Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Jeffery J. Zimmerman
Richard B. Evans
Locke A. Karriker
This experiment was designed as a longitudinal study in which pigs were followed for up to 202 days post inoculation (DPI). On day 0, 109 3-week-old pigs were intramuscularly inoculated with PRRSV strain VR-2332. Negative control pigs (n = 56) were sham inoculated with MEM by the intramuscular route. Thereafter, at approximately 2-week intervals, samples were collected from all animals and a subset of randomly selected animals was euthanized and tissues collected. The presence and amount of virus was assessed using qRT-PCR, standard virus isolation, and bioassay. Detection of PRRSV in serum by qRT-PCR showed that most pigs cleared the viremia by 42 DPI, but some pigs continued to test positive up to 154 DPI. Lymphoid tissue was qRT-PCR positive through 202 DPI in one or more pigs at each sampling point. Infectious virus was recovered from serum and lymphoid tissue by virus isolation on MARC-145 cell culture in a few pigs up to 28 DPI. Swine bioassays based on lymphoid tissue homogenate showed that infectious virus was present in these tissues up to 175 DPI. These results suggest that infectious virus is able to persist in populations for a longer period of time than previously thought. RT-PCR was the most sensitive assay for detecting PRRSV, but the discrepancy between PCR and bioassay results indicated that PCR is detecting non-infectious virus.;A subset of 89 PRRSV-inoculated pigs (donor pigs) and 46 negative control pigs were selected to estimate the risk of PRRSV transmission via ingestion of muscle. Beginning on DPI 28, serum, lymphoid tissues, and muscle ( M. longissimus dorsi) samples were collected from euthanized pigs. A total of 7 of 89 (7.7%) serum samples, 62 of 89 (69.6%) lymphoid tissues samples, and 13 of 89 (14.6%) muscle samples were positive by qRT-PCR. Swine transmissibility studies were performed by feeding thirteen 3-week-old PRRSV-naive pigs (recipient pigs) qRT-PCR-positive muscle, and monitored by qRT-PCR for evidence of PRRSV viremia. No transmission of PRRSV to recipient pigs via consumption of muscle samples was observed.;To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n = 10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post inoculation (DPI). Negative control pigs (n = 10) were IM inoculated with minimum essential medium (MEM). At ∼2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (Elispot), PRRSV viremia (qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories, Inc.). All pigs were viremic by 7 days post inoculation (DPI), with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC = 0.97), the N ELISA (AUC = 0.96), and the M 3' ELISA (AUC = 0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It may be concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA are the good predictors of prior exposure to PRRSV, but provide little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes appears to be a poor prognosticator of PRRSV infection status.;In addition, three assays were evaluated for their ability to detect antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) in porcine muscle transudate ("meat juice") samples. Serum samples were assayed at a dilution of 1:40, and muscle transudate samples were assayed at 5 dilutions (1:2, 1:5, 1:10, 1:20, 1:40) using a commercial PRRSV antibody enzyme-linked immunosorbent assay (ELISA). Additionally, muscle transudate samples were tested using an indirect fluorescent antibody test (IFAT) at 5 dilutions (1:2, 1:5, 1:10, 1:20, 1:40). Attempts to assay muscle transudate samples for neutralizing antibodies using a modified fluorescent focus neutralization assay were unsuccessful.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Ramón Miguel Molina Barrios
Molina Barrios, Ramón Miguel, "Porcine reproductive and respiratory syndrome virus: understanding and managing persistent infection" (2008). Retrospective Theses and Dissertations. 15733.