamente amostradas 251 propriedades com criação de bovinos, sendo que nestas, eram sorteadas 40 ou 20 fêmeas acima de 24 meses para diagnóstico de tuberculose e 15 ou 10 fêmeas acima de 24 meses para diagnóstico sorológico de brucelose, dependendo do total de fêmeas existentes. O rebanho foi considerado positivo para brucelose quando havia pelo menos um animal com diagnóstico positivo. Para tuberculose, em propriedades com mais de 99 fêmeas acima de 24 meses, era necessário pelo menos dois animais positivos para a propriedade ser classificada como positiva, enquanto que em propriedades com até 99 fêmeas acima de 24 meses, um animal positivo classificava a propriedade como positiva. Foi aplicado questionário epidemiológico e com base nesse documento, foram feitas as análises univariada e regressão logística para identificar os fatores de risco associados à condição de foco e calculado o valor de odds ratio para quantificar o risco. A prevalência aparente de focos de brucelose foi 11,2% (IC95% = 7,8;15,8) e de tuberculose foi 14,1% (IC95% = 10,2%; 19%). A prevalência aparente de animais positivos para brucelose foi 2,5% (IC95% = 1,5%; 4,1%) e para tuberculose foi 2,7% (IC95% = 1,6;4,5%). Como fatores de risco associados à brucelose ter mais de 57 bovinos no rebanho, apresentou valor de OR = 4,2 (IC95% = 1,9; 9,5). Para tuberculose, exploração leite apresentou valor de OR = 2,6 (IC95% = 1,3; 5,3), ajustado por aquisição de bovinos, OR = 2,4 (IC95% = 1,2; 5,0). Os esforços para redução da prevalência de brucelose não surtiram efeito mesmo após 10 anos de aplicação de medidas de controle preconizadas pelo PNCEBT, sugerindo-se a sua reformulação na área de estudo. / A study was conducted to characterize the epidemiological situation of bovine brucellosis and tuberculosis in Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim and São João da Boa Vista área, State of São Paulo. A total of 251 farms were randomly selected and in each of them 40 or 20 cows over 24 months were selected for tuberculosis diagnosis and 15 or 10 females over 24 months for brucellosis diagnosis depending on the existing total females. The herd was considered positive for brucellosis when there was at least one positive animal. For tuberculosis in herds above 99 females over 24 months at least two positive animals to classify the herd as positive. As long as in farms with up to 99 females over 24 months, a positive animal classified the property as positive. An epidemiological questionnaire was applied and based on this document, univariate and multivariate logistic regression were performed to identify risk factors associated with the diseases, based on odds ratio calculation. The apparent prevalence of brucellosis positive herds was 11.2% (95% CI = 7.8, 15.8) and tuberculosis positive herds was 14.1% (95% CI = 10.2%, 19%). The apparent prevalence of positive animals for brucellosis was 2.5% (95% CI = 1.5%, 4.1%) and tuberculosis was 2.7% (95% CI = 1.6, 4.5%). Having more than 57 cattle in the herd, with an OR = 4.2 (95% CI = 1.9, 9.5) was associated with brucellosis. Milk farm type with an OR = 2.6 (95% CI = 1.3, 5.3), and cattle purchase, with an OR = 2.4 (95% CI = 1.2, 5.0) were associated with tubercullosis. Efforts to reduce the prevalence of brucellosis do not reached the expected effects even after 10 years of implementation of control measures recommended by the PNCEBT. Therefore, the reformulation of the Program is recommended in the study area.

