Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17Α was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4+, CD8+, and γδ T cell receptor-positive (TCR+) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies.

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