Assessing the impact of various tuberculin PPD brands on bovine tuberculosis diagnosis.

Although several brands of tuberculin purified protein derivatives (PPDs) are available for diagnosing bovine tuberculosis (bTB), comparative studies to determine their diagnostic accuracy are infrequent. In Ecuador we compared two different PPD brands for bTB diagnosis using skin testing and measuring skin thickness increase. Additionally, we evaluated four PPD brands, including those used for skin testing, in the Bovine Tuberculosis Interferon Gamma Test (IFN-γ test) measuring IFN-γ induction in whole blood. The study included 17 naturally tuberculosis-infected PPD and IFN-γ test positive bovines. Both the field and laboratory results showed significant differences in classifying the 17 bovines as bTB positive or negative. We hypothesize that several factors, such as the genetic background of the cows, sensitization to environmental mycobacteria, M. bovis strains involved in the bTB infection, and the manufacturing procedures of the PPDs, could have influenced the immune reaction toward the different tuberculin PPD brands. Our study emphasizes the necessity for comparative studies aimed at determining the diagnostic accuracy of PPD brands for bTB diagnosis as well as the development of standardized methods for PPD production and potency determination.

Live attenuated Mycobacterium bovis strains combined with the encapsulated H65 antigen as a vaccine strategy against bovine tuberculosis in a mouse model.

Mycobacterium bovis is an etiological agent of bovine tuberculosis (bTB) that also infects other mammals, including humans. The lack of an effective vaccine for the control of bTB highlights the need for developing new vaccines. In this study, we developed and evaluated an M. bovis strain deleted in the virulence genes phoP, esxA and esxB as a vaccine candidate against bTB in BALBc mice. The evaluated strains were the new live vaccine and BCG, alone or in combination with ncH65vD. The immunogen ncH65vD is a fusion protein H65, encapsulated together with vitamin D3, within the oily body of a nanocapsule composed of an antigen-loading polymeric shell. All vaccines conferred protection against the M. bovis challenge. However, no significant differences were detected among the vaccinated groups regarding bacterial loads in lungs and spleen. Mice vaccinated with the mutant strain plus ncH65vD showed negative Ziehl Neelsen staining of mycobacteria in their lungs, which suggests better control of bacteria replication according to this protection parameter. Consistently, this vaccination scheme showed the highest proportion of CD4 + T cells expressing the protection markers PD-1 and CXCR3 among the vaccinated groups. Correlation studies showed that PD-1 and CXCR3 expression levels in lung-resident CD4 T cells negatively correlated with the number of colony forming units of M. bovis in the lungs of mice. Therefore, the results suggest a link between the presence of PD-1 + and CXCR3 + cells at the site of the immune response against mycobacteria and the level of mycobacterial loads.

Condemnation of bovine carcasses due to tuberculosis-gross lesions in Rio Grande do Sul, Brazil: Associated risk factors.

Surveillance of bovine tuberculosis (bTB) lesions in animals at slaughterhouses is useful for controlling and eradicating the disease, besides providing epidemiological information. This study aimed to identify risk factors for bovine tuberculosis (bTB) condemnation in cattle at slaughterhouses in Rio Grande do Sul, Brazil. A logistic regression analysis was conducted using data on bTB-related condemnations. Variables examined included animal origin, number of slaughtered animals, season, inspection level (state or municipality), animal sex, and slaughterhouse location. A total of 297,817 Animal Transport Guides were evaluated, representing the transportation of 3497,521 animals. Among these, 6097 (2.05%) had at least one animal condemned due to bTB lesions. Risk factors for condemnation included larger batch sizes, female animals, slaughterhouses, and animal origin. The higher condemnation frequency in females and regions with dairy farms suggests links to milk production. Variation in condemnation rates by inspection level and slaughterhouse highlights the need for standardized procedures in identifying bTB lesions. Identifying these risk factors enables targeted interventions to enhance disease control and eradication efforts.

Deletion in KARLN intron 5 and predictive relationship with bovine tuberculosis and brucellosis infection phenotype.

