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.

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.

Global Distribution and Evolution of Mycobacterium bovis Lineages.

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 genomes from 23 countries and at least 24 host species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3, and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis (South Siberian infected skeletons at ∼2,000 years before present) and indicates that extant M. bovis originated between 715 and 3,556 years BP, with later emergence in the New World and Oceania, likely influenced by trades among countries.

Global distribution and evolution of mycobacterium bovis lineages

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 genomes from 23 countries and at least 24 host species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3, and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis (South Siberian infected skeletons at ~2,000 years before present) and indicates that extant M. bovis originated between 715 and 3,556 years BP, with later emergence in the New World and Oceania, likely influenced by trades among countries.

Global distribution and evolution of mycobacterium bovis lineages

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 genomes from 23 countries and at least 24 host species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3, and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis (South Siberian infected skeletons at ~2,000 years before present) and indicates that extant M. bovis originated between 715 and 3,556 years BP, with later emergence in the New World and Oceania, likely influenced by trades among countries.

Genome sequencing of Mycobacterium pinnipedii strains: genetic characterization and evidence of superinfection in a South American sea lion (Otaria flavescens).

BACKGROUND: Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis Complex (MTBC), is capable of infecting several host species, including humans. Recently, ancient DNA from this organism was recovered from pre-Columbian mummies of Peru, sparking debate over the origin and frequency of tuberculosis in the Americas prior to European colonization. RESULTS: We present the first comparative genomic study of this bacterial species, starting from the genome sequencing of two M. pinnipedii isolates (MP1 and MP2) obtained from different organs of a stranded South American sea lion. Our results indicate that MP1 and MP2 differ by 113 SNPs (single nucleotide polymorphisms) and 46 indels, constituting the first report of a mixed-strain infection in a sea lion. SNP annotation analyses indicate that genes of the VapBC family, a toxin-antitoxin system, and genes related to cell wall remodeling are under evolutionary pressure for protein sequence change in these strains. OrthoMCL analysis with seven modern isolates of M. pinnipedii shows that these strains have highly similar proteomes. Gene variations were only marginally associated with hypothetical proteins and PE/PPE (proline-glutamate and proline-proline-glutamate, respectively) gene families. We also detected large deletions in ancient and modern M. pinnipedii strains, including a few occurring only in modern strains, indicating a process of genome reduction occurring over the past one thousand years. Our phylogenomic analyses suggest the existence of two modern clusters of M. pinnipedii associated with geographic location, and possibly host species, and one basal node associated with the ancient M. pinnipedii strains. Previously described MiD3 and MiD4 deletions may have occurred independently, twice, over the evolutionary course of the MTBC. CONCLUSION: The presence of superinfection (i.e. mixed-strain infection) in this sea lion suggests that M. pinnipedii is highly endemic in this population. Mycobacterium pinnipedii proteomes of the studied isolates showed a high degree of conservation, despite being under genomic decay when compared to M. tuberculosis. This finding indicates that further genomes need to be sequenced and analyzed to increase the chances of finding variably present genes among strains or that M. pinnipedii genome remodeling occurred prior to bacterial speciation.

Micobactérias diagnosticadas em gatos domésticos no sertão da Paraíba / Mycobacteriae diagnosed in domestic cats in the backland of Paraiba, Brazil

Descrevem-se dois casos de infecção por Mycobacterium sp. em gatos domésticos no sertão da Paraíba, Nordeste do Brasil. Os animais apresentavam emagrecimento progressivo, dispneia, tosse e linfadenomegalia generalizada em um dos casos, com evolução clínica de um e sete meses, respectivamente. Macroscopicamente as lesões estavam restritas aos linfonodos superficiais e/ou profundos e parênquima pulmonar, caracterizadas por nódulos multifocais ou multifocais a coalescentes, amarelados, irregulares que ao corte apresentavam áreas multifocais a coalescentes amareladas, friáveis e caseosa, além de áreas multifocais brancacentas e firmes. Histologicamente, verificou-se linfadenite e pneumonia granulomatosa caracterizada por áreas centrais de necrose e mineralização central circundadas por macrófagos, células epiteliais e tecido conjuntivo fibroso. No primeiro caso havia ainda hepatite granulomatosa discreta e aleatória. Em ambos os casos, foram visualizados bacilos álcool-ácido resistentes livres e no citoplasma de macrófagos na coloração de Ziehl-Neelsen. Houve imunomarcação para Mycobacterium sp. no citoplasma de macrófagos em ambos os casos. Na PCR, identificou-se apenas o gênero Mycobacterium no Caso 1 e Mycobacterium bovis no Caso 2. O diagnóstico de infecção por Mycobacterium sp. nos felinos foi realizado com base nos achados anatomopatológicos e imuno-histoquímica. Pela PCR foi realizado o diagnóstico etiológico de micobacteriose no Caso 1 e tuberculose por M. bovis no Caso 2. O diagnóstico das doenças causadas por micobactérias em felinos, principalmente aquelas em que M. bovis esteja envolvido, é de extrema importância para a saúde pública, destacando o potencial zoonótico desta espécie, pois muitos animais infectados podem ser assintomáticos ou apresentarem sinais clínicos inespecíficos.(AU)