This thesis is about making inference on the host status of feral ferrets in New Zealand for Mycobacterium bovis, the aetiological agent of bovine tuberculosis. The central question addressed is whether the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to persist in ferret populations in the absence of external, non-ferret sources of infection (inter-specific transmission). The question is tackled in three parts�firstly using model selection to identify suitable models for estimating the force of M. bovis infection in ferret populations; secondly applying statistical hypothesis testing to the results of planned manipulative field experiments to test the relationship between M. bovis infection in brushtail possums and that in ferrets; and thirdly using modelling to estimate intra-specific disease transmission rates and the basic reproductive rate (Ro) of M. bovis infection in ferrets. The model selection approach clearly identified the hypothesis of oral infection related to diet was, as modelled by a constant force of infection from the age of weaning, the best approximation of how M. bovis infection was transmitted to ferrets. No other form of transmission (e.g., during fighting, mating, or routine social interaction) was supported in comparison. The force of infection (λ) ranged from 0.14 yr-1 to 5.77 yr-1, and was significantly higher (2.2 times) in male than female ferrets. Statistical hypothesis testing revealed transmission of M. bovis to ferrets occurred from both brushtail possums and ferrets. The force of M. bovis infection in ferrets was reduced by 88% (λ=0.3 yr-1 vs. λ=2.5 yr-1) at sites with reductions in the population density of sympatric brushtail possum populations. A smaller decline in the force of infection resulting from the lethal cross-sectional sampling of the ferret populations was also demonstrated. The modelling approach estimated the basic reproductive rate (Ro) of M. bovis infection in ferrets in New Zealand to vary from 0.17 at the lowest population density (0.5 km-2) recorded to 1.6 at the highest population density (3.4 km-2) recorded. The estimates of Ro were moderately imprecise, with a coefficient of variation of 76%. Despite this imprecision, the Ro for M. bovis infection in ferrets was significantly less than unity for all North Island sites surveyed. Hence it is inferred ferrets are spillover hosts (0<Ro<1) for M. bovis infection in these environments. That is, M. bovis infection will progressively disappear from these ferret populations if the source of inter-specific transmission is eliminated. The estimates of Ro for M. bovis infection in South Island ferret populations were above one (the level required for disease establishment) for a number (5/10) of populations, though the imprecision made it impossible to ascertain whether Ro was significantly greater than one. The estimated threshold population density (Kt) for disease establishment was 2.9 ferrets km-2. It is inferred that, given sufficient population density (>Kt), the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to establish in ferrets in the absence of interspecific transmission. In these areas, ferrets would be considered maintenance hosts for the disease. Active management (e.g., density reduction or vaccination) of ferrets would be required to eradicate M. bovis from ferret populations in these areas, in addition to the elimination of sources of inter-specific transmission, particularly brushtail possums.

The persistent increase of bovine tuberculosis (bTB) over the past twenty years has put a substantial strain on both the British economy and the welfare of livestock. However, the development of an effective bTB vaccine has been continually hindered by the lack of knowledge on the immune response following Mycobacterium bovis (M. bovis) infection. In collaboration with the TB Research Group at the Veterinary Laboratories Agency (VLA, Surrey), this thesis is part of a much wider strategy managed by the Department of Environment, Food and Rural Agency (DEFRA) aimed at elucidating the immunopathogenesis of M. bovis and to develop more effective infection control measures. The specific focus of this thesis was to enable a stronger understanding of the bovine immune response over different periods of M. bovis infection and to apply this new knowledge in evaluating the efficacy of a novel BCG vaccination. Time Course Study: Knowledge of time dependent cytokine expression following M. bovis infection would aid vaccine development by revealing potential correlates of protection. Interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α), interleukin (IL) 4 and 10 expression were analysed using quantitative (q) PCR in formalin fixed bovine lymph nodes following five, twelve and nineteen weeks of M. bovis infection. A strong pro-inflammatory/ T helper 1 (TH1) lymphocyte response was evident at five weeks post M. bovis infection, represented by IFN-γ and TNF-α expression (log2 copies of 6.5 and 2.15, respectively) in the absence of IL4. Between five and twelve weeks of infection, a significant increase was observed in IL10 (log2 copies from 5.97 to 8.27, p<0.01, Mann Whitney test), accompanied by an increase in both IFN-γ (log2 7.53) and TNF-α (log2 3.94). This data conformed to a recently described aspect of TH1 lymphocytes, a ‘self-limiting’ nature in which cells produced both IFN-γ and IL10 with the aim of controlling the heightened pro-inflammatory response. The role of IL10 as an immunosuppressive became evident when comparing cytokine expression between four different types of thoracic lymph node; the left bronchial (LB), cranial mediastinal (CRM), caudal mediastinal (CM) and cranial