| Bovine tuberculosis is a chronic consumptive zoonosis casued by Mycoabcterium tuberculosis complex. The main host of this disease is the cattle, but wild animals and humans can also be infected through sick animals. Bovine tuberculosis has brought huge economic losses to the development of animal husbandry in our country. Its popular not only seriously affects the sustainable development of the livestock industry, but also on human health and TB control. With the development of animal husbandry, the bovine tuberculosis increased in recent years. Bovine tuberculosis has become increasingly serious threat to the development of the dairy industry, and bovine tuberculosis prevention and control are also getting more and more attention. However, the prevalence of bovine tuberculosis in China is not very clear, and it is one of the biggest obstacles in the control of bovine tuberculosis. In addition, the sensitivity and specificity of the clinical detection methods for the bovine tuberculosis, such as skin test, ELISA,γinterferon detection, remain to be further enhanced. Therefor we need an accurate, fast and standard tuberculosis detection method to provide effective results for the control and eradication of bovine tuberculosis. This study was aimed to the bovine tuberculosis molecular epidemiology and novel diagnosis target of Mycobacterium tuberculosis. The main results were summarized as follows:1. The difference genomic regions among the Mycobacterium tuberculosis H37Rv, Mycobacterium bovis and BCG were obtained from NCBI’s Nucleotide database,129 ORFs totally. We designed the primers for the full gene sequence, then amplified genes and clonned to the vector pET28a. By sequencing, we finally constructed 66 recombinant plasmid successfully. it lay a foundation for the further identifing the function and screening diagnosis target.2. E.coli bacteria and supernatant were used to adsorb the non-specific antibody in the the positive sera preserved by our lab. After transforming the correct recombinant plasmid into E.coli, we artificial pointed the correct E.coli in the solid medium. When it growth well, the colony transfer on the NC membrance. Then the membrance hybidized with the adsorbed positive serum, and signal was capture by chemiluminescence method. After preliminary screening, we found 11 recombinant E.coli bacterials can react with positive serology.8 of them deleted in the genome of M.bovis compared to the M.tuberculosis, they were Rv1585C、Rv0222、Rv3120、Rv2657C、Rv3620C、Rv1255C、Rv3428 and Rv2647; 3 of them deleted in the geome of BCG compraed to the M.bovis and M.tuberculosis. 3. After induction and expression,11 positive recombinant bacteria were soluble idengtified.The results showed that 8 proteins in soluble expression, another three for the inclusion bodies.Each protein was purified by Ni ions affinity chromatography. Finally, we obtained 7 purified soluble recombinant proteins, they were Rv0222、Rv1255C、Rv2647、Rv2657C、Rv0310、Rv1772、Rv3404C.4. The serum immune response between recombinant proteins and positive and negative TB serum preserved in the laboratory was further validated by Western blot and ELISA. All purified proteins had been reacted to the TB serum.But the results failed to find significantly difference between positive and negative serum.5.147 copies of TST positive samples were collected totally,59 samples were bovine lung and lymphoid tissue from Suzhou,57 samples were nasal swabs from Huanggang,31 samples were milk from Huanggang, too. When examing the bovine lung and lymphoid, we could not observe obvious lesions. However,20 strains were isolated from the lung and lymphoid tissue, and isolation rate was 33%. This showed that not all skin test positive cattle had clinical symptoms and high volume of tissue carrier. For the nasal swabs and milk samples from Huanggang, we could not isolate strain from them. It illustrated the nasal swabs and milk had a low bacterial content compared with tissue sample.6. A random sample of 568 slaughterhouses were obtained, of which 281 were from Xinjiang,189 from Shanxi,98 samples were yak. The sample tissues were all lung and lymph tissues, from which we have isolated bacteria and cultured them. The results showed that 6 strains were isolated from 281 tissues of Xinjiang; 3 strains from 189 tissues of ShanXi, the separation rates were 2.1% and 1.6% respectively. None strain was isolated from the 98 yak tissues.7. The thermal lysis product of 17 isolated positive culture were genotyped by Spoligotyping method. Through the genotyping of Mycobacterium, the prevalence of M. bovis was characterized with regional. |
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