The investigation of environmentally regulated proteins has led to a better understanding of host-pathogen interactions and identified novel vaccine candidate antigens for several bacterial pathogens. In an effort to identify such proteins in Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), a genomic expression library was differentially screened with sera from rabbits that had been immunised with live M. paratuberculosis (alpha-live) as well as sera from rabbits immunised with heat-killed M. paratuberculosis (alpha-killed). These experiments identified seven recombinant plaques that were uniquely recognised by the alpha-live sera. Sequence data showed that five of these clones overlapped with each other and contained a common open-reading frame encoding a 25-kDa protein, termed Csp1. The 25-kDa antigen shows weak similarity to a secreted Corynebacterium glutamicum protein. The remaining two clones overlapped with each other and contained two partial open-reading frames, both encoding proteins with strong homology to polyketide synthase from various species of mycobacteria. Antisera were produced against a peptide of the polyketide synthase gene product designated Pks7. Csp1-specific antibodies were affinity purified from the alpha-live sera. These purified antibodies demonstrated that Csp1 was present within infected macrophages. Collectively, these data identify novel M. paratuberculosis antigens that may be important in pathogenesis.