Improvement of serological-based diagnostics for Mycoplasma hyopneumoniae infections in swine will require identification of mycoplasma-specific antigens and their use in an ELISA-based assay format as well as development of more current technologies. The overall goal of this project was to develop an improved serology-based assay on M. hyopneumoniae-specific antigens, follow pig responses to these antigens during infection over an extended period of time, and utilize these antigens in a bead-based assay. This will improve the specificity, reproducibility, and sensitivity of current serological assays. We identified eight M. hyopneumoniae-specific proteins that lack cross-reactivity to other swine mycoplasma species. These antigens were cloned and expressed as recombinant proteins for further analysis. Each protein posed unique problems in expression levels, solubility, etc. The proteins are being assessed by a commercial partner for their usefulness in a standard ELISA style assay. The proteins have also been bound to beads, and testing is underway with our defined sera and sera from clinical samples. The defined sera were developed by challenging pigs (4 per group) with one of the following mycoplasmas, M. hyopneumoniae, M. hyosynoviae, M. hyorhinis, and M. flocculare, and monitoring the infection for a 118 day period. Serum samples were taken throughout the study, oral fluids were collected on a monthly basis, and at necropsy histopathology was performed on the M. hyopneumoniae challenged pigs. Serological responses indicated a slow conversion except for the M. hyorhinis-challenged pigs. Cross-reactions were noted in the M. hyopneumoniae sera with M. hyorhinis Tween 20 antigens and in the M. flocculare sera with M. hyorhinis Tween 20 antigens. Oral fluids from M. hyopneumoniae challenged pigs showed a positive IgA response with the Tween 20 ELISA. The ELISA and bead-based assays with the purified antigens are still being tested. (Contact: Chris Minion, VMPM, Iowa State University, [email protected])