Using Pig Oral Fluid To Monitor Porcine Reproductive And Respiratory Syndrome Virus Neutralizing Antibody And To Assess The Nanoparticle Vaccine Candidate

Porcine Reproductive and Respiratory Syndrome (PRRS) has been a devastating disease for pork producers since it was formally identified in1991. It is currently the most economically significant disease impacting pig production worldwide. Oral fluid is a mixture of saliva and mucosal transudate which contains specific antibodies derived from both serum and salivary glands. Pig oral fluid samples for disease surveillance are gaining importance due to ease of collection and low-cost.PRRSV neutralizing antibody (NA) is important to clear the virus, also is a useful indicator to evaluate the herd immunity against PRRS. Shedding of PRRSV and the presence of virus-specific antibody in oral fluid samples has been reported. However, no attempts have been made so far to standardize a technique to detect NA titers in oral fluid samples against PRRSV. The aim of this study was to develop PRRSV NA assay to determine NA titers in pig oral fluid samples. At first, we standardized the PRRSV NA assay using pen-based oral fluid samples collected over a period of three months from PRRSV modified live vaccine (PRRS-MLV) received swineherd, and also using oral fluid and serum samples collected from individual boars vaccinated (PRRS-MLV) or infected with a virulent PRRSV strain. Our results suggested that PRRSV NA titer of greater than eight in oral fluid samples is virus specific, and it could be detected from28days post-vaccination or infection. To validate the assay, we used104pen-based oral fluid and five representative serum samples from each pen of unknown history, and100sera from repeatedly vaccinated sows and oral fluid samples of their respective litters belong to four different swine breeding farms. Our results demonstrated that PRRSV NA titers in oral fluid samples are associated with serum titers, and maternally derived PRRSV specific NA titers could be detected in the litters at the time of weaning.Neither of current vaccines could protect pigs completely against heterologous virus infection. The other objective of this study was to analyze the role of oral fluid samples in assessing the efficacy of candidate vaccines in the laboratory. We inactivated PRRSV-2332by binary ethylenimine (BEI) method, and entrapped UEA (Ulex Europaeus Agglutinin) along with inactivated PRRSV antigens in PLGA (poly lactide-co-glycolide) nanoparticle (NP-VR) as a vaccine. Two doses of NP-VR vaccine was coadministered intranasally with four selected mucosal adjuvants to pigs individually and cross-protective immune response of these vaccine candidates were investigated in a virulent heterologous PRRSV-MN184challenge study. Group8(NP-VR+adjuvant4) was selected as a vaccine candidate for further investigation according to the results of immune correlates at mucosal sites (lung, tracheobronchial lymph node (TBLN), bronchoalveolar lavage (BAL) fluid), serum and oral fluid. Such as,(1) low PRRSV RNA load in mucosal sites, complete clearance of heterologous replicating PRRSV from circulation in serum as early as DPC9, and complete clearance of PRRSV RNA load in oral fluid at DPC15;(2) generation of high levels of PRRSV neutralizing antibody titers in the lungs and BAL fluid, and PRRSV specific NA was detected in oral fluid samples at DPC15;(3) significantly increased PRRSV specific IgG antibody response in the lungs and BAL fluid, and high level of IgA and IgG response in the serum and oral fluid. This is the first time using oral fluid samples along with serum and tissue samples we assessed the efficacy of PRRSV vaccine candidates based on PRRSV load, NA titers, and specific IgA and IgG response. All these results provided an important reference to the vaccine efficacy. In order to take full advantage of oral fluid in monitoring PRRSV immunity, further investigations are required.In conclusion, we have standardized and validated the pig oral fluid-based PRRSV NA assay, which has94.3%specificity and90.5%repeatability. We used pig oral fluid to assess PRRSV vaccine candidates based on PRRSV load, NA titers and specific IgA and IgG response, which is found to be useful and provide an important reference to the vaccine efficacy. Thus, pig oral fluid could be used as a diagnostic sample to monitor PRRSV response.