tracheobronchial (CRT) nodes. The LB and CRM lymph nodes produced significantly higher levels of IFN-γ expression (log2 copies between 8.2 and 10) as compared to the CM and CRT (log2 copies between 2.6 and 5.5, p<0.001, Mann Whitney test). Further analysis of the data as a profile of cytokine expression for each lymph node type revealed that IFN-γ was dominantly expressed within the LB and CRM nodes, whereas within the CM and CRT nodes, IL10 was the dominant cytokine. The former nodes also displayed a higher level of pathological damage (represented by mean percentage area coverage of granuloma, 33.6 and 20%, respectively) as compared to the CM (13%) and the CRT lymph node types (10.8 %). This suggests conflicting roles for IFN-γ and IL10 in the development of immune-associated pathology. Following nineteen weeks of infection, the expression levels of IFN-γ, TNF-α and IL10 reduced (log2 6.22, 3.02 and 7.03, respectively) implying a loss of the cellular response. The later stages of bovine tuberculosis have been shown within the literature to display characteristics of a humoral rather than cell mediated response. However, within this study at nineteen weeks post infection IL4 (an important cytokine in the development of the humoral response) remained undetectable. The results from this study therefore confirm the importance of the cell mediated immune profile in response to M. bovis infection as well as the integral role of IFN-γ in both protection and pathology. It also further demonstrates the involvement of IL10 in controlling the IFN-γ response and highlights this cytokine as being potentially important in future immunologybased vaccination studies. BCG Vaccination Study: The current vaccine used against human tuberculosis, BCG, has provided variable results on protection against infection in experimental bovine studies. The BCG bacterium has lost a comparatively large quantity of genomic DNA through attenuation since its primary production in 1921, of which the majority represented genes encoding antigenic proteins. MPB70 and MPB83 are differentially expressed between BCG sub-strains due to a single nucleotide polymorphism in the alternative sigma factor K (SigK). BCG Pasteur has been shown to produce low levels of these antigenic proteins; however complementation of BCG Pasteur with a copy of sigK from BCG Russia resulted in up-regulating expression. It was therefore hypothesised that the recombinant BCG (sigK) Pasteur would prove more efficient in controlling M. bovis infection by inducing a stronger protective immune response post vaccination. IFN-γ, TNF-α, IL 4 and 10 expression were analysed using qPCR within the freshly dissected lymph nodes of five experimental cattle groups; BCG Pasteur vaccinated M. bovis challenged, BCG (sigK) Pasteur vaccinated challenged, non-vaccinated infected, non-vaccinated noninfected and BCG Pasteur vaccinated non-infected. Five weeks following infection, a strong IFN-γ mRNA response was detected in both the non-vaccinated and vaccinated cattle (mean log2 copies between 9.6 and 10.5 as compared to between 7.84 and 8.58 in the non-infected cattle). M. bovis infection also induced a significant reduction in IL10 mRNA levels in both vaccinated and non-vaccinated cattle (mean log2 14.4 in the infected groups compared to 15.5 in the non-infected cattle, p<0.005, Mann Whitney test) although there was little difference in TNF-α expression (mean log2 copies between 11.06 and 11.8 in all five groups). Interestingly, IL4 mRNA was detectable only within the two non-infected control groups (mean log2 12.4), further supporting the concept of a strong cell mediated response after five weeks of infection. Vaccination prior to challenge had an effect on IFN-γ mRNA levels only, as both the BCG Pasteur and BCG (sigK) Pasteur vaccinated groups displayed a smaller increase in IFN-γ mRNA following challenge (mean log2 10.3 and 9.6, respectively) as compared to the nonvaccinated group (mean log2 10.5). This reflected the role of vaccination in priming the immune response to enable more rapid elimination of the bacteria and subsequently inducing a lesser pro-inflammatory response. Interestingly, the BCG Pasteur vaccinated group appeared to control the immune response to a greater extent, as IFN-γ mRNA was significantly similar to that observed in the non-vaccinated non-infected group (mean log2 8.58, p>0.05, Mann Whitney test). In addition to the qPCR data, levels of IFN-γ and TNF-α protein (represented by the number of cells producing these proteins) were also analysed by immunohistochemistry. IFN-γ protein in the five experimental groups displayed the same pattern as that observed for IFN-γ mRNA expression (p<0.001, Spearmans correlation coefficient). However, analysis of TNF-α protein revealed significant differences between the five groups (p<0.005, Kruskal Wallis test) in contrast to that observed for the mRNA levels (p>0.05, Spearmans correlation coefficient) suggesting that posttranscriptional controls may play an important role in TNF-α translation. The difference in IFN-γ mRNA and protein expression between the two vaccination groups was also reflected within the pathological data. Although both BCGs reduced levels to below that of the non-vaccinated group (represented by mean percentage area coverage of granuloma, 59%), the BCG Pasteur group displayed less pathology (mean 6%) compared to the BCG (sigK) Pasteur cattle (mean 35%). It was suggested that the increased antigenic repertoire of the recombinant BCG (sigK) Pasteur did result in a stronger stimulation of the immune response post vaccination but that, as a consequence the bacterial threat was eliminated more rapidly.

