Pathogenic Investigation Of Bovine Tuberculosis And Identification Of In Vivo Induced Antigen Genes Of M. Tuberculosis

Bovine tuberculosis (bTB) which is mainly caused by Mycobacterium bovis is a chronic wasting zoonosis. Dairy cows are the most susceptible hosts. In the past years, Chines dairy sector is experiencing rapid expansion. Meanwhile, the prevalence of bovine tuberculosis is correspondingly increasing. This situation is not only threatening the sustainable development of dairy industry, but also the food safety and human health. Thus this study was aimed to isolate tuberculous mycobacteria from both cow and humans infected by mycobacteria and investigate the epidemiological link between cow and human tuberculosis. The evidence would be helpful to make a combined control program to stop TB in animals and humans.In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in animal infections. The general principle is that the sera from naturally infected animals and absorbed with bacteria grown in vitro were hybridized with blotted colonies containing the gene libraries of the pathogen. The positive colonies contain the genes which could be specially expressed in vivo and might be potential targets to novel vaccines, drugs and diagnostic methods.By using conventional methods, a total of 806 nasal swabs, 312 tissue samples of cows were cultured and 15 isolates were obtained. By biochemical test, multiplex PCR, spoligotyping and MIRU, 13 isolates of M. tuberculosis, 1 isolates of M. bovis were identified. Two positive cows were slaughtered. The gross and histopathogenical examination was performed and confirmed the tuberculosis. The bacterial culture with lesioned lung tissue obtained one M. tuberculosis and one M. bovis as expected. Meanwhile, 84 isolates from human tuberculosis patients from Tuberculosis Hospital and Tuberculosis Prevention and Treatment Institute were genotyped and all of the isolated belonged to M. tuberculosis. The retrospective investigation of the patient files for the past three years in the hospital revealed a prevalence of 0.34 % (17/5011) M. bovis infection.For screening in vivo induced antigen genes, the sera from cows infected by tuberculous bacteria were collected. The sera were classified into high, medium and low grade according to the titers of antibodies to tuberculous mycobacteria. Each of 4 sera with the same levels of antibody was pooled. M. tuberculosis was cultured and harvested. The serum pool was adsorbed by the bacterial body, culture supernatant, and bacterial lysis of both M. tuberculosis and E. coli. And ELISA was used to evaluate the adsorption effect. The M. tuberculosis library was transformed E. coli BL21(DE3) and blotted on velvet cloth. The adsorbed serum pool was used to hybridize the colonies on the velvet cloth and the positive colonies were developed with HRP coupled goat anti-rabbit IgG and DAB substrate. A total of 8000 colonies were screened and 42 suspected positive colonies were sequenced. However, no positive colony was confirmed. The reason remained to be investigated.