Currently, diagnosis of PRRSV infection is by virus antigen, nucleic acid or antibody detection in serum samples. Thus, serum is the standard sample for diagnostic evaluation. However, blood sample collection is a labor-intensive procedure and may cause negative effect on animal health. In contrast, oral-fluid samples provide a cost effective and non-invasive alternative to serum samples. Particularly, it is more suitable for sampling in large epidemiologic studies. Since oral fluid contains lower amount of antibody, a more sensitive oral fluid-based assay is needed for detection of PRRSV infection. The fluorescent immunomicrosphere assay (FMIA) has advantage over traditional ELISA test format, including improved sensitivity and the ability of multiplex, i.e., detect PRRSV antigen and host antibody response to several viral proteins simultaneously. In this study, we developed a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of PRRSV specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (Type I and Type II) were used as antigen and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs were achieved greater than 92% sensitivity and 91% specificity. In serum samples (n = 1639), the FMIAs reached greater than 98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of antibody response in infected pigs. In oral fluid, N protein was more sensitive for the detection of early infection (7 and 14 dpi), but nsp7 detected higher and longer antibody response after 28 days post infection. In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days post infection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will revolutionize the way we survey for swine herds and improve our ability to cheaply, efficiently track PRRSV infections in both population and individual animals.
For more information about this project, please contact Dr. Ying Fang at South Dakota State University, Brookings, SD 57007, Phone: 605-688-6647, e-mail: [email protected].
