A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononucler cells (PBMCs) from control uninfected, clinical Johne's disease positive, and subclinical Johne's positive Holstein cows. PBMCs from control, cows and an early clinical Johne's disease positive cow responded similarly to the general mitogen ConA, with activation of 119 genes, of these, 104 were identical between the two animals. Stimulation of PBMCs from the control uninfected animal with M. paratuberculosis had little effect on immune cell gene expression (activation of 6 genes involved in phagocytosis). Stimulation of PBMCs from the clinical Johne's disease positive animal with M. paratuberculosis, however caused significant (fold change > 1.5, P < 0.05) down-regulation in expression of over 30 immune cell genes and activation of many others (>100). Analysis of a second, more severely affected animal validated these initial results. Exposure of PBMCs from the subclinical Johne's disease positive cows to M. paratuberculosis resulted in significant (fold change >1.5, P < 0.05) activation of numerous genes. In sharp contrast to the clinical infected animal PBMCs, there were no genes whose expression was repressed following exposure of PBMCs from the subclinical animals to M. paratuberculosis. In fact, the pattern of immune cell gene activation observed in PBMCs from the subclinical Johne's positive cows exposed to M. paratuberculosis was extremely similar to that of PBMCs exposed to ConA, a general T cell mitogen. Thus, within the confines of a small sample set, the overall response of PBMCs from cows with clinical Johne's disease to M. paratuberculosis are dramatically different than the response of PBMCs from cows with subclinical Johne's disease. In addition, our results demonstrate that the response of PBMCs from subclinical Johne's disease positive cows to M. paratuberculosis is extremely similar to PBMCs stimulated with ConA.