Kalirin (gene: KALRN) is a Rho-GEF kinase linked to neurodegenerative diseases in humans. Unexpectedly, various polymorphisms in KALRN gene were previously associated with resistance to bacterial infections in ruminants. In this study, we evaluated the effect of the rs384223075 (RS-075) deletion in KALRN intron 5 on the occurrence of Mycobacterium bovis and Brucella abortus infections in cattle. We performed two separate case-control association analyses: one for bovine tuberculosis (bTB) using 308 Holstein and Jersey cows from three herds with prevalence between 5 and 15% for this infection; and another for brucellosis using 140 Holstein and beef crossbred cows from two herds with high prevalence for brucellosis (> 30%). In the bTB analysis, the RS-075 deletion frequency was higher among cases than controls (p = 0.0001), and the absence of the RS-075 deletion allele was associated with negative PPD-skin test results (p = 0.0009) at genotype level. On the contrary, RS-075 was not associated with Brucella spp. serological status (p = 0.72) but, unexpectedly, the deletion allele was more frequent among controls than cases in the beef crossbred herd (0.31 vs. 0.14, p = 0.02). In concordance with this observation, in vitro assays showed that the RS-075 deletion could be linked to an enhanced cellular response to bacterial antigens and unspecific stimulation in mononuclear cells derived from beef crossbred cows, specifically the reactive nitrogen species production (p = 0.008) and proliferation capacity (p = 0.018). This study is consistent with other reports that support an important role of the KALRN gene and its polymorphisms in the host response to intracellular pathogens.

Impact on condemnations of bovine carcasses for tuberculosis and cysticercosis after changes in federal legislation in the state of Rio Grande do Sul (2014-2020) / Impacto nas condenações de carcaças bovídeas por tuberculose e cisticercose após as modificações na legislação federal no estado do Rio Grande do Sul (2014-2020)

The inspection of products of animal origin, carried out by veterinary agricultural inspectors, is based on the Brazilian legislation described in the Regulation of Industrial and Sanitary Inspection of Products of Animal Origin of the Ministry of Agriculture, Livestock and Supply. On March 29, 2017, Decree No. 9,013 was published, which repeals Decree No. 30,691, of 1952, with this, major changes occurred in the legislation described in RIISPOA, such as the criteria used for judging carcasses. The objective of this study was to verify whether these changes had an impact on condemnations of bovine carcasses because of cysticercosis and tuberculosis in the State of Rio Grande do Sul. The number of animals slaughtered and the number of carcasses condemned because of tuberculosis and cysticercosis between 2014 and 2020 were obtained from the database of the Agricultural Defense System (SDA), the official data system of the Secretariat of Agriculture, Livestock and Rural Development (SEAPDR), it was possible to observe an increase in the number of cases of carcasses condemned because of cysticercosis and a reduction of animals affected by the disease in the period after the changes in the regulation. The number of carcasses condemned because of tuberculosis, as well as the number of animals affected by the disease, remained stable after the changes in the regulation. The results showed that the changes in federal legislation had a real impact on the historical series of condemnations of bovine carcasses due to cysticercosis in the state of Rio Grande do Sul. In carcasses affected by tuberculosis, the impact of carcass condemnation on the historical series after changes was not significant.

