We performed the first epidemiological study of the Mycobacterium bovis (M. bovis) in Amazonas state, Brazil. The goal of this project was, through a One Health Approach, to clarify the burden of M. bovis in Amazonas State. To achieve the goal our study addressed four fundamental questions regarding the epidemiology of bovine Tuberculosis (bTB): 1) What is the M. bovis prevalence and profile in cattle and buffalo in Amazonas State?; 2) What are the major risk factors for animal-to-animal bTB transmission in the area?; 3) How can the detection of animals (cattle and buffalo) and herds infect by M. bovis be improved?; and 4) Does consumption of raw milk from cattle and buffalo represents a risk for zoonotic Tuberculosis (zTB) to human population in Amazonas state? The study used a two-phases cross-sectional design. During the first phase, from July 2016 to February 2018, a total of 214 animals (91 buffalo and 123 cattle) that were considered bTB suspects had tissues collected for laboratory confirmation using culture, polymerase chain reaction (PCR), spoligotyping, and whole genome sequencing (WGS). During the second phase, from February 2019 to August 2019, a total of 250 samples of raw milk were collected, 91 milk samples from cattle and 159 from buffalo, for analysis in pools using shotgun metagenomic sequencing (SMS). Our results demonstrated that Amazonas state presented the highest bTB prevalence ever reported, both in cattle (3%) and buffalo (11.8%), in Brazil. Additionally, M. bovis presents a distinct genetic profile in the state when compared with the rest of the country with surprising identification of ancient strains (Lb1) never described in South America. Species (Bubalus bubalis), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by M. bovis in the state. Sole adoption of Tuberculin Skin Test (TST) protocols failed to detect aged animals and herds infected by the M. bovis, but the antibody assay was able to detect infected animals and herds missed by the Comparative Cervical Tuberculin test (CCT). Mycobacterium tuberculosis complex (MTC) species genetic material were identified in all pools of raw milk. Genetic material consistent with M. bovis were identified in seven pools of raw milk (1 cattle and 6 buffalo). Buffalo presented significantly higher M bovis infection prevalence than cattle and significantly a higher contamination (rate) in raw milk compared with raw milk from cattle. The findings support the original hypothesis that M. bovis represents a potential risk to public health in the area. Combination of epidemiological, clinical, and laboratorial data are measures recommended to strengthen bTB programs in Amazonas and Brazil. The popular practice of consumption of raw milk and its derivatives in Amazonas state poses a high risk of transmitting M. bovis to humans. Therefore, education measures to increase awareness of zTB in key public and private stakeholders are necessary to nurture the engagement and collaboration needed to effectively address the challenge to end TB. Finally, we recommended further molecular studies to clarify the origins of M. bovis in the region, its possible spread to the local wildlife, and active surveillance for M. bovis in TB patients to assess the impact on human health in the region.
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