| The diagnostic methods of Bovine tuberculosis include pathogen identification, tuberculin skin test,IFN-γ release assay and serological test. Mycobacteria isolation and identification is the gold standarddiagnostic tool for bovine TB, but the sensitivity of it is not high enough. The tuberculin skin test andIFN-γ release assay are based on the detection of T cell mediate immune responses in TB-infected cattlethat induced by Mycobacterium antigens including PPD-B and PPD-A. The tuberculin skin test andIFN-γ release assay showed high sensitivity and were recommended by OIE. However, the PPD-B andPPD-A is a poorly defined mixture of proteins, lipids and carbohydrates obtained from virulent M. bovisculture, of which is difficult to remain uniform from batch to batch. Moreover, use of virulent M. bovisduring the production of PPD-B may potentially harm workers. Importantly, shared antigeniccomponents in PPD-B and non-pathogenic environmental mycobacterium can reduce the specificity oftuberculin skin test and IFN-γ release assay. To overcome the drawbacks of the traditional diagnosticmethods, and establish new more effective diagnostic method, we focus on screening new M. bovisspecific antigens to substitute PPD as stimuli in skin test and IFN-γ release assay.Twelve recombinant proteins including CFP10, ESAT6, CE, TB10.4, TB27.4, Rv3872, MX,MPT63, MPT64, MPB70, MPB83and Ag85B were expressed and purified. All of these proteins wereexchanged into PBS buffer, and removed LPS using Triton X-114. The purity of each protein is higherthan90%, and the concentration of LPS of each protein is lower than2EU/mg. Each recombinantprotein showed good T cell and B cell activities, and could be potential used in the diagnosis of bovineTB. Two kinds of combinations (CFP10/ESAT6/TB10.4protein cocktail andCFP10/ESAT6/Rv3872/MPT63protein cocktail) were developed and used as stimuli in the skin test.Cattle were double-blind tested to assess the efficiency of protein cocktail-based skin tests. The resultsshowed that the CFP10/ESAT6/TB10.4protein cocktail-based skin test can differentiate theTB-infectedcattle from M. avium-infected ones, and shows a high degree of agreement with the traditionaldiagnostic method (κ>0.75). When compared to traditional diagnostic methods, the relative sensitivityand relative specificity of the protein cocktail-based skin tests were higher than87%and97%,respectively. The protein cocktail-based skin tests correlate to traditional measures of bovine TBevaluation, including skin test and interferon gamma release assay. The CE-based IGRA was optimizedand evaluated in field trial. The correlation between the OD450nmvalues obtained after the bloodstimulation with CE and PPD-B was high (spearman r=0.9229). The correlation between the OD450nmvalues obtained after the blood stimulation with CE and the increase in skin thickness observed afterinoculation of. PPD-B was high (spearman r=0.6893). The sensitivity and specificity of the CE-basedIGRA were69-77%and98%, respectively. The recombinant protein CE showed good activities andcould be used as stimuli in IGRA.To assess the efficacy of different diagnostic methods used in the different stages of TB disease,twelve cattle were devided into four groups (3cattle per group), and infected with M. bovis, M. avium, M. bovis BCG and inoculated with PBS, respectively. All cattle were detected using tuberculin skin test,IFN-γ release assay and serological diagnostic methods at regulaer intervals. The results showed thePPD-B-based tuberculin skin test and IFN-γ release assay could detect the M. bovis-infection at96h,and could last to58w, but could‘t differentiate the M. bovis-infected animal from BCG vaccinated ones,further more the M. avium-infected animal could react with PPD-B. The M. bovis-infected cattle couldelicit IgG antibodies to MPB70and MPB83at2-58w, and different animal showed different levelshuamoral responses. The protein cocktail CFP10/ESAT6/TB10.4-andCFP10/ESAT6/MPT63/Rv3872-based skin test and the CE-based IFN-γ release assay coulddifferentiate the M. bovis-infected cattle for M. avium-infected or BCG-vaccinated ones, it means the M.bovis specific antigen could be used in the diagnose of bovine TB. |
