Little is known about regulatory mechanisms of immunity in cattle infected with M. paratuberculosis. Although M. paratuberculosis activates both humoral and cell-mediated immunity, chronic states of infection are associated with this disease. This appears to be due to the ability of this intracellular pathogen to survive and replicate within resident macrophages of infected hosts. Acquired resistance to mycobacterial infection is thought to be mediated by activation of macrophage microbicidal activity through sensitized T cells. Since T cells can stimulate bacteriocidal and/or bacteriostatic activity in macrophages via secretion of various cytokines, it is likely that this activation pathway is arrested in some manner during mycobacterial infection. We have evaluated the effects of subclinical and clinical Johne's disease in cattle on transcription of TNF in vitro. Peripheral blood mononuclear cells from noninfected control cows subclinically (low/intermittent shedders) and clinically (high/chronic shedders) infected animals were incubated in RPMI-1640 medium with 10 mcg concanavalin A/ml and 10 ng phorbol myristate acetate (stimulated) or medium alone (nonstimulated) for 18 hours. Total RNA was extracted from cultured cells by guanidium isothiocyanate lysis, slot-blotted onto nitrocellulose membrane and probed with a digoxigenin labeled-mouse TNF cDNA insert followed by a digoxigenin labeled-chicken beta-actin cDNA insert. Tumor necrosis factor mRNA expression was significantly reduced in both nonstimulated (28%) and stimulated 48-57% cell cultures from clinically infected cows compared to subclinical and noninfected control animals. In addition, expression of TNF mRNA in cells isolated from clinical cows was significantly lower for cells stimulated in vitro with ConA/PMA compared to nonstimulated cell cultures. Serum TNF measured in samples taken at the same time cell isolations were performed was undetectable regardless of infection status. In contrast, serum IL-6 was negatively correlated with level of infection with lower serum IL-6 observed in Johne's infected cows compared to noninfected controls. These results indicate that TNF transcription is reduced during clinical disease suggesting that a corresponding decrease in translation of TNF could be causative factor in the loss of protective immunity at this stage of infection. Alternatively, a reduction in the level of TNF transcription could be protective mechanism invoked by cells to safeguard against the toxic effects of TNF during periods of chronic inflammation.