Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.