|Royaee, Atabak - ARS BELTSVILLE MD|
Submitted to: Agricultural Experiment Station Publication
Publication Type: Experiment Station
Publication Acceptance Date: December 1, 2003
Publication Date: December 1, 2003
Citation: Lunney, J.K., Royaee, A. 2003. Porcine Reproductive and Respiratory Syndrome (PRRS): Mechanisms of disease and methods for the detection, protection and elimination of the PRRS virus. Agricultural Experiment Station Publication. Technical Abstract: Principal leaders at Illinois AES: Federico A. Zuckermann and Tony L. Goldberg, Department of Veterinary Pathobiology, University of Illinois, Urbana, IL Principal leaders at BARC: Joan K. Lunney and Atabak R. Royaee, APDL, ANRI, BARC, ARS, USDA, Beltsville, Maryland. Cooperating Agencies in the Research reported by the Illinois AES: Department of Veterinary Pathobiology, Illinois Veterinary Diagnostic Laboratory at Urbana and Illinois AES. Collaborating agencies and principal leaders outside Illinois: Dr. Fernando A. Osorio, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln (UNL) PROGRESS OF THE WORK AND PRINCIPAL ACCOMPLISHMENTS: Objective 3. Characterize the different components of the immune response during acute and persistent infection and the implications of this response in pathogenesis and diagnosis 1. Characteristics of the Immune Response of Pigs to PRRS virus a. Development of immunity to vaccination (IL AES). The immune response of pigs to infection with wild-type virus or vaccination with a conventional modified live virus (MLV) vaccine against the arterivirus Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is characterized by an initial, weak interferon-gamma (IFN-g)response that increases gradually over a period of months (Meier et al., 2003). However a conflicting report has appeared (Batista et al., 2003, AASP) suggesting that the kinetics of the T-cell mediated IFN-' response to PRRS virus in not as we have described (i.e., gradual). In order to solve this discrepancy we conducted an additional experiment in which a group of 5 naïve pigs was immunized once at 10 weeks of age with the PRRS MLV produced by BI, instead of twice as we had done in our previous studies, in order to mimic the study done by Batista et al. Animals were maintained in isolation to prevent a subsequent exposure to PRRS virus. The kinetics of the IFN-g response was monitored for a period of 9 months. The result of this new study has confirmed that, as we showed previously, a weak IFN-g response to PRRS virus is initially detectable within a few weeks after vaccination by ELISPOT. Although, similar to the report by Batista, the response appeared to wane at 10 weeks after vaccination, the frequency of IFN-g-secreting cells (SC) then rebounded and, with fluctuation, the IFN-g SC increased gradually in intensity after a period of months without a booster immunization. The kinetics and fluctuation of the response as measured by the ELISPOT, was confirmed by ELISA determinations of the concentration of IFN-g in cell-free culture supernatants of parallel bulk cultures. These results suggest that the development of the IFN-' response is under complex regulation. The nature of plausible mechanisms that affect and determine the unusual kinetics of this response is being examined. b. Immune gene expression profile in response to PRRS virus (IL AES and BARC). To understand the mechanisms responsible for the regulation of the immune response to PRRS virus, we conducted studies aimed at establishing the expression of profile of genes known to play an important role in the development and regulation of immunity. Levels of several immune-regulatory genes (IFN-a, IFN-g, IL-1a, TNF-a, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-15, IL-18, IL-12 p35/p40, IL-23 and IL-25) were monitored by real time RT-PCR on cDNA prepared from PBMC isolated from pigs vaccinated with PRRS MLV. In one study blood was collected from 9pigs at week 0, 2, 5, 9, 11 and 13 following vaccination with PRRS MLV. Peripheral blood mononuclear cells (PBMC) were prepared, cultured with PRRSV, pelleted and stored in Trizol prior to RNA isolation. Gene expression was calculated relative to week 0. Our data indicated that, following vaccination, it takes up to 5 weeks for pigs to develop a statistically significant (p<0.05) IFN-a protein and mRNA response to PRRSV. No changes in IL-12 p35/p40 gene expression were seen. A vaccination boost at week 7 further increased the frequency of virus-specific IFN-g-SC. A significant up-regulation of TNF-' indicated the involvement of innate immunity to PRRSV starting early in the infection, yet there was limited up-regulation of IFN-a. A second experiment was conducted to determine the effect of the administration of an exogenous source of IFN-' at the time of vaccination with a PRRS MLV on the cellular cytokine protein and mRNA responses. Our results showed that the co-administration of IFN-' resulted in remarkable changes in the cytokine gene expression profile observed at 4 weeks after vaccination. Most notable were the changes in the expression of inflammatory cytokines. Administration of IFN-' resulted in a faster return to homeostasis (pre-vaccination levels) of the expression of mRNA for IL-6, IL-8 and IL-10. Remarkably, the spontaneous production of IL-10 (as determined by ELISA) at 2 weeks post vaccination was significantly lower in the PBMC isolated from pigs that received IFN-' along with the vaccine as compared to that of the pigs immunized with the vaccine alone. As found in previous experiments, the co-administration of IFN' resulted in an enhancement of the IFN-g response to the vaccine. These results indicate that the mechanism of the enhancing effect of IFN-a on the IFN-g response to the PRRS MLV vaccine might be through the induction of changes in the cytokine microenvironment present during the development of the immune response to this virus. Studies will be continued along this line to discern regulatory mechanisms influencing the development of the IFN-g response to PRRS virus. c. Mechanisms of protective immunity against PRRS virus (IL AES and UNL). The nature of the immune mechanism responsible for mediating protective immunity against PRRS virus remains unclear. Our previous studies have suggested a correlation between the intensity of the IFN-g response and protective immunity. On the other hand, studies by Osorio et al. have suggested a correlation between the presence of neutralizing antibodies and protective immunity. In attempt to clarify this issue a field study was conducted to determine whether strong cellular immunity and humoral responses, particularly, high titers of neutralizing antibodies to PRRSV, helped improve the reproductive performance of female swine in a commercial herd endemically infected with PRRS virus. Incoming replacement gilts were exposed two weeks after entry onto the farm to either: 1) a wild-type virus recovered from the study farm or 2) a heterologous commercially available vaccine virus via intramuscular injection. A proportion of gilts exposed to homologous virus were subsequently "boosted" with killed vaccine. Cell-mediated immunity (measured by IFN-g ELISPOT) and humoral immunity (titers of circulating antibodies, neutralizing and non-neutralizing, measured using the FFN and commercial IDEXX ELISA tests, respectively) were measured in all animals at fixed intervals. Statistical analyses revealed that a higher IFN-' response was associated with a lower proportion of stillborn pigs per litter. There was no relationship between humoral or cellular immune response and the recovery of PRRS virus RNA from the tonsil tissue. ELISA antibodies were not predictive of any production variables. Importantly, the use of a heterologous killed PRRS virus vaccine, following exposure to live virus, significantly boosted the cell-mediated immune response. These results indicate that exposure of replacement gilts to live wild-type PRRS virus followed by proper monitoring can be a successful way of inducing cellular immunity in gilts in the field. Furthermore, strong cellular immunity appears to be critical in protecting infected gilts from clinical disease. However, the risks of infecting replacement animals with field virus on the lifetime reproductive performance of the animal, and the overall health of the herd, remain to be determined.