Mycobacterium paratuberculosis is the etiologic agent of Johne's disease in cattle, sheep and goats. The diagnosis of Johne's disease in animals is hampered by 1) a lack of a rapid, specific, and sensitive serologic test, 2) slow growth of the organism by fecal culture [4-6 weeks in cattle and goats; usually no growth in culture from sheep], 3) a lack of sensitive PCR direct from fecal samples (not reliable in low and medium shedding cases). In order to improve the diagnosis of Johne's disease in these animals, we are applying the primers described by Vary et al., to set up a PCR and an enhanced chemiluminescence detection system for M. paratuberculosis. The DNA was purified from blood monocytes or milk macrophages and was used as a template in the PCR. The PCR products were run on an agarose gel electrophoresis and transferred to nitrocellulose and hybridized with ECL 3'-oligolabelled probe. We have detected PCR positive samples from 3 of 22 samples from cattle milk macrophages, 8 of 31 samples from sheep blood monocytes, and 7 of 21 samples from sheep milk macrophages. The possible application of this technique for diagnosis of M. paratuberculosis in animals and for culling the infected animals are discussed.