Genetically Related Mycobacterium bovis Strains Displayed Differential Intracellular Growth in Bovine Macrophages.

Molecular typing of bacterial isolates provides a powerful approach for distinguishing Mycobacterium bovis (M. bovis) genotypes. It is known that M. bovis strain virulence plays a role in prevalence and spread of the disease, suggesting that strain virulence and prevailing genotypes are associated. However, it is not well understood whether strain virulence correlates with particular genotypes. In this study, we assessed the in vitro intracellular growth of 18 M. bovis isolates in bovine macrophages as an indicator of bacterial virulence and sought a relationship with the genotype identified by spoligotyping. We found 14 different spoligotypes-11 were already known and three spoligotypes had never been reported before. We identified 2 clusters that were phylogenetically related, containing 10 and 6 strains, respectively, and 2 orphan strains. Intracellular growth and phagocytic rates of 18 M. bovis strains were heterogeneous. Our results suggest that M. bovis intracellular growth and phagocytosis are independent of the bacterial lineage identified by spoligotyping.

Population structure of Mycobacterium bovis isolates from cattle in Mexico.

The molecular fingerprints of 878 isolates of Mycobacterium bovis collected from cattle between 2009 and 2010 in different regions of Mexico were used in this study. One hundred and ninety-four spoligotypes were observed in total with a high degree of heterogeneity. Sixty-four percent of the isolates grouped into just nine spoligotypes, and 27% fell into only two spoligotypes: SB0673 and SB0669; 149 were orphan spoligotypes. The two predominant spoligotypes were found in almost all states in Mexico, especially in central Mexico, where there is a high concentration of dairy cattle; however, some spoligotypes were closely associated with restricted geographical areas. The hypothetical evolutionary relationship among spoligotypes was estimated using the spoligoforest program in the spolTools webpage. Four trees with connected components and nine unconnected nodes were found. The biggest tree had SB0140 strain as a root, suggesting this as the oldest strain in the tree. However, the relationship of this spoligotype with SB0673 and SB0669 was weak. The discriminatory power of spoligotyping for this M. bovis sample of isolates was 0.94, and the recent transmission index (RTI) 0.83, suggesting a high rate of recent transmission of some strains of M. bovis in the population. This parameter indicates that new measures are required to stop the dissemination of tuberculosis in cattle.

[Molecular epidemiology of cattle and human tuberculosis in Mexico]. / Epidemiología molecular de las tuberculosis bovina y humana en una zona endémica de Querétaro, México.

OBJECTIVE: The purpose of the study was to determine the role of bovine TB in cases of human TB. MATERIAL AND METHODS: Two-hundred and fifty-five samples from symptomatic patients were included in the study. All samples were cultured in Stonebrink and Lowënstein-Jensen media and analyzed using a nested PCRMPB70. The molecular analysis was performed by spoligotyping. RESULTS: From 255 samples, 74 were PCR-positive and 20 were culture-positive. From 94 samples positive to PCR or to isolation, 66 (70%) showed a spoligotype compatible with M. tuberculosis, and 13 (13.8%) with M. bovis. Four fingerprints of M. bovis from humans were identical to the fingerprints of M. bovis from cattle in the same region. CONCLUSIONS: Our study shows that M. bovis plays an important role in the epidemiology of TB in humans and that TB in cattle represents a risk to public health.

Epidemiología molecular de las tuberculosis bovina y humana en una zona endémica de Querétaro, México / Molecular epidemiology of cattle and human tuberculosis in Mexico

OBJETIVO: Determinar el papel de la tuberculosis bovina en la tuberculosis humana. MATERIAL Y MÉTODOS: Se analizaron 255 muestras de pacientes sintomáticos, sembradas en medios de Stonebrink y Lõwenstein-Jensen y analizadas por PCRMPB70 anidada y luego por spoligotyping. RESULTADOS: De las 255 muestras, 74 fueron positivas a la PCR y 20 al aislamiento: de las primeras, 58 (78 por ciento) mostraron espoligotipo de M. tuberculosis y 5 (6.7 por ciento) de M. bovis; de las segundas, 8 (47 por ciento) revelaron espoligotipo de M. tuberculosis y 8 (47 por ciento) de M. bovis. De las 94 muestras positivas al aislamiento o PCR, 66 (70 por ciento) correspondieron a M. tuberculosis y 13 (13.8 por ciento) a M. bovis. Los patrones moleculares de cuatro muestras de M. bovis de seres humanos fueron idénticos a los de las cepas de M. bovis de ganado. CONCLUSIONES: Se demuestra que M. bovis juega un papel importante en la epidemiología de la tuberculosis humana y representa un riesgo para la salud pública.

OBJECTIVE: The purpose of the study was to determine the role of bovine TB in cases of human TB. MATERIAL AND METHODS: Two-hundred and fifty-five samples from symptomatic patients were included in the study. All samples were cultured in Stonebrink and Lowënstein-Jensen media and analyzed using a nested PCRMPB70. The molecular analysis was performed by spoligotyping. RESULTS: From 255 samples, 74 were PCR-positive and 20 were culture-positive. From 94 samples positive to PCR or to isolation, 66 (70 percent) showed a spoligotype compatible with M. tuberculosis, and 13 (13.8 percent) with M. bovis. Four fingerprints of M. bovis from humans were identical to the fingerprints of M. bovis from cattle in the same region. CONCLUSIONS: Our study shows that M. bovis plays an important role in the epidemiology of TB in humans and that TB in cattle represents a risk to public health.

Molecular epidemiology of Mycobacterium bovis: usefulness in international trade.

Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.

Polymorphism of the PE domain of PE/PE_PGRS sequences in clinical isolates of Mycobacterium bovis in Mexico.

Polymorphism of the PE domain of PE/PE_PGRS sequences was studied in Mycobacterium bovis isolates from different Mexican states. Samples were analyzed by spolygotyping and RFLP using IS6110 and a 235-bp fragment of the PE domain of PE/PE_PGRS as probes. With the PE probe, three different genotypes were observed, one being predominant in all states. These results confirm the high conservation of the PE domain and suggests a potential role for PE sequence as a stable genetic marker for bovine tuberculosis.