Pathogenic Investigation And Molecular Epidemiological Of Bovine Tuberculosis

Background: With the rapid expansion of China's milk industry, bovine tuberculosis (TB) poses a serious threat to the development of a flourishing dairy industry and its prevention and control have received increasing attention. However, the prevalence of bovine TB in China remains unclear, and this is one of the biggest obstacles to its prevention and control. Molecular epidemiology techniques have been used for genotyping the domestic Mycobacterium tuberculosis complex (MTBC) isolates. These include spacer oligonucleotide typing (spoligotyping) and variable number tandem repeat mycobacterial interspersed repetitive unit (VNTR-MIRU) methods. These approaches have great significance for understanding the genotypes and distribution of the MTBC complex, as well as the feasibility of the application of the two methods in China.Methods and Main Results: We studied 1067 bovine PPD (protein purified derivative, PPD)-positive throat swabs and their corresponding serum samples collected from different regions of China. The collected PPD-positive samples were inoculated on the modified L(o|¨)wenstein-Jensen medium for the isolation and culture of Mycobacterium. Acid-fast bacilli were identified using traditional biochemical methods, polymerase chain reaction (PCR) amplification and multiplux-PCR. They were distinguished as MTBC and non-MTB strains. An indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect specific antibodies of bovine TB, and to analyze and compare correlations between the antibody positive rate and the rate of isolation of MTBC. Spoligotyping and VNTR-MIRU were used for genotyping MTBC strains to determine the national prevalence of major epidemic strains and genotypes. One hundred eleven strains of acid-fast positive bacilli were cultured from the 1067 throat swabs, including 43 stains of MTBC (14 strains of M. bovis and 29 of M. tuberculosis) and 68 strains of non-MTB bacteria. The coincidence rate of the positive rate of cultured MTBC with the positive rate of indirect ELISA antibody was 60% (43/72), much higher than the positive rate of PPD allergy (3.9%; 43/1067; P < 0.01). Genotyping of the 43 strains of MTBC isolates using spoligotyping and VNTR-MIRU showed that the 43 isolates had 26 genotypes, with 16 strains having a unique genotype. The major prevalent genotype of M. bovis was the BCG family. The 'Beijing family' genotype strains were the major epidemic strains of MTBC infecting cattle. Five strains had the same spoligotyping pattern as the Beijing family, showing hybridization with only nine spacers between positions 35～43. Only two strains hybridized with eight spacers between positions 35～43. In other words, the Beijing genotype showed only one difference between regions. Combined application of spoligotyping and VNTR-MIRU typing would effectively improve the molecular epidemiological investigation and monitoring of the etiology of bovine TB in China.Conclusion: This was a preliminary investigation of the situation of bovine TB infection across China. The MIRU-VNTR technique facilitates universal access and can play an important role in the epidemiological research of bovine TB.