Serology due to its low cost and ease of sample collection is the most common tool used to monitor the status of M. hyopneumoniae infection within a herd. In addition, testing replacement animals for entry into M. hyopneumoniae negative herds to ensure continued negative status is critical for herd health. Two ELISA assay technologies are currently used for M. hyopneumoniae diagnostics and consist of either a competitive inhibition assay based on a monoclonal antibody to an internal protein in M. hyopneumoniae, or indirect ELISAs that consist of a mixture of membrane-derived proteins as the test antigen. Recent reports from the field increasingly find that the current ELISAs provide conflicting results that are not easily resolved. This is particularly true for younger animals where antibody titers to M. hyopneumoniae infections are slow to develop. As a result, the need for an improved assay has become increasingly apparent. This project will develop a new ELISA with improved sensitivity and specificity over currently available tests providing the swine industry with a more effective tool for M. hyopneumoniae screening and monitoring on a herd basis. We will screen proteins expressed in E. coli and purified for the ability to be recognized by M. hyopneumoniae-specific sera. A cocktail of these positive proteins will then be used for validation of the ELISA. In addition, we will identify proteins recognized by M. hyopneumoniae-specific monoclonal antibodies (Mabs). We have expressed and purified approximately 40 M. hyopneumoniae membrane-associated proteins and have screened them using rabbit and swine hyperimmune sera. This is the first step in the ELISA development, to identify antigens from other mycoplasma species (M. hyorhinis, M. hyosynoviae, and M. flocculare) that cross-react with M. hyopneumoniae antigens so that we can eliminate them from our protein cocktail for the ELISA antigen, thus reducing or eliminating false positive reactions due to cross reactivity. We have also identified the proteins identified by the two Mabs 80.1 and D79, Mhp511 and Mhp677 (P46), respectively. These two Mabs are thought to be specific for M. hyopneumoniae, but one protein, Mhp511, showed significant cross reactivity with all antisera tested. This suggests that D79.1-7 must recognize a portion of the Mhp511 protein that is unique to M. hyopneumoniae, but other portions of the protein are cross reactive to other swine mycoplasma species and should not be used in a serological based assay.
Contact Information; Chris Minion,, Iowa State University, 515-294-6347.