Epidemiological Investigation Of Bovine Paratuberculosis And Research On Detection Method For ELISA Antibody In Parts Of Xinjiang Area

Bovine paratuberculosis is a chronic hyperplastic enteritis that caused by Mycobacterium tuberculosis subsp.Paratuberculosis(MAP).The main manifestation of the disease is the presence of serous secretions in the intestine,accompanied by long-term diarrhea and poor physical condition.It endangered the lives of the infected cattle occasionally,which causing huge economic losses to the breeding industry.The disease is defined as a compulsory notification of infectious diseases abroad and designated as a Class II animal epidemic.The disease,which often occurred in cattle farms in Xinjiang,encountered the difficulty of clinically differentiation with other diseases and diagnosis.This study has provided technical support for the diagnosis and control of the disease by carrying out antibody detection on serum of cattle farms in different areas of Xinjiang,using molecular biological diagnosis on clinical suspected cases,and exploring the establishment of diagnostic method for enzyme-linked immunosorbent assay(ELISA).Purpose:to investigate the positive results of serum antibody of bovine paratuberculosis in different areas of Xinjiang,by using molecular biology methods to diagnose suspected cases of bovine paratuberculosis,so as to understand clinical epidemic and incidence of the disease;to screen the important antigen protein of MAP and establish an indirect ELISA method for detection of antibody to the disease,so as to provide reference for the establishment of the diagnosis method of bovine paratuberculosis.Methods: 1.Collect 681 blood samples from cattle farms in 13 regions including Shihezi,Shawan,Kuitun,Xinjiang for antibody detection;2.Perform PCR test on 64 clinically suspected cow fecal samples;3.Analyze the bovine paratuberculosis samples Epitope of important antigen protein of Mycobacterium to construct tandem recombinant protein MAP3290-1595 expression vector,transformed into E.coli and induced expression by IPTG;4.Verify the reactivity of recombinant protein MAP3290-1595 by Western Blot and indirect ELISA;The optimal conditions of indirect ELISA method based on recombinant protein were screened.Result:1.The average positive rate of serum antibody of bovine paratuberculosis in different areas of Xinjiang was 6.17%(42/681)and the differences existed in different areas;2.Positive rate of PCR for clinical suspected cases was 81.25%(52/64).The positive gene fragments were identified by sequencing as mycobacterium avium subsp.Paratuberculosis,and the 7 sequences were 100% homologous;3.The expression and purification of tandem recombinant protein MAP3290-1595 was successfully induced with a concentration of 0.430 mg / mL and a purity greater than 90%;4.It is confirmed by western blot and indirect ELISA methods that the recombinant protein MAP3290-1595 has good reactogenicity with the positive serum of bovine paratuberculosis.5.Preliminary screening of the optimal conditions for the indirect ELISA method established by recombinant protein.Conclusion:1.Bovine paratuberculosis infection was presented in cattle farms in different regions of Xinjiang,and the proportion of mycobacterium paratuberculosis in cattle with clinical diarrhea was high;2.The recombinant protein map3290-1595 of M.paratuberculosis was successfully expressed,and an indirect ELISA method for the detection of map antibody was established,which provided basic data for the diagnosis and control of the disease in Xinjiang.