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose e tuberculose bovinas na região de Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim e São João da Boa Vista. Foram aleatoriamente amostradas 251 propriedades com criação de bovinos, sendo que nestas, eram sorteadas 40 ou 20 fêmeas acima de 24 meses para diagnóstico de tuberculose e 15 ou 10 fêmeas acima de 24 meses para diagnóstico sorológico de brucelose, dependendo do total de fêmeas existentes. O rebanho foi considerado positivo para brucelose quando havia pelo menos um animal com diagnóstico positivo. Para tuberculose, em propriedades com mais de 99 fêmeas acima de 24 meses, era necessário pelo menos dois animais positivos para a propriedade ser classificada como positiva, enquanto que em propriedades com até 99 fêmeas acima de 24 meses, um animal positivo classificava a propriedade como positiva. Foi aplicado questionário epidemiológico e com base nesse documento, foram feitas as análises univariada e regressão logística para identificar os fatores de risco associados à condição de foco e calculado o valor de odds ratio para quantificar o risco. A prevalência aparente de focos de brucelose foi 11,2% (IC95% = 7,8;15,8) e de tuberculose foi 14,1% (IC95% = 10,2%; 19%). A prevalência aparente de animais positivos para brucelose foi 2,5% (IC95% = 1,5%; 4,1%) e para tuberculose foi 2,7% (IC95% = 1,6;4,5%). Como fatores de risco associados à brucelose ter mais de 57 bovinos no rebanho, apresentou valor de OR = 4,2 (IC95% = 1,9; 9,5). Para tuberculose, exploração leite apresentou valor de OR = 2,6 (IC95% = 1,3; 5,3), ajustado por aquisição de bovinos, OR = 2,4 (IC95% = 1,2; 5,0). Os esforços para redução da prevalência de brucelose não surtiram efeito mesmo após 10 anos de aplicação de medidas de controle preconizadas pelo PNCEBT, sugerindo-se a sua reformulação na área de estudo. / A study was conducted to characterize the epidemiological situation of bovine brucellosis and tuberculosis in Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim and São João da Boa Vista área, State of São Paulo. A total of 251 farms were randomly selected and in each of them 40 or 20 cows over 24 months were selected for tuberculosis diagnosis and 15 or 10 females over 24 months for brucellosis diagnosis depending on the existing total females. The herd was considered positive for brucellosis when there was at least one positive animal. For tuberculosis in herds above 99 females over 24 months at least two positive animals to classify the herd as positive. As long as in farms with up to 99 females over 24 months, a positive animal classified the property as positive. An epidemiological questionnaire was applied and based on this document, univariate and multivariate logistic regression were performed to identify risk factors associated with the diseases, based on odds ratio calculation. The apparent prevalence of brucellosis positive herds was 11.2% (95% CI = 7.8, 15.8) and tuberculosis positive herds was 14.1% (95% CI = 10.2%, 19%). The apparent prevalence of positive animals for brucellosis was 2.5% (95% CI = 1.5%, 4.1%) and tuberculosis was 2.7% (95% CI = 1.6, 4.5%). Having more than 57 cattle in the herd, with an OR = 4.2 (95% CI = 1.9, 9.5) was associated with brucellosis. Milk farm type with an OR = 2.6 (95% CI = 1.3, 5.3), and cattle purchase, with an OR = 2.4 (95% CI = 1.2, 5.0) were associated with tubercullosis. Efforts to reduce the prevalence of brucellosis do not reached the expected effects even after 10 years of implementation of control measures recommended by the PNCEBT. Therefore, the reformulation of the Program is recommended in the study area.

