Document Type

Thesis - University Access Only

Award Date

2006

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

Abstract

Porcine reproductive and respiratory syndrome ( P RRS) continues to be the major viral disease of economic consequence to the swine industry in the United States. Pigs persistently infected with porcine reproductive and respiratory syndrome virus (PRRSV) are the major impediment to the control and elimination of PRRSV within herds. Three experiments are presented in this thesis to address the diagnosis of acute and persistent infections in adult and young animals and to determine the role of viral quasi-species in mediating persistent infections. The purpose of the first study was to; 1) Evaluate the progression of PR RSV infection in non-pregnant breeding age gilts, 2) Determine the effectiveness of routine diagnostic assays for detection of PR RSV {VI and nRT-PCR) and antibodies (IFA, ELISA and SVN) to characterize acute and persistent infections, 3) Evaluate the usefulness of ante-mortem samples (serum and tonsil biopsies) as diagnostic specimens; and 4) Determine the duration of transmission to naive gilts. Two groups of principal gilts (n=5/group) were housed separately and infected with PR RSV strain VR-2332. Principal pigs were bled at 0, 4, 7, 14, 28, 42, 56, 70, 84, 98 and 112 days post-inoculation (dpi) and tonsil biopsies were collected at 7, 14 dpi and every 2 weeks thereafter through 84 dpi. One sentinel pig was introduced into each group of principals at 14 dpi and every 2 weeks thereafter to 84 dpi. Viremia was initially detected at 4 dpi by both VI and nRT-PCR and persisted until 14 dpi by V I and 28 dpi by nRT-PCR. Antibodies to PR RSV were identified in 80% of the gilts by 14 dpi with both IFA and ELISA. ELISA values peaked at 28 to 42 dpi and declined through out the experiment with 40% of the gilts being seropositive at 112 dpi with one principal gilt never becoming ELISA positive. Compared to ELISA values, antibodies detected by I FA remained could be demonstrated through out the experiment and 70% of the principals were still seropositive at 112 dpi. PARS viral RNA was detected in biopsies in 100% of the principals at 7, 14 and 28 dpi and 20, 40, 40 and 20% at 42, 56, 70 and 84 dpi, respectively. Transmission of infectious virus from principal gilts to sentinel gilt occurred at 14, 28 and 42 dpi. Monitoring and detection of the virus in established breeding herds and replacement gilts/sows is crucial for the industry to control confinement/nursery outbreaks. While the combination of serology and RT-PCR provide important tools for the diagnosis of PR RSV, neither test alone is sufficient to identify individual persistently infected pigs. In the second study, the goal was to determine the primary site(s) of PRRSV replication prior to the appearance of viremia. Determining the primary and secondary target tissues of this virus will provide crucial information towards our understanding of the acute and persistent pathogenesis of this disease. The objective of this study was to determine the role of lymphoid and non-lymphoid tissue in acute and persistent infections of PRRSV and what tissues were of greatest value in diagnosis of acute and persistent infections. Seventy-seven, 3- week-old pigs were separated into two experimental groups. Mock-inoculated pigs were in one room and the remaining pigs were separated into three rooms and inoculated with PRRSV strain SD 92-23983. Pigs were euthanized at 6 and 12 hpi, 1, 2, 4, 7 and 14 dpi and every 14 days thereafter to 126 dpi. Blood and 18 different tissues were collected and examined for infectious virus, viral nucleic acid, viral antigen and microscopic lesions. Pigs inoculated with the PRRSV developed mild clinical signs and antibodies were detected by ELISA and neutralization assays. PRRS viral nucleic acid was detected in serum and palatine tonsil as early as 6 hpi. Infectious virus was detected primarily in tonsil and lymph nodes early in infection and in all tissues examined by 7 dpi. Infectious virus and viral antigens were not detected in tissues after 28 dpi, but viral nucleic acid persisted primarily in tonsil and lymph nodes throughout the experimental period. These results suggest that lymphoid tissues are prominent targets in acute PRRS infections and virus becomes persistently established in tonsil or lymph nodes. We also report the first detection of PRRS virus in lingual tonsil. The objective of the third study was to investigate the evolution of ORF5 in pigs from O to 126 dpi. Three-week old pigs were either mock-inoculated or inoculated with a plaque-purified virus, strain SD 92-23983 and direct PCR products from serum, lung, palatine tonsil and inguinal lymph node were cloned and sequenced from pigs euthanized at intervals from 6 hpi to 126 dpi. Of 622 clones, the most common change was at aa 33 (2 1 /45 Gly to Ser; 2/45 Gly to Asn). Only two aa changes V29 to A and D30 to N were seen in immunoepitope A and 3 aa changes in epitope B two L4 1 and the other at N44. N-glycosylation sites were eliminated in four clones at amino acids 32 and 34, one at amino acids 44 and 5 new glycosylation sites resulted from amino acids substitutions at 30, 32, 33 and 34. Results of this study suggest that ORF5 gene continues to evolve from O to 126 dpi in infected pigs, but the significance of these changes as related to persistence could not be established.

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome

Swine -- Virus diseases

Format

application/pdf

Number of Pages

179

Publisher

South Dakota State University

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