A inspeção de produtos de origem animal, realizada por médicos veterinários fiscais agropecuários, é baseada na legislação brasileira descrita no Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal (RIISPOA) do Ministério da Agricultura, Pecuária e Abastecimento (MAPA). No dia 29 de março de 2017 foi publicado o Decreto nº 9.013, que revoga o Decreto nº 30.691, de 1952, com isso grandes alterações ocorreram na legislação descrita no RIISPOA, como os critérios de julgamento de carcaças. O objetivo desse estudo foi verificar, se essas alterações tiveram impacto nas condenações de carcaças de bovídeos por cisticercose e tuberculose no Estado do Rio Grande do Sul (RS). O número de animais abatidos e o número de carcaças condenadas por tuberculose e cisticercose entre 2014 e 2020 foram obtidos do banco de dados do Sistema de Defesa Agropecuária (SDA), sistema oficial de dados da Secretaria da Agricultura Pecuária e Desenvolvimento Rural (SEAPDR), foi possível observar aumento do número de casos de carcaças condenadas por cisticercose e redução de animais afetados pela enfermidade no período após as alterações no regulamento. O número de carcaças condenadas por tuberculose, assim como o número de animais afetados pela doença se manteve estável após as mudanças no regulamento. Os resultados encontrados mostraram que as modificações na legislação federal trouxeram impacto real na série histórica de condenações de carcaças bovídeas por cisticercose no âmbito do Estado do Rio Grande do Sul enquanto nas carcaças afetadas por tuberculose o impacto das condenações não foi significativo.

Apparent prevalence and risk factors for bovine tuberculosis in the state of Paraná, Brazil: an assessment after 18 years since the beginning of the Brazilian program.

Bovine tuberculosis (bTB) impacts considerably animal production and one health worldwide. To describe the prevalence, risk factors, and spatial pattern of the disease in the state of Paraná, Brazil, a cross-sectional study was conducted from September 2018 to February 2019. The area was divided into seven regions. Within each region, farms were randomly selected, and a predetermined number of cows was selected and tested by a comparative cervical tuberculin test. 17,210 animals were tested across 1757 farms. Herd prevalence of bTB-infected herds in Paraná was 2.5% [1.87-3.00%]. It has varied from 0.8 to 3.98% among seven regions, with clustering being detected in the west, central, and northeast areas. Animal prevalence was 0.35% [0.21-0.59%] and has varied from 0.08 to 0.6% among the pre-set regions. No major shifts in the prevalence of bTB were detected since 2007. Large-sized herds, dairy production, and feeding with whey were detected to be correlated with the presence of bTB. Exclusively among dairy herds, veterinary assistance from cooperatives, possession of self-owned equipment to cool milk, and feeding with whey were correlated with the disease. Considering these results, it is recommended that the state of Paraná seek to implement a surveillance system for the detection of bTB-infected herds transforming them into free ones, if possible, incorporating elements of risk-based surveillance. Health education is also recommended to inform farmers about the risks of introducing animals without testing and of feeding raw whey to calves.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

One Health and surveillance of zoonotic tuberculosis in selected low-income, middle-income and high-income countries: A systematic review.

BACKGROUND: Little is known about zoonotic tuberculosis (zTB) due to Mycobacterium bovis burden across the globe. The aim of this study was to describe zTB surveillance programs in selected WHO signatory countries and to assess the relationship of the disease with the country's income level and the risk of M. bovis transmission. METHODS: We searched the main articles databases and grey literature for guide documents published between 1980 and 2019. For inclusion, the articles and guide documents had to be in English, French, Portuguese, Spanish, or Italian. Only original articles and narrative and systematic reviews were accepted and the guide documents were required to be available on official websites. We excluded articles that did not focus on epidemiology, control and surveillance. We used bovine TB cases in livestock and wildlife populations as a proxy for the country's risk of zTB using data from the World Organization for Animal Health (OIE) published from 2015 to 2018. Countries were classified according to income level (World Bank's classification) and strength of zTB surveillance. The study was registered in PROSPERO under number CRD42018090603. FINDINGS: We included 13 articles and 208 guide documents including data from 119/194 countries (61.3%). We found a lack of surveillance data about zTB in over half (89.9%) of the 119 WHO signatory countries. Most surveillance systems perform passive surveillance and are not integrated into the One Health perspective, which was operating in 4/119 (3.4%) countries, all high-income. Many of these countries (71/119, 59.7%) have M. bovis circulating in their cattle herds, but only ~10% of them have implemented zTB surveillance activities. INTERPRETATION: Our findings highlight weaknesses in zTB surveillance worldwide, with a consequent lack of information that could support an adequate understanding of disease burden, especially in countries at major risk for M. bovis transmission. To meet this challenge, efforts will be needed to promote intersectoral policies, implementing the One Health strategy.