Realizou-se um inquérito epidemiológico para estimar a prevalência da tuberculose bovina no Estado de Rondônia. O Estado foi estratificado em três circuitos produtores, e em cada um dos circuitos escolheu-se aleatoriamente as propriedades a serem visitadas, totalizando 904 propriedades e 19.640.animais amostrados. Um número pré-estabelecido de animais foram aleatoriamente escolhidos e submetidos ao teste cervical comparativo, de modo que em propriedades com até 100 fêmeas com idade superior a 24 meses, foram testadas 20 fêmeas ou menos e, em propriedades com mais de 100 fêmeas com idade superior a 24 meses, foram testadas 40 fêmeas. Foram considerados focos as propriedades com um animal reagente entre os 20 amostrados e nas propriedades com 40 fêmeas amostradas que tiveram dois animais reagentes. As prevalências de focos e animais para o Estado foram, respectivamente, 2,3% [1,5; 3,5] e 0,1% [0,1; 0,2], e para os circuitos pecuários foram: Circuito 1 (Norte-Oeste-Sul), 1,7% [0,7; 4,0] e 0,1% [0; 0,4], Circuito 2 (Nordeste), 3,0% [1,6; 5,7] e 0,2% [0,1; 0,3] e Circuito 3 (Sudeste), 2,3% [1,1; 4,7] e 0,1%[0; 0,2]. Além dos testes tuberculínicos, aplicou-se um questionário para levantamento dos fatores de risco para tuberculose bovina. As variáveis foram submetidas à analise univariada e aquelas que apresentaram p < 0,20, foram submetidas à regressão logística. A aquisição de animais sem tuberculinização foi associada a condição de foco de tuberculose (OR = 7,1 [1,6;31,1], p=0,009). As baixas prevalências tanto de focos como de animais no Estado de Rondônia, sugerem a adoção de medidas de erradicação, previstas no PNCEBT. A obrigatoriedade de execução de testes tuberculínicos para trânsito interestadual independentemente da finalidade e a regulamentação de rastreamentos de novos focos a partir de amostras coletadas em frigoríficos tornam-se imprescindíveis para detecção de focos residuais e subsequente erradicação da tuberculose bovina. / An epidemiological survey was conducted to assess the prevalence of bovine tuberculosis in the State of Rondônia, Brazil. The State was divided into three epidemiological regions and, in each of them, a total of 904 farms were randomly chosen and 19,640 animals were sampled. A fixed number of animals were randomly chosen and submitted to the cervical comparative test, so that in farms with less than 100 bovine females over 24 months of age, 20 females, or less (if the herd was < 20), were tested. In farms with over 100 bovine females over 24 months of age, 40 females were sampled. We have considered positive farms when at least one positive animal among the 20 sampled or two positive animals in the farm with 40 animals sampled were found. The prevalence of infected herds and positive animals in Rondonia State were, respectively, 2.3% [1.5; 3.5] and 0.1% [0.1; 0.2]. In each epidemiological region, the results were: Region 1 (North west - South) = 1.7% [0.7; 4.0] and 0.1% [0; 0.4]; Region 2 (Northeast) = 3.0% [1.6; 5.7] and in the Region 3 (Southeast) = 2.3%[1.1; 4.7] and 0.1% [0; 0.2]. In addition to the tuberculin tests a questionnaire was applied in each visited farm in order to determine the risk factors for bovine tuberculosis. The variables were submitted to univariate analysis and those with p < 0.20) were submitted to logistic regression. The acquisition of animals without tuberculin tests was associated to tuberculosis (OR = 7.1 [1.6; 31.1], p = 0.009). The low prevalence of bovine tuberculosis in Rondonia State suggests the adoption of eradication measures, provided by the National Program for the Eradication and Control of Bovine Brucellosis and Tuberculosis (PNCEBT). Compulsory enforcement tuberculin tests for interstate transit regardless of the purpose and regulation of new outbreaks tracing from abattoir samples makes it essential to detect residual foci and subsequent eradication of bovine tuberculosis.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-18T13:39:28Z No. of bitstreams: 1 Luiz Carlos Fontes Baptista Filho.pdf: 748065 bytes, checksum: c624b21e9ec70bfdd8c60bd08dac6e9f (MD5) / Made available in DSpace on 2016-10-18T13:39:28Z (GMT). No. of bitstreams: 1 Luiz Carlos Fontes Baptista Filho.pdf: 748065 bytes, checksum: c624b21e9ec70bfdd8c60bd08dac6e9f (MD5) Previous issue date: 2012-03-17 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Enzootic Leukosis (EBL) and Bovine Tuberculosis (BT) compromise the immune status of the herds, especially the immunosuppressive potential of bovine leukosis virus (BLV). Several factors interfere with leukocyte values of cattle, but little is known about the effects of BT on them. The aim with the study was to evaluate the leukogram of tuberculinized cattle, with a view to the use of leukocytes as an epidemiologic tool in combating EBL in dairy herds of Pernambuco. 