Measurements of paratuberculosis-specific cellular mediated immune responses (CMI) by the interferon-gamma (IFN-γ) test are sensitive to the time interval from sampling to culture. This is because de novo IFN-γ production by antigen-specific T-cells requires antigen presentation and co-stimulation by antigen presenting cells during culture. The general recommendation is a maximum time lapse of 8 hours from blood sampling to culture, which is a major obstacle to the routine use of the IFN-γ test in diagnostic or surveillance programs. The aim of the present study was to investigate if additions of the co-stimulatory cytokines IL-2 and/or IL-12 to bovine whole blood cultures could potentiate the waning Johnin PPD-specific IFN-γ production in day-old samples. Potentiating the whole blood IFN-γ test with IL-12 rescued antigen-specific IFN-γ production in day-old samples and produced IFN-γ levels that were comparable to those of fresh stimulated samples. Addition of IL-12 to cultures without antigen did not induce non-specific IFN-γ production. Furthermore, addition of IL-12 to fresh or day old samples did not induce non-specific IFN-γ responses to PPD in animals that were IFN-γ test negative. Addition of IL-2 was not able to induce comparable increases in IFN-γ responses in day-old samples, and although a combination of IL-2 and IL-12 induced higher levels of antigen-specific IFN-γ than IL-12 alone, this cocktail also induced high levels of IFN-γ in absence of antigen.Thus, the IL-12 potentiated IFN-γ test makes possible the study of antigen-specific CMI responses in stabilized blood samples for an extended period of time allowing samples submitted to the laboratory by ordinary mail services to be tested. Presently, the IL-12 potentiated IFN-γ test is being used to estimate the level of calf-exposure to M. paratuberculosis by yearly screening of 45 heifers in the 15-24 months age group in 18 dairy herds with an ongoing management-based eradication programme.