AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay

Sera from cattle naturally infected with Mycobacterium avium subsp, paratub erculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infecte d with Mycobacterium bovis were tested for the presence of antibodies again st paratuberculosis antigens, An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp, paratuberculosis ant igens on a hyperimmune antiserum against M. avium subsp, avium proteins in order to remove cross-reacting antigens, This absorbed-antigen ELISA recogn ized 66% of animals with paratuberculosis (37 of 56), while none of the ani mals with naturally occurring bovine tuberculosis (TB) had detectable antib odies. However, the animals with experimental bovine TB also responded in t his ELISA, Similar results were found in a commercial ELISA, showing that n either of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against pur ified AhpC and AhpD, which are proteins constitutively expressed by M. aviu m subsp, paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to A hpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 4 8), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. Non e of the animals with bovine TB had detectable antibodies against any of th e purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to different iate between paratuberculosis and bovine TB in ELISA.