Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii

Abstract

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

FIG. 1.

Delayed-type-hypersensitivity responses to M. bovis PPD as determined by the caudal fold test. Caudal fold skin test responses (means ± standard errors) by control (n = 10), M. kansasii-infected (n = 4), and M. bovis-infected (n = 9) cattle to M. bovis PPD. Responses represent the change in skin thickness relative to preinjection CFT values. The letters a to c indicate responses that differ (P < 0.05).

FIG. 2.

Longitudinal nitrite production to mycobacterial antigens. Blood mononuclear cells were isolated from control (n = 10), M. kansasii-infected (n = 4), and M. bovis-infected (n = 9) cattle at the indicated time points after challenge. Isolates were cultured for 72 h with medium alone, 1 μg/ml rESAT-6-CFP-10, or 5 μg/ml PPDs, and supernatants were harvested for analysis of nitrite (Griess reaction) as an indication of NO production. Values are presented as mean (±standard error) responses to rESAT-6-CFP-10 (A) or M. bovis PPD (B) stimulation minus the response to medium alone. The letters a to c indicate responses that differ (P < 0.05) for each respective time point after challenge.

FIG. 3.

Longitudinal IFN-γ production to mycobacterial antigens. Blood mononuclear cells were isolated from control (n = 10), M. kansasii-infected (n = 4), and M. bovis-infected (n = 9) cattle at the indicated time points after the challenge. Isolates were cultured for 48 h with medium alone, 1 μg/ml rESAT-6-CFP-10, or 5 μg/ml PPDs, and supernatants were harvested for the analysis of IFN-γ by ELISA. Values are presented as mean (±standard error) responses to rESAT-6-CFP-10 (A) or M. bovis PPD (B) stimulation minus the response to medium alone. Letters a to c indicate responses that differ (P < 0.05) for each respective time point after challenge. The same letter indicates that the responses did not differ.

FIG. 4.

Effects of the skin test on in vitro IFN-γ production by individual M. kansasii-sensitized cattle in response to mycobacterial antigens. Blood mononuclear cells were isolated from M. kansasii-infected cattle at the indicated time points after challenge (x axis) and cultured for 48 h with medium alone, 1 μg/ml rESAT-6-CFP-10, or 5 μg/ml PPDs, and supernatants were harvested for analysis of IFN-γ by ELISA. Values are presented as individual responses (in the key, numbers next to symbols refer to the animals' numbers) to rESAT-6-CFP-10 (A) or M. bovis PPD (B) stimulation (stim.) minus the response to the medium alone. Arrows below “CFT” and “CCT” indicate time points of injection of PPDs for the caudal fold and comparative cervical skin tests, respectively.

FIG. 5.

Western blot analysis of antibody responses to MPB83. Preparative immunoblots of M. bovis MPB83 antigen probed with sera from cattle experimentally infected with either M. kansasii, M. bovis, or serum from a noninfected animal (control). Molecular mass is indicated to the left of the top blot (uniform for each blot), days after infection are shown along the top, animal number and infection status are shown on the right, and time points of injection of PPD(s) for skin tests are shown at the bottom. NS, no sample.

FIG. 6.

Relative intensities of responses to MPB83 as measured by MAPIA. Sera collected from M. bovis-infected (circles; n = 9) and M. kansasii-infected (triangles; n = 4) cattle 112 days after inoculation were evaluated by MAPIA for reactivity to mycobacterial antigens. For comparison, the intensities of bands of reactivity to MPB83 were determined by densitometry and are presented as absorbency values.