Mycobacterium avium subspecies paratuberculosis (Map) genomic library expressed in Escherichia coli was screened for the identification of novel interferon-gamma (IFN-γ) inducing antigens using peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. Two positive clones which induced IFN-γ in the PBMC were identified from 1,200 colonies examined. Three recombinant proteins, referred to as Map10, Map39 and Map41 according to their respective molecular sizes, were produced based on nucleotides sequences obtained from these clones. All recombinant proteins significantly elicited IFN-γ production by PBMC from cattle infected with Map. Two of the three recombinant proteins, Map39 and Map41, appeared to be members of the PPE protein family of Map, due to the sequence homology of the deduced amino acids with those of the M. tuberculosis PPE family protein. M. avium subsp. avium also possessed similar PPE family protein genes, but no cross-reactions were observed among the other mycobacterial species tested. Monoclonal antibodies (Mab) against Map39 and Map41 strongly reacted with corresponding antigens in immunoblotting with the sonicated extract and PPD of Map, suggesting that these two PPE antigens are actually produced by this organism. Most of IFN-γ producing cells stimulated by these PPE antigens were CD4 positive cells, and they co-cultured with the dendritic cells which were stimulated by PPE antigens secreted IFN-γ far higher than that with macrophages. IL-10 was also detected in the culture supernatant of PBMC stimulated by PPE antigens besides IFN-γ, and addition of anti bovine IL-10 Mab increased IFN-γ production. These cytokine inductions by PPE antigens may be implicated in immunopathogenesis of Johne's disease.