Lack of Exposure to Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis in Chilean Cervids, and Evidence of a New Mycobacterium-Like Sequence.

Screening of serum and fecal samples from huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from southern Chile for Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP) found all but four samples Mycobacterium-negative. The positive sequences showed only 92-93% similarity with MAP and were from remote Isla Riesco populations.

Replication and transmission features of two experimental vaccine candidates against bovine tuberculosis subcutaneously administrated in a murine model.

Cattle vaccination is an attractive approach in compliance with control and eradication programs against Bovine Tuberculosis (bTB). Today, there is no anti bTB vaccine licensed. Two vaccine candidates, MbΔmce2 and MbΔmce2-phoP previously designed were evaluated in BALB/c mice, including the parental M. bovis NCTC10772 and a M. bovis hypervirulent Mb04-303 strains as controls. Sentinel mice (non-inoculated) cohoused with subcutaneous inoculated mice. Persistence, visible tuberculosis lesions (VTL) in lungs and spleens and bacillary load were investigated subcutaneously delivered at 60 and 90 days after inoculation (dpi) as well as their potential transmission to naïve mice. While a 100% survival was observed at 90 dpi without VTL in all groups, transmission was not evidenced in the sentinels mice. Vaccine candidates and control strains were isolated from the spleen of all inoculated mice, while Mb04-303 was isolated from the lungs of one inoculated mouse. Vaccine candidate's attenuation considering survival, lung bacillary load and VTL was confirmed, administrated by the subcutaneous route. Future experiments are necessary to demonstrate whether the persistence of both mutants in the spleen, with low CFU, remains over time to increase the potential increasing risk of dissemination to organs and subsequent transmission to other animals by airborne or other routes.

Análise da correlação dos índices de estrutura e recursos humanos do serviço veterinário oficial em 2019 e as incidências acumuladas (2017 ­ 2019) de doenças de notificação obrigatória em bovinos e equinos no Brasil / Analysis of the correlation between the structure and human resources indexes of the official veterinary service in 2019 and the accumulated incidences (2017 ­ 2019) of notifiable diseases in cattle and horses in Brazil

As doenças de notificação obrigatória em bovinos podem gerar impactos sociais e econômicos significativos na cadeia pecuária brasileira, além de consequências negativas no mercado internacional devido a embargos sanitários. Para auxiliar no entendimento de como um sistema de vigilância epidemiológica com mais recursos pode gerar mais credibilidade para o país, foram realizadas análises de correlação entre a notificação de doenças e a estrutura veterinária disponível nos Órgãos Executores de Sanidade Agropecuária (OESAs), a partir dos dados contidos no Sistema Nacional de Informação Zoossanitária (SIZ), entre os anos de 2017 e 2019. Com base nos dados do serviço veterinário, foram produzidos o Índice de Estrutura Física Oficial (IEFO) e o Índice de Recursos Humanos do Serviço Oficial (IRHSO). Foi realizada análise de correlação entre a notificação de doenças de bovinos com a capacidade de estrutura física e recursos humanos de vigilância epidemiológica disponíveis no Serviço Veterinário brasileiro. Os estados AP, RR e SC foram os que mais notificaram brucelose e tuberculose no período e estão entre os melhores índices de estrutura e recursos humanos do país. A análise dos índices mostrou que a raiva não possui correlação significativa com estrutura e recursos humanos do serviço, entretanto, brucelose e tuberculose possuem correlação positiva com estrutura veterinária oficial disponível para a vigilância em bovinos. Portanto, melhorias na estrutura podem refletir no incremento dos índices de notificação das doenças de bovinos, assim como na qualidade de suas informações.

