AhpC and AhpD are detoxifying enzymes involved in the protection against oxidative stress. The enzymes are constitutively expressed in M. avium subsp. paratuberculosis as opposed to other mycobacteria. The enzymes are immunogenic and can differentiate between animals infected with bovine tuberculosis and paratuberculosis in ELISA. The aim of the current study was to further characterise the immune response against these proteins including epitope mapping of AhpC using overlapping synthetic peptides. Experimentally infected calves with subclinical disease were used for measuring IFN-g production, lymphocyte proliferation and T-cell epitope mapping. Naturally, infected cattle and goats in addition to immunised rabbits where used for B-cell epitope mapping. Several of the infected calves had IFN-g production and lymphocyte proliferation responses against AhpC and AhpD at various time points after infection. The calves with the strongest responses were used to identify T-cell eptiopes. An epitope causing lymphocyte proliferation was located near the C terminal end of the protein while several peptides induced low levels of IFN-g production. Two B-cell eptiopes were localised and these were recognised both by infected cattle, infected goats and immunised rabbits showing consistency between the species in recognition of B-cell epitopes. One of the B-cell epitopes was in the same area as the epitope that induced lymphocyte proliferation. These results show that AhpC and also AhpD are immunogenic proteins that can identify cattle with subclinical paratuberculosis and may thus be useful in diagnostic testing.