Porcine Respiratory Disease Complex (PRDC) is a significant economic problem for swine producers. PRDC outbreaks can cause elevated mortality, decreased feed efficiency, higher cull rates, increased days to market, and increased treatment costs. This syndrome is caused by the interaction of multifactorial etiologies, including the participation of porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus (PCV2) in the disease progression. In aid of PRDC prevention and control, producers and veterinarians desire multiplex tests that can readily detect multiple pathogens at the same time, in the same sample, and at a reasonable cost. In this study, we generated a panel of recombinant viral antigens and monoclonal antibodies (mAbs) for the diagnosis of PRRSV, SIV and PCV2 infections. Using these reagents, we developed a multiplex fluorescent immunomicrosphere assay (FMIA) for detection of PRRSV, SIV and PCV2 simultaneously using serum or oral fluid samples. To meet the demanding of on-site field test, we also antibodies in the field. At the meanwhile, standard reagents and protocols have established through this study. They have been sharing with major diagnostic laboratories and swine disease researchers. Development of rapid, multiplex, cost effective diagnostic tests will be important in population-based epidemiological studies, which provide important data on the early identification of susceptible groups in the population and evaluation of vaccination and herd management strategies. These assays present advantages of simplicity, rapidity, cost-effectiveness, and potentially increase the sample number of representative individual animals in a large population. The dipstick test is user-friendly format, and can be performed on-site in a swine farm by untrained personnel. It is expected that the dipstick test can be used as an on-site initial screening test to determine the disease status of a swine population in the field, and the multiplex FMIA will be used as a laboratory confirmation test for the accurate identification of various pathogens in PRDC. Importantly, these tests are more suitable in the PRDC surveillance program at a population level. Technologies developed in this study could apply to other swine pathogens, such as porcine respiratory corona virus (PRCV), Mycoplasma hyopneumoniae (M. hyo.), Swine Brucellosis. Our ultimate goal is to develop a rapid test to detect various swine pathogens simultaneously to help preventing and controlling of PRDC. For more information, please contact Dr. Ying Fang, South Dakota State University, Phone: 605-688-6647, E-mail: [email protected].