Notifiable diseases in cattle can generate significant social and economic impacts on the Brazilian livestock chain, in addition to impacts on the international market due to sanitary embargoes. To help understand how an epidemiological surveillance system with more resources can generate more credibility for the country, correlation analyzes were carried out between the notification of diseases and the veterinary structure available in the Executing Bodies of Agricultural Health (OESAs), based on the data contained in the National System of Zoosanitary Information (SIZ), between the years 2017 to 2019. Based on public data from the veterinary service, the Official Physical Structure Index (IEFO) and the Official Service Human Resources Index (IRHSO) were produced. Correlation analysis was performed between the notification of bovine diseases with the capacity of physical structure and human resources for epidemiological surveillance available in the Brazilian Veterinary Service. AP, RR and SC were the states that most notified brucellosis and tuberculosis in the period and are among the best indices of structure and human resources in the country. The analysis of correlation indices showed that the rabies disease does not have a significant correlation with the structure and the human resources of the service, however, brucellosis and tuberculosis does have a positive correlation with the official veterinary structure available for surveillance in cattle. Therefore, Improvements in the structure can reflect in the increase of the notification rates, as well as in the quality of its information.

Isolation and molecular identification of Mycobacterium bovis in tissue lesions of cattle slaughtered in slaughterhouses located in the State of Ceará / [Isolamento e identificação molecular de Mycobacterium bovis em lesões teciduais de bovinos abatidos em matadouros-frigoríficos localizados no estado do Ceará]

The objective of this research was to identify Mycobacterium bovis in lesions suggestive of tuberculosis in bovine carcasses in the State of Ceará, by means of bacteriological and molecular diagnostic tests. Between August 2017 and January 2019, the State inspection service (SIE) inspected 59,512 cattle, of which 7.4% (44 / 59,512) presented suggestive lesions. Of these animals, 68 samples were sent, of which 4.5% (31/68) located in the lung, 2.9% (20/68) in lymph nodes, 2.0% (14/68) in the liver, and 0.4% in the carcass (3/68). When performing bacteriological isolation, 15.9% (7/44) of bovines showed colony growth in the samples. The smears of the isolates were submitted to Zielh-Neelsen staining and all confirmed acid-fast bacilli. The polymerase chain reaction identified all isolates 100% (7/7) as M. bovis. The association of diagnostic techniques allowed to identify the presence of the agent in the State and the molecular analysis proved to be a beneficial technique in the monitoring of bovine tuberculosis and can be used as an auxiliary method in the bovine tuberculosis control and eradication program in the State of Ceará.

O objetivo do trabalho foi pesquisar Mycobacterium bovis em lesões sugestivas de tuberculose nas carcaças de bovinos no estado do Ceará, por meio dos testes de diagnóstico bacteriológico e molecular. Entre agosto de 2017 e janeiro de 2019, o Serviço de Inspeção Estadual (SIE) inspecionou 59.512 bovinos; destes, 7,4% (44/59.512) apesentaram lesões sugestivas. Desses animais foram enviadas 68 amostras, das quais 4,5% (31/68) estavam localizadas no pulmão, 2,9% (20/68) nos linfonodos, 2,0% (14/68) no fígado e 0,4% (3/68) na carcaça. Ao realizar o isolamento bacteriológico, 15,9% (7/44) dos bovinos evidenciaram crescimento de colônias nas amostras. Os esfregaços dos isolados foram submetidos à coloração de Zielh-Neelsen e todos eles confirmaram bacilo álcool-ácido resistente. A reação em cadeia da polimerase identificou todos os isolados, 100% (7/7), como M. bovis. A associação das técnicas de diagnóstico permitiu identificar a presença do agente no estado, e a análise molecular demonstrou ser uma técnica benéfica no monitoramento da tuberculose bovina, podendo ser utilizada como um método auxiliar no programa de controle e erradicação da tuberculose bovina no estado do Ceará.

Reduced levels of serum 25(OH)D3 are associated with tuberculosis positive cattle under conditions of high natural exposure to Mycobacterium bovis.

