The phage library of Mycobacterium avium subspecies paratuberculosis(Map) strain ATCC19698, which had been constructed using the Zap Express cloning vector (Infect. Immun. 73:3778-82, 2005), was screened with sera from calves infected with Map in order to detect the Map antigens with eliciting humoral immunity. The serum used for screening of the phage library was absorbed with plaques from non-recombinant phages, Escherichia coli and Mycobacterium phlei organisms to remove cross-reacting antibodies. After the screening of about 4 x 10^5 plaques, we have finally cloned one recombinant phage expressing Map antigen which strongly react with serum antibodies from infected calves. The Map DNA insert cloned into the phage vector was excised out of the phage into the form of the phagemid vector with E. coli strain XLOLR, and a part of 5' end of the insert DNA was sequenced using T3 universal primer. The sequence results indicated that the insert Map DNA contained Map gene encoding "echA12_2", an enoyl-CoA hydratase protein (echA), which plays a role for effective ATP synthesis in stationary phase survival. Therefore, the coding sequence of Map echA gene was amplified from DNA of Map strain ATCC19698 by PCR, and cloned into pQE plasmid vectors to obtain the recombinant Map-echA protein (rMap-echA). Sera from experimentally infected calves strongly reacted with the rMap-echA with immunoblotting, whereas sera from uninfected cattle did not. Although the homologous gene of M. avium subspecies avium (Maa) showed 99% nucleotide sequence homology with echA12_2 gene of Map, ELISA using the rMap-echA indicated high specificity without any cross-reaction to bovine hyperimmune sera against Maa and other related Mycobacterium species. These results suggest that the ELISA using the rMap-echA antigen may be useful for the diagnosis of Johne's disease.