In vivo induced antigen technology (IVIAT) is a method that utilizes sera from infected animals (in vivo) absorbed versus unabsorbed with protein from in vitro grown bacteria to differentially screen expression libraries in E. coli. This characterizes antigens and potential virulence determinants expressed specifically by the pathogen in association with the disease process. A modification of this process has been used to differentially screen an arrayed expression library of Mycobacterium avium subspecies paratuberculosis (Mptb) and identify genes apparently up-regulated in vivo. Protein preparations were generated from Mptb organisms retrieved from infected tissues of Johne's disease affected animals and separately from Mptb grown in artificial culture medium. These protein preparations were used to raise hyper-immune sera in rabbits. Replicates of 384 well plate arrays of an E. coli expression library of Mptb were probed with the sera. A number of differences were observed the most striking of which were from four clones (pMK19, pMK22, pMK20, pMK40) which represented two independent copies each of two separate genes. Of these pMK19 and pMK22 each contained a major in-frame fragment of the gene katG, the catalase-peroxidase, which was only detected by anti-sera to the in vivo (disease) protein extracts. This confirmed independent evidence from RNA studies and support this approach as a useful method for identifying clearly up-regulated (or down-regulated) genes of Mptb in the in vivo environment. pMK20 and pMK40 each contained a substantial in-frame fragment of the same gene sequence, representing an as yet uncharacterised potential protein antigen associated with the in vivo disease state, which has no presently characterized protein homologues. Assessment of more extensive expression libraries continues.