Diagnostic implications of antigen-induced gamma interferon, nitric oxide, and tumor necrosis factor alpha production by peripheral blood mononuclear cells from Mycobacterium bovis-infected cattle

Abstract

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-gamma), nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) 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-gamma, NO, and TNF-alpha responses. Infection-specific increases in NO, but not in IFN-gamma or TNF-alpha, 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-gamma, NO, and TNF-alpha responses 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 IFN-gamma and TNF-alpha responses, was influenced by infective strains of M. bovis. The TNF-alpha, NO, and IFN-gamma responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-alpha, like IFN-gamma, may prove useful as indices for the diagnosis of bovine tuberculosis.

FIG. 1.

Comparison of the results of the Griess reaction with those of HPIC for the measurement of nitrite in 48-h culture supernatants. The measurements (n = 49) utilized in this analysis were from four time points (prechallenge [day 0] and 34, 68, and 124 days postchallenge) and stimuli (nonstimulated [medium alone] and stimulated with PPDa, PPDb, or PWM). The symbols (+) represent individual responses, and the line was generated by linear regression analysis (r = 0.94, P < 0.0001; y = 0.57x + 70.87).

FIG. 2.

Spontaneous production of nitrite by nonstimulated PBMC during the experimental period. Nitrite production (means ± SEMs) in supernatants from 48-h nonstimulated cultures was measured by the Griess reaction. Asterisks (***) indicate that results for the postchallenge response differed (P < 0.001) from those for the prechallenge (day 0) response.

FIG. 3.

Effects of challenge dose and time relative to challenge on IFN-γ, nitrite, and TNF-α responses of cells in vitro. Blood mononuclear cells were isolated from cattle immediately prior to challenge (day 0) and at 34 days, 68 days, and 124 days after challenge with 10³ (closed triangles, n = 10) or 10⁵ (open boxes, n = 10) CFU of M. bovis strain 95-1315 organisms. The cells were cultured for 48 h with medium alone or with 5 μg of PPDb per ml. IFN-γ and TNF-α concentrations in culture supernatants were quantified by ELISA, and nitrite concentrations were quantified by the Griess reaction. Values are presented as mean (± SEM) responses to M. bovis PPDb stimulation minus the response to medium alone. An asterisk (*) indicates that the values for the responses for cattle receiving 10⁵ CFU of PBMC differed (P < 0.05) from the values for the responses for cattle receiving 10³ CFU of M. bovis organisms.

FIG. 4.

Effects of challenge strain on nitrite responses. Blood mononuclear cells collected immediately before challenge (day 0) and at 0 days, 34 days, 68 days, or 124 days after being challenged with M. bovis strain 95-1315 (closed triangles, n = 10) or M. bovis strain HC-2045T (open boxes, n = 10) were cultured with 5 μg of PPDb per ml, 5 μg of PPDa per ml, 10 μg of M. bovis strain 95-1315 WCS per ml, or 10 μg of M. bovis strain HC-2045T WCS per ml. Nitrate concentrations in 48-h culture supernatants were quantified by the Griess reaction. The P values for the effects of the strains are indicated on each graph.