Diagnostic Implications of Antigen-Induced Gamma Interferon, Nitric Oxide, and Tumor Necrosis Factor Alpha Production by Peripheral Blood Mononuclear Cells fromMycobacterium bovis-Infected Cattle

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-), nitric oxide (NO), and tumor necrosis factor alpha (TNF-) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-, NO, and TNFresponses. Infection-specific increases in NO, but not in IFN- or TNF-, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-, NO, and TNFresponses in antigen-stimulated cells from cattle receiving 10 5 CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10 3 CFU of M. bovis organisms. The NO response, but not the IFNand TNF- responses, was influenced by infective strains of M. bovis. The TNF-, NO, and IFN- responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-, like IFN-, may prove useful as indices for the diagnosis of bovine tuberculosis. First described by Robert Koch in 1891, the tuberculin skin reaction has been the principal means of tuberculosis diagnosis for both humans and domestic animals (23). For cattle, the caudal fold skin test (CFT) is the primary approved test for tuberculosis within the United States. The CFT relies on in vivo reactivity to Mycobacterium bovis purified protein derivative (PPDb) injected intradermally into a fold of skin at the base of the tail. Cattle classified as reactors or suspect with this test are often retested by using the comparative cervical skin test, in which PPDb is injected at one site and M. avium PPD (PPDa) is injected at a separate site. The comparative cervical skin test, while technically more challenging than the CFT, provides an added ability to distinguish M. avium (including M. avium subsp. paratuberculosis) responders from M. bovis responders. An in vitro method of tuberculosis diagnosis has also been developed (41) and approved for use in the United States as a complementary test (i.e., in conjunction with the skin test) (22). The in vitro assay detects gamma interferon (IFN-) produced differentially by peripheral blood mononuclear cells (PBMC) exposed to no antigen (i.e., background response), PPDa, PPDb, or mitogen (e.g., pokeweed mitogen, PWM) (40). The assay is particularly suitable for diagnostic laboratories, as whole blood cultures are used, thus circumventing the need for cumbersome cell separation techniques. More recently, recombinant antigens specific for virulent tubercle ba