Mycobacterium paratuberculosis (M. paratuberculosis) is a facultative intracellular bacteria and the causal agent of Johne's disease in cattle. M. paratuberculosis and other Mycobacteria (in general) have the ability to survive and proliferate within the phagosomes of host macrophage cells by arresting vesicle maturation. How Mycobacteria are able to accomplish this unique survival tactic is not well understood. Recently, our group has taken a functional genomics approach to determine if phagocytosis of Mycobacteria affects macrophage gene expression and how these interactions may lead to reduced phagosome maturation. DDRT-PCR was used, as an initial unbiased high throughput-screening tool that allows the macrophage to "tell" us which genes may be of interest during the general process of phagocytosis and during the phagocytosis of M. paratuberculosis specifically. We have compared the RNA expression profiles of macrophage cells at a 60-minute time point during a negative control of no phagocytosis with the expression profiles of RNA from macrophages during phagocytosis of E. coli, M. paratuberculosis and latex beads (a positive phagocytosis control). To date we have identified over 380 separate amplicons representing genes whose expression appears to change during the general process of phagocytosis. A subset of amplicons has been cloned and subjected to dot-blot and Northern blot hybridizations to confirm differential expression observed by DDRT-PCR. Amplicons exhibiting differential expression by Northern hybridization were then subjected to direct DNA sequencing and BLAST analysis for probable identification. Our initial results indicate that macrophage gene expression profiles change dramatically during the general process of phagocytosis and that gene expression profiles during the phagocytosis of M. paratuberculosis are distinctly different from gene expression profiles during phagocytosis of a readily degradable bacteria such as E. coli. Genes discovered thus far appear involved in a variety of functions such as energy metabolism, calcium binding, cell signaling and macrophage migration.