1.000 serum samples from 33 herds of cattle coming from various municipalities in the state were submitted to the serodiagnosis of EBL (AGID), being 920 cattle previously tuberculinized by the simultaneously comparative technique (SCT). Blood smears were prepared of all tuberculinized cattle, and the total and differential leukocyte count was performed using conventional techniques. The evaluation of the influence of BT in leukocyte counts was performed by comparing the results of four experimental groups, according to the results of the AGID and SCT (gTB group - 41 to the positive SCT; group gLEB - the 151 AGID positive, group gNEG - 379 to the negative TSC and AGID, group gINT - 43 positive to both tests). The prevalence rates of TB and LEB were, respectively, 11% (99/920) and 28% (282/1000), with 88% (29/33) of herds contributing at least one animal positive for one or both tests. From the 920 cattle examined, 43 (4,7%) were positive to both tests simultaneously. The mean values of leukocytes and lymphocytes (x 103/mm3 of blood) of the experimental groups were, respectively: gTB 9,6 ± 2,5 and 5,9 ± 1,9; gLEB 13,3 ± 6,3 and 9,1 ± 6,0; gNEG 11,5 ± 3,8 and 7,6 ± 5,1; and gINT 11,8 ± 4,3 and 8,0 ± 3,5. There were significant differences (p<0,05) between leukocytes and lymphocytes, when confronted gTB group (lower values) and gLEB (higher values) with the gNEG group, while gINT group did not differ (p>0,05) in none of the parameters analyzed in relation to the group gNEG. It is concluded that the leukogram is influenced by bovine BT, and health programs to combat LEB in dairy herds in the state of Pernambuco, and include the white blood cell count as an epidemiologic tool wish to consider the strategic prior tuberculin test the herds involved, being discarded cattle that are experiencing imunoalergic test positivity. / Leucose Enzoótica (LEB) e Tuberculose bovina (TB) comprometem o estado imunitário dos rebanhos, especialmente pelo potencial imunodepressor do Vírus da Leucose Bovina (VLB). Inúmeros fatores interferem nos valores leucométricos dos bovinos, porém, pouco se sabe dos efeitos da TB sobre os mesmos. O objetivo com o estudo foi avaliar o leucograma de bovinos tuberculizados, com vista ao uso da leucometria como ferramenta epidemiológica no combate à LEB em rebanhos leiteiros de Pernambuco. Amostras séricas de 1.000 bovinos procedentes de 33 rebanhos de diversos municípios do estado foram submetidas ao sorodiagnóstico da LEB (IDGA), sendo 920 bovinos previamente tuberculinizados pela técnica simultânea comparada (TSC). Esfregaços sanguíneos foram confeccionados de todos os bovinos tuberculinizados, sendo a contagem total e diferencial dos leucócitos realizada usando técnicas convencionais. A avaliação da influência da TB no leucograma foi realizada pelo confronto dos resultados de quatro grupos experimentais, em função dos resultados da IDGA e TSC (grupo gTB - 41 positivos ao TSC; grupo gLEB - 151 positivos à IDGA; grupo gNEG - 379 negativos ao TSC e IDGA; grupo gINT - 43 positivos a ambos os testes). As taxas de prevalência da TB e da LEB foram, respectivamente, 11% (99/920) e 28% (282/1000), com 88% (29/33) dos rebanhos contribuindo com ao menos um animal positivo para um ou ambos os testes. Dos 920 bovinos examinados, 43 (4,7%) apresentaram positividade simultânea a ambos os testes. Os valores médios dos leucócitos e linfócitos (x 103/mm3 de sangue) dos grupos experimentais foram, respectivamente: gTB 9,6 ± 2,5 e 5,9 ± 1,9; gLEB 13,3 ± 6,3 e 9,1 ± 6,0; gNEG 11,5 ± 3,8 e 7,6 ± 5,1; e gINT 11,8 ± 4,3 e 8,0 ± 3,5. Houve diferença significativa (p<0,05) entre os leucócitos e linfócitos, quando confrontados os grupos gTB (valores menores) e gLEB (valores maiores) com o grupo gNEG, enquanto o grupo gINT não diferiu (p>0,05) em nenhum dos parâmetros analisados em relação ao grupo gNEG. Conclui-se que o leucograma dos bovinos sofre influência da TB, devendo programas sanitários de combate à LEB em rebanhos leiteiros do estado de Pernambuco e que incluam a leucometria como ferramenta epidemiológica estratégica preconizar a prévia tuberculinização dos rebanhos envolvidos, sendo descartados os bovinos que apresentarem positividade ao teste imunoalérgico.