Serum vitamin D (25(OH)D3) concentrations of < 30 ng/mL in cattle are insufficient to induce an adequate immune response against intracellular pathogens, which suggests that the efficacy of the immune response may be highly dependent on the bioavailability of 25(OH)D3. This study shows an overview of both in vitro and in vivo 25(OH)D3-mediated immune modulation amongst dairy cattle naturally exposed to M. bovis. Tuberculin status was confirmed by interferon gamma release assay (IGRA), and natural exposure was demonstrated by polymerase chain reaction (PCR). Tuberculin (-) cattle have a higher serum concentration of 25(OH)D3 (X¯= 87.12 ng/mL) when compared to tuberculin (+) cattle (X¯ = 45.86 ng/mL). Reduced serum 25(OH)D3 levels are associated with the presence of bovine TB, and serum 25(OH)D3 levels of > 80 ng/mL are necessary to counteract infection by M. bovis. Kill assays were performed to evaluate in vitro 25(OH)D3 modulation of intracellular M. bovis growth in bovine macrophages, which showed that reduced serum 25(OH)D3 levels are associated with diminished mycobactericidal capacity in this experimental model. On the other hand, increased 25(OH)D3 in culture media enhances phagocytosis and nitric oxide production, which in turn improves capacity to combat M. bovis.

Vaccination of Holstein heifers with Mycobacterium bovis BCG strain induces protection against bovine tuberculosis and higher milk production yields in a natural transmission setting.

Bovine tuberculosis (TB) is a chronic disease caused mainly by Mycobacterium bovis, a zoonotic pathogen that has a worldwide distribution causing serious economic losses for milk and meat producers. In Chile, the disease in dairy cattle has a heterogeneous distribution, where the Metropolitan Region concentrates the highest animal prevalence and the main challenge for the national control and eradication programme. In this epidemiological context, vaccination with the M. bovis Bacillus Calmette-Guerin (BCG) vaccine might be a useful strategy for disease prevention and control. The objective of this study was to assess the efficacy and impacts on productivity and fertility of vaccination with the BCG Russia strain in 11 month-old heifers from a dairy farm, under a natural transmission condition. Sixty-two animals were vaccinated via the subcutaneous route with the equivalent of one human dose of BCG, and 60 control animals received saline. Subsequently, blood sampling was performed at 3, 6, 9, 12, 15 and 18 months post-inoculation, and infection status was determined using the IFNγ release assay (IGRA) with the DIVA (differentiate infected from vaccinated animals) antigens ESAT-6, CFP-10 and Rv3615c. Efficacy was calculated as the percentage of reduction in the incidence of infection attributable to vaccination, which showed a statistically significant level of overall protection of 66.5%. No adverse effects on fertility and production were recorded. In contrast, we observed beneficial effects of vaccination on several milk production parameters, with the milk yield in the first 100 days after calving in the BCG group significantly higher compared to unvaccinated heifers (p < .05). These results suggest that BCG vaccination of heifers in a natural transmission setting might result in both sanitary and productive benefits, supporting its implementation as a new strategy for TB prevention in a high prevalence area of Chile.

Milk Contamination by Mycobacterium tuberculosis Complex, Implications for Public Health in Amazonas, Brazil.

ABSTRACT: In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. The highest rate of consumption of raw milk and its derivatives in Brazil occurs in Amazonas. This state also has the highest prevalence of tuberculosis in both humans and livestock. We assessed the contamination of cow's milk and buffalo's milk with MTC in Amazonas, focusing on Mycobacterium bovis, the species most commonly found in cattle and buffalo. In 2019, 250 samples of raw milk (91 from cattle, 159 from buffalo) were collected before processing from three milk plants in the state of Amazonas. The samples were placed into 21 pools and analyzed using shotgun metagenomic sequencing and taxonomic classification with Kraken 2 and MegaBLAST. To confirm the identity of mycobacterial species found, BLASTN was used to identify specific genomic positions in the TbD1 and RD1 regions and flanking RD4 region. MTC genetic material was identified in all pools of raw milk. Genetic material consistent with M. bovis was identified in seven pools of raw milk (1 from cattle, 6 from buffalo). Buffalo's milk had significantly higher MTC reads than did cow's milk. The common practice of consumption of raw milk and its derivatives in Amazonas presents a risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources also should be directed toward elimination of bovine tuberculosis in cattle and buffalo herds in Amazonas and the rest of Brazil.

Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: A call for action on wildlife in Brazil.

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Using a previously sequenced dataset of 71 M. bovis genomes from cattle, we found no recent transmission events between nearby farms and the park based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Genetic diversity of Mycobacterium bovis evaluated by spoligotyping and MIRU-VNTR in an intensive dairy cattle breeding area in Mexico.

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis. In Mexico, dairy cattle play an important role in the persistence and spread of the bacillus. In order to describe M. bovis genetic diversity, we genotyped a total of 132 strains isolated from slaughtered cattle with bTB suggestive lesions between 2009 and 2010 in Hidalgo, Mexico, using a panel of 9-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) and spoligotyping. We found 21 spoligotypes, and 124 isolates were grouped in 13 clusters. The most frequent spoligotypes were SB0121 (49, 37.1%) and SB0673 (27, 20.5%); three new spoligotypes were reported SB02703, SB02704 and SB02705. We observed 37 MIRU-VNTR patterns, 107 isolates were grouped in 12 clusters and 25 isolates were unique. Spoligotypes SB0121, SB0673, SB0140, SB0145 and SB0120 showed marked subdivision applying MIRU-VNTR method; meanwhile, spoligotypes SB0971 and SB0327 showed single MIRU-VNTR profiles. The Hunter-Gaston discriminatory index (HGDI) was 0.88, 0.78 and 0.90 for 9-loci MIRU-VNTR, spoligotyping and both methods, respectively. Additionally, allelic diversity (h) analysis showed high diversity for QUB3232, QUB26 and QUB11b with h = 0.79, 0.66 and 0.63, respectively. Overall, high genetic variability was observed among M. bovis isolates. Thus, the use of 9-loci MIRU-VNTR panel is enough to describe genetic diversity, evolution and distribution of M. bovis. This study supports the use of these tools for subsequent epidemiological studies in high incidence areas.

Frequência de lesões sugestivas de tuberculose em bovinos abatidos no estado do Espírito Santo e perdas econômicas aos produtores / Frequency of suggestive lesions of tuberculosis in bovine slaughtered in the state of Espírito Santo and economic losses for farmers

A bovinocultura de corte tem papel fundamental na economia do Brasil, que atualmente é um dos maiores produtores, consumidores e exportadores de carne bovina, e consequentemente se faz necessária a inspeção sanitária desses animais abatidos no país. A legislação vigente determina que quando as carcaças possuem indicativos de doenças, como a tuberculose bovina, podem ser condenadas ou aproveitadas condicionalmente. A tuberculose bovina é uma zoonose crônica causada pelo bacilo Mycobacterium bovis que carece de estudos para estabelecer a sua frequência em frigoríficos no país, bem como as consequentes perdas econômicas aos pecuaristas decorrentes da condenação de carcaças e vísceras. Assim, esse trabalho objetivou analisar a frequência de condenação de carcaças de bovinos do Estado do Espírito Santo, em um frigorífico que condena todos os casos de tuberculose, durante os anos de 2019 a 2021, e estimar as perdas econômicas aos produtores. Verificou-se que entre os 156.800 animais abatidos, 120 (0,076%) apresentaram lesões sugestivas de tuberculose, o que ocasionou uma perda econômica de R$ 461.160,00 (U$ 85.338,40). Conclui-se que a tuberculose é endêmica e responsável por importantes perdas econômicas para a cadeia produtiva da carne nesse estado. Também se destacou a importância da inspeção sanitária em abatedouros-frigoríficos de bovinos para reduzir os riscos à saúde pública.

