Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and M. bovis

Abstract

The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO(+) CD25(+) T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

Fig 1

Secretion of gamma interferon (IFN-γ; Abs450nm) in a whole-blood assay by control noninfected calves (control) or calves infected with live Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), M. kansasii, or M. bovis. (A) Whole blood was incubated for 18 h with concanavalin A (ConA), pokeweed mitogen (PWM), or whole-cell sonicates of M. avium subsp. avium (MavS), M. kansasii (MkS), or M. bovis (MboS). (B) Whole blood was incubated with a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS) or pools of recombinant M. avium subsp. paratuberculosis proteins (MAP1, MAP2, MAP3). Data are expressed as means ± standard errors of the means (SEM). Significant (P < 0.05) differences between the infection groups and within each in vitro treatment are represented by different letters.

Fig 2

Secretion of interleukin-12 (IL-12) (A), IL-4 (B), and IL-10 (C) by PBMCs isolated from control noninfected calves (control) or calves infected with live Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), M. kansasii, or M. bovis. PBMCs were incubated for 24 h with concanavalin A (ConA) or a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS). Data are expressed as means ± SEM. Significant differences (P < 0.05) between the infection groups and within each in vitro treatment are represented by different letters.

Fig 3

Percentages of CD4 (A) and CD8 (B) T cells from PBMCs isolated from control noninfected calves (control) or calves infected with live Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), M. kansasii, or M. bovis. Cells are expressed as CD4 or CD8 populations with coexpression of CD25 and CD26 subpopulations, after stimulation with a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS). Data are expressed as means ± SEM. Significant differences (P < 0.05) between the groups are represented by different letters.

Fig 4

Percentages of γδ (gd) T cells (A) and B cells (B) from PBMCs isolated from control noninfected calves (control) or calves infected with live Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), M. kansasii, or M. bovis. Cells are expressed as total γδ T-cell or B-cell populations with coexpression of CD25 and CD26, after stimulation with a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS). Data are expressed as means ± SEM. Significant differences (P < 0.05) between groups are represented by different letters.

Fig 5

Percentage of CD45RO cells from PBMCs isolated from control noninfected calves (control) or calves infected with live Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), M. kansasii, or M. bovis. Cells are expressed as the total CD45RO population (A), after stimulation with either concanavalin A (ConA), a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS), or pools of recombinant M. avium subsp. paratuberculosis proteins (MAP1, MAP2, MAP3). Subpopulation (%) of CD45RO⁺ CD25⁺ CD26⁺ within CD4, CD8, and γδ T cells (B) after stimulation with a whole-cell sonicate of M. avium subsp. paratuberculosis (MPS). Data are expressed as the mean ± SEM. Significant differences (P < 0.05) between groups are represented by different letters.