Mycobacterium avium ssp. paratuberculosis (MAP) is a facultative intracellular pathogen, residing in subepithelial macrophages. Clearance of MAP critically depends upon an appropriate pro-inflammatory and cytotoxic Th-1 immune response leading to activation and/or lysis of persistently infected macrophages to promote bacterial killing. However, work in vivo has shown that the appropriate Th-1 immune response occurring early in MAP infection is lost, followed by an ineffective, antibody-mediated Th-2 response. Our overall hypothesis is that once MAP persists within naive macrophages, it reduces the ability of infected macrophages to react to normal T cell signaling thereby macrophages fail to be activated and destroy MAP, and fail to properly signal T cells to respond. To test this hypothesis, we investigated the effect of MAP infection on CD40 signaling, a main pathway used by T cells to activate macrophages. Using Q-RT-PCR analysis, we recently demonstrated that once bovine monocyte-derived macrophages (MDM) are infected with MAP, they fail to up-regulate the expression of iNOS and IL-12p40 encoding genes in response to CD40 ligand (CD40L). Our recent studies investigate possible mechanisms that are responsible for MAP-specific changes in CD40L-mediated gene expression. Using flow cytometric analysis we demonstrated that failure of infected macrophages to respond to CD40L was not due to down-regulation of CD40 on the cell surface of MAP-infected MDM. Studies with specific inhibitors revealed that the CD40L-mediated increase in IL-12p40 and iNOS gene expression is dependent upon activation of p38. However, western blot analysis revealed that interference with CD40L-mediated increases in gene expression does not appear to be due to prevention of early and rapid p38 activation. Paradoxically, while p38 activation in CD40L-stimulated MDM cells is only transient, we observed a sustained p38 activity in MAP-infected MDM cells upon CD40L stimulation, suggesting p38 plays a role in MAP interference with CD40 signaling. Additionally, our data revealed the possibility of a third potential mechanism. We observed a dramatic increase in IL-10 gene expression in MAP-infected MDM upon CD40L stimulation relative to uninfected cells. IL-10 has been shown to negatively regulate IL-12p40 gene expression. Therefore, we hypothesize that MAP-induced IL-10 expression at early times following CD40 signaling interferes with subsequent IL-12p40 and iNOS expression. Continuing studies are underway to uncover the role of sustained p38 activity and enhanced IL-10 expression for MAP interference with CD40 signaling in infected macrophages.