Many antigens within the protein/peptide complement of Johnin purified protein derivative (PPD) and cell free culture supernatants of Mycobacterium avium subspecies paratuberculosis are capable of eliciting a strong interferon-g release response by lymphocytes from ruminants early in the course of Johne's disease. Since these preparations are complex, ill defined and hard to reproduce it is of value to identify individual gene products that may contribute to the overall response. An E. coli based expression system has been used to express and purify a range of protein antigens using poly-histidine tags. The purified recombinant proteins have been used to stimulate whole blood preparations and using the BOVIGAM^TM assay (CSL Animal Health Ltd) to determine interferon-g release in a range of animal species, of differing infection states and at different time points post-infection. This has been performed within the reference frame of a time course of bacteriological, immuno-assay and clinical observations. Interferon-g release has been observed in response to four of fourteen antigens assessed. Two antigens, AhpC and BrfA, elicited strong responses in a small number of animals across a number of time points. A number of these antigens, including AhpC, have been assessed by expression in a baculovirus/insect cell culture system. These materials currently show no benefit over the E. coli derived materials. The purification/assay system proved robust with no animals showing responses to either control recombinant proteins or potential contaminants from the E. coli system. Qualitative comparison of native purified antigens and antigens the expressed and purified from an E. coli or M. smegmatis background will be performed. This approach is being utilized in conjunction with proteomic analysis and expression library screening.