Development Of A Diagnosis Method Of PPD_B-P(E/C)-IFN-γ And Isolation And Identification Of Mycobacterium Bovis

Bovine tuberculosis is one of chronic consumption of infectious zoonotic diseases mainlycaused by Mycobacterium bovis(M. bovis). As one of the three major infectious diseasesthreatening human health, it is an urgent public health problem and social problem in the world,it causes economic losses in livestock farming and poses a serious threat to public health andfood safety. So it is necessary to develop a new diagnostic method with high sensitivity andspecificity for bovine tuberculosis, and provide technological support for the control anderadiczation of tuberculosis.In present study, first, prevalence of bovine tuberculosis in Xinjiang Uygur AutonomousRegion was investigated. And last56strains of M. bovis from Xinjiang was isolated andidentified using two level-multiplex PCR. Second, a new diagnosis method of PPDB-PE/C-IFN-γwas developed, in which ESAT-6(PE) and CFP-10(PC) proteins were as stimulation antigens.(1)551whole blood samples were collected in Xinjiang Uygur Autonomous Regiondetected with IFN-γ test. The results showed that,65of the551samples are M. bovis positivegiving a positive rateas11.8%and166samples are M. avium positive giving a positive rate as30.1%.(2) From56positive cows in Xinjiang diagnosed by SITT, SICTT or IFN-γ, lungs or lymphnodes tissues were collected. Then56Mycobacterium strains were isolated by cultivation on theSodium pyruvate solid mediums. Finally these strains are all confirmed as M. bovis by theMTC’s two level-multiple PCR.(3) Development of a diagnosis method of PPDB-PE/C-IFN-γ for bovine tuberculosis: PEandPCwere prokaryotically expressed and purified. Bovine whole blood cells from56positives and460negatives were respectively stimulated with PPDB(as control), PE, PCand PE/C(PEand PCequally mixed) in vitro, then IFN-γ levels being induced and released to plasma were determinedby indirective ELISA. Results were statistically analyzed and demonstrated by the ROC curves,by which the optimal cut-offs for determination of positives, sensitivities and specificities ofdiagnosis corresponding to PPDB-IFN-γ, PE-IFN-γ, PC-IFN-γ and PE/C-IFN-γ were respectivelydetermined. And additionally, sensitivities and specificities of diagnosis for PPDB-PE-IFN-γ,PPDB-PC-IFN-γ, PPDB-PE/C-IFN-γ were also analyzed when PPDBwas respectively used in combination with PE, PCand PE/C(in series and parallel). The molecular weight of PEand PCexpressed in present study were27.8ku and16.4ku respectively. The optimal cut-off ofPPDB-IFN-γ was0.02, where the sensitivity and specificity were94.6%and92.2%; PE, PC, PE/Cshowed the same optimal cut-off as0.01. Of which, PE/C-IFN-γ gave the best sensitivity andspecificity as92.9%and95.7%. Of the combined applications, PPDB-PE/C-IFN-γ demonstratedthe highest sensitivity and specificity corresponding89.3%and96.5%when used serially as wellas98.2%and91.3%parallelly. The sensitivity of PE/C-IFN-γ is lower than PPDB-IFN-γ by1.7%,however the specificity is higher by3.5%. For PPDB-PE/C-IFN-γ, the sensitivity is higher thanPPDB-IFN-γ by3.6%when used in parallel, and the specificity higher by4.3%when in series.Innovation point: The diagnosis method of PPDB-PE/C-IFN-γ is characterized by both highsensitivity and specificity, and can exclude the interference from nontuberculosis Mycobacteria.