Cattle farming has a fundamental role in the economy of Brazil, which is currently one of the largest producers, consumers, and exporters of beef, and consequently it is necessary to inspect meat from cattle slaughtered in the country. The current legislation establishes that when carcasses have signs of diseases, such as bovine tuberculosis, should be condemned or conditionally used. Bovine tuberculosis is a chronic zoonosis caused by Mycobacterium bovis that requires epidemiological studies to establish its frequency in abattoirs and the economic losses due condemnation of viscera and carcasses for beef farmers. Thus, this study aimed to analyze the frequency of condemnation of bovine carcasses slaughtered in the state of Espírito Santo, in an abattoir that condemned all cases of tuberculosis, during the years 2019 to 2021, and to estimate the economic losses to farmers. Among the 156,800 animals slaughtered, 120 (0.076%) had lesions suggestive of tuberculosis, which caused an economic loss of U$ 85,338.40 (R$ 461,160.00). Bovine tuberculosis is endemic and responsible for important economic losses to the meat production chain in this state. Additionally, the importance of inspecting beef in abattoirs to reduce the risks for public health was highlighted.

The Rv2807 target gene: a determining factor to directly detect Mycobacterium bovis from suspected bovine tuberculosis lesions / Gene alvo Rv2807: um fator determinante para detectar Mycobacterium bovis diretamente de lesões suspeitas de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis, a species belonging to the Mycobacterium tuberculosis complex (MTC) group. Direct bTB diagnosis from suggestive lesions can be performed by nested q-PCR targeting the Rv2807 gene present in the MTC group, as well as the TbD1 gene, present in M. bovis. In this context, the aim of the present study was to assess the importance of considering positive MTC results for the Rv2807 target gene obtained through the nested real time polymerase chain reaction (nested q-PCR) applied to samples obtained directly from suspected bTB lesions. A total of 174 samples of suggestive bTB caseous lesions were obtained during cattle slaughter in slaughterhouses in the state of Mato Grosso, Brazil. DNA was extracted from the lesions and nested q-PCR was performed to detect both MTC and M. bovis. Both samples positive for the Rv2807 (41/174) and TbD1 (29/174) were submitted to bacterial culturing (23/41), and the DNA of the isolates (23) was extracted and submitted again to nested q-PCR. The Rv2807 gene (MTC) was previously amplified by nested q-PCR directly from the lesions, although the TbD1 gene specific for M. bovis was not amplified previously in four of the successfully isolated samples (4/23), only following isolation, and only the Rv2807 gene was amplified before and after isolation. In conclusion, the target gene Rv2807(MTC) exhibited higher positivity in the analyzed samples compared to the TbD1 gene (M. bovis).

A tuberculose bovina (bTB) é uma zoonose causada pelo Mycobacterium bovis, uma espécie pertencente ao grupo do complexo Mycobacterium tuberculosis (MTC). O diagnóstico direto de bTB a partir de lesões sugestivas pode ser realizado por nested q-PCR visando o gene Rv2807 presente no grupo MTC, bem como o gene TbD1, presente em M. bovis. Nesse contexto, o objetivo do presente estudo foi avaliar a importância de considerar os resultados de MTC positivos para o gene alvo Rv2807 obtidos através da reação em cadeia da polimerase nested real time (nested q-PCR) aplicada a amostras obtidas diretamente de lesões suspeitas de bTB. Um total de 174 amostras de lesões caseosas sugestivas de bTB foram obtidas durante o abate de bovinos em frigoríficos do estado de Mato Grosso, Brasil. DNA foi extraído das lesões e nested q-PCR foi realizado para detectar tanto MTC quanto M. bovis. Ambas as amostras positivas para Rv2807 (41/174) e TbD1 (29/174) foram submetidas a cultura bacteriana (23/41), e o DNA dos isolados (23) foi extraído e submetido novamente à nested q-PCR. O gene Rv2807 (MTC) foi previamente amplificado por nested q-PCR diretamente das lesões, embora o gene TbD1 específico para M. bovis não tenha sido amplificado anteriormente em quatro das amostras isoladas com sucesso (4/23), apenas após o isolamento, e apenas o gene Rv2807 foi amplificado antes e após o isolamento. Em conclusão, o gene alvo Rv2807 (MTC) apresentou maior positividade nas amostras analisadas em relação ao gene TbD1 (M. bovis).