This report describes two cases of domestic cats infected with Mycobacterium sp. in the backland of Paraiba, Northeast Brazil. The animals manifested nonspecific clinical signs, characterized by progressive weight loss, dyspnea, cough and generalized lymphadenomegaly in one cases, and clinical evolution of one and seven months respectively. Macroscopically the lesions were restricted to superficial and/or deep lymph nodes and pulmonary parenchyma, characterized by multifocal or multifocal to coalescent, yellowish and irregular nodules that presented multifocal areas with yellowish, friable and caseous as well as multifocal to coalescent, white and firm areas. Histologically, it was observed lymphadenitis and granulomatous pneumonia in both cases, varying in intensity of inflammatory response and degree of mineralization. The granulomas were constituted by macrophages and epithelioid cells, some with marked necrosis and central mineralization, surrounded by a large amount of connective tissue. In the first case there were also discrete and random granulomatous hepatitis. In both cases alcohol-acid-resistant bacilli were visualized inside the cytoplasm of macrophages in Ziehl-Neelsen staining; there was immunostaining for Mycobacterium sp. in the cytoplasm of macrophages. Through PCR was identified only the genus Mycobacterium in Case 1, and Mycobacterium bovis in Case 2. The diagnosis of Mycobacterium sp. In the cats was performed based on anatomopathological findings and immunohistochemistry. The PCR was performed the etiological diagnosis of mycobacteriosis in Case 1 and tuberculosis by M. bovis in Case 2. The diagnosis of diseases caused by mycobacteria in cats, especially those in which M. bovis was involved, is extremely important for public health highlighting the zoonotic potential of this species, since many infected animals may be asymptomatic or show nonspecific clinical signs.(AU)

Complete Genome Sequencing of Mycobacterium bovis SP38 and Comparative Genomics of Mycobacterium bovis and M. tuberculosis Strains.

Mycobacterium bovis causes bovine tuberculosis and is the main organism responsible for zoonotic tuberculosis in humans. We performed the sequencing, assembly and annotation of a Brazilian strain of M. bovis named SP38, and performed comparative genomics of M. bovis genomes deposited in GenBank. M. bovis SP38 has a traditional tuberculous mycobacterium genome of 4,347,648 bp, with 65.5% GC, and 4,216 genes. The majority of CDSs (2,805, 69.3%) have predictive function, while 1,206 (30.07%) are hypothetical. For comparative analysis, 31 M. bovis, 32 M. bovis BCG, and 23 Mycobacterium tuberculosis genomes available in GenBank were selected. M. bovis RDs (regions of difference) and Clonal Complexes (CC) were identified in silico. Genome dynamics of bacterial groups were analyzed by gene orthology and polymorphic sites identification. M. bovis polymorphic sites were used to construct a phylogenetic tree. Our RD analyses resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. M. bovis SP38 along with strain 35 represent the first report of CC European 2 in Brazil, whereas two other M. bovis strains failed to be classified within current CC. Results of M. bovis orthologous genes analysis suggest a process of genome remodeling through genomic decay and gene duplication. Quantification, pairwise comparisons and distribution analyses of polymorphic sites demonstrate greater genetic variability of M. tuberculosis when compared to M. bovis and M. bovis BCG (p ≤ 0.05), indicating that currently defined M. tuberculosis lineages are more genetically diverse than M. bovis CC and animal-adapted MTC (M. tuberculosis Complex) species. As expected, polymorphic sites annotation shows that M. bovis BCG are subjected to different evolutionary pressures when compared to virulent mycobacteria. Lastly, M. bovis phylogeny indicates that polymorphic sites may be used as markers of M. bovis lineages in association with CC. Our findings highlight the need to better understand host-pathogen co-evolution in genetically homogeneous and/or diverse host populations, considering the fact that M. bovis has a broader host range when compared to M. tuberculosis. Also, the identification of M. bovis genomes not classified within CC indicates that the diversity of M. bovis lineages may be larger than previously thought or that current classification should be reviewed.

Mycobacterium pinnipedii in a stranded South American sea lion (Otaria byronia) in Brazil.

We report tuberculosis in a stranded South American sea lion (Otaria byronia) in Brazil caused by Mycobacterium pinnipedii, a member of Mycobacterium tuberculosis complex.

Mycobacterium kansasii isolation from captive South American coati (Nasua nasua).

Three of six captive South American coatis (Nasua nasua) presented with respiratory distress and died despite treatment. Postmortem examination performed on two of these animals revealed granulomatous pleuropneumonia associated with acid-fast bacilli. Because of the possible diagnosis of mycobacterial infection, the three remaining asymptomatic coatis were anesthetized. Tracheal washes were sampled and submitted for microbiology, and the animals were euthanatized and postmortem examinations performed. One of these asymptomatic adult males had whitish granulomas in multiple organs and tissues. Additionally, the isolate from this male's tracheal wash was identified as Mycobacterium kansasii by molecular analysis. To the authors' knowledge, this is the first report of M. kansasii infection in Nasua nasua.