Epidemiological Investigation Of Mycobacterium Avium In Cattle And Development Of Diagnostic Antigen For Bovine Tuberculosis

Bovine Tuberculosis(BT)is a zoonotic disease mainly caused by Mycobacterium bovis(M.bovis)infection.With the rapid development of the cattle industry in my country,the problem of bovine tuberculosis has become more and more serious,which not only brings losses to the cattle industry,but also endangers human life and health.Clinical quarantine methods for bovine tuberculosis mainly include interferon gamma test(IFN-γ),tuberculin intradermal allergy(TST)and antibody test.Because antibody detection is mainly suitable for advanced tuberculosis cattle,clinical application is less.The antigen used for IFN-γ detection and TST is mainly the protein derivative of Mycobacterium bovis,which cross-reacts with Mycobacterium avium,resulting in false positive,so the control test of Mycobacterium avium needs to be added.Expression and purification of the antigen protein of Mycobacterium bovis,and fusion or mixing them to establish a specific diagnostic antigen for bovine tuberculosis with good sensitivity and high specificity.Omit the control experiment of avian mycobacteria in the diagnosis process,reduce the quarantine steps and costs,and promote the quarantine and purification of bovine tuberculosis.In this experiment,the epidemiological investigation of mycobacterium avian in our country was carried out by etiology and serological methods.To establish a fluorescence quantitative detection method for Mycobacterium avian tuberculosis,Mycobacterium para-tuberculosis and Mycobacterium human porcine,and to detect the etiology of cattle feces,nasal swabs and milk samples from cattle farms in China.The bovine serum collected from2018 to 2019 was tested for IFN-γ and paratuberculosis antibody for avian tuberculosis.Esat-6,CFP-10,RV3615 and RV3020 gene fragments were amplified by PCR from Mycobacterium bovis AF2122/97 genome.Recombinant plasmids pET-28a-ESAT-6,pET-28a-CFP-10,pET-28a-RV3615 and pET-28a-RV3020 were constructed.The fusion genes ECR(ESAT-6,CFP-10,RV3615)and ECR30(ESAT-6,CFP-10,RV3615,RV3020)were designed and sent to the company for plasmid construction.The positive recombinant plasmid was extracted,transformed into competent cells and induced expression.SDS-PAGE analysis showed that the recombinant proteins ESAT-6,RV3615,ECR and ECR30 existed in the form of inclusion bodies,and the recombinant proteins CFP-10 and RV3020 existed in the soluble form.The 6 recombinant proteins had good antigenic properties,and the 6 target proteins were successfully eluted by Ni-NTA chromatography column.The sensitivity of cocktails protein antigen 1(ESAT-6,CFP-10,RV3615)and fusion protein ECR were 90.1% and 85.9%,respectively,and the specificity was 100%.The sensitivity and specificity of cocktail protein antigen 2(ESAT-6,CFP-10,RV3615,RV3020)and fusion protein ECR30 were 80% and 70%,respectively.In the selection of working concentration,the optimal working concentration of 4 proteins was 80 μg.A total of 120 cattle were randomly selected from bovine TB positive field for IFN-γ test.35 cattle were positive for IFN-γ test and 33 cattle were positive for cocktail antigen 1.In IFN-γ testing,it was found that 8 cattle were not determined by clinically standard testing methods,all of which were positive.To sum up,the infection of Mycobacterium avian is widespread in cattle farms in our country,which seriously interferes with the quarantine of bovine tuberculosis.A control test of Mycobacterium avian must be added to the quarantine of bovine tuberculosis.The specificity of the protein antigen prepared in this study was 100% in IFN-γ and s ICTT detection.The sensitivity was lower than that of standard test results.The sensitivity of SICTT in IFN-γ test was higher than that of SICTT.The sensitivity of cocktail antigen in IFN-γ test was 90%,and the sensitivity of fusion protein was lower than that of cocktail antigen.There is a high proportion of co-infections between Mycobacterium bovis and Mycobacterium avian in cattle,and standard detection methods cannot be distinguished.The protein antigen developed in this research can eliminate interference for accurate diagnosis.