Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovisand by M. aviumsubsp. aviumand M. aviumsubsp. paratuberculosis

ABSTRACTImmunological diagnosis of Mycobacterium bovisinfection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-?)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. aviumexposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. aviumor experimentally challenged with Mycobacterium aviumsubsp. aviumor Mycobacterium aviumsubsp. paratuberculosiswere compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-a), IFN-?, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P< 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. aviumsubsp. paratuberculosisinduced significant (P< 0.05) IFN-? and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-? responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P< 0.05) the corresponding responses of noninfected, M. avium-infected, and M. aviumsubsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. aviumand M. aviumsubsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. aviumand/or M. aviumsubsp. paratuberculosis.