Aims Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease. To survive within host macrophages, the pathogen secretes a battery of proteins to interfere with the immunological response of the host. One of these proteins is tyrosine phosphate A (PtpA), which has been identified as a secreted protein critical for survival of its close relative M. tuberculosis within infected macrophages. Methods and results In this study, the immune response to recombinant PtpA used as an antigen was investigated in a cohort of ∼1000 cows infected with MAP compared to negative control animals using ELISA. The sera from MAP-infected cows had significantly higher levels of antibodies against PtpA when compared to uninfected cows. Conclusions The data presented here indicate that the antibodies produced against PtpA are sensitive enough to detect infected animals before the appearance of the disease symptoms. Significance and impact of study The use of PtpA as an antigen can be developed as an early diagnostic test. Moreover, PtpA is a candidate antigen for detection of humoral immune responses in cows infected with MAP.