Document Type

Thesis - University Access Only

Award Date

2008

Degree Name

Master of Science (MS)

Department / School

Biology

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is caused by a single stranded positive sense RNA virus. The virus is widespread and economically devastating to the swine industry. Transmission through insemination of tainted semen, biosccurity breaches, and pig-to-pig contact have led to the need for better understanding of superior diagnostics and elimination techniques. The first objective of this study was to evaluate the feasibility of using PCR to detect PRRSV in pooled samples through the estimation of the sensitivity of RT-PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twentynine boars were inoculated with a low virulent PRRSV isolate. Scrum, blood swab, and semen samples were collected every 2 to 3 days for two weeks. The data showed that scrum was the best sample to detect PRRSV during acute infection, with the blood swab performed in a close second. PRRSV was not detected in semen samples in most cases. Pooling samples in pools of 3 and 5 resulted in a decrease in sensitivity, particularly within the first 3 days after infection. Sensitivity was reduced by 6% and 8% when scrum or blood swab samples were run in pools of 5, respectively. The second objective of this study was to use a previously developed density gradient centrifugation technique to purify boar semen of PRRSV. However, semen was purified by using a "scaled up" method from a 15 ml tube to a 50 ml tube for this study, allowing for a more expedient and useful method. A majority of the samples were purified from PRRSV using this method but further refinements or other methods will be necessary to ensure PRRSV is eliminated consistently from all PRRSV positive semen samples. The final objective of this study was to measure seminal cytokine levels in PRRSV infected (n=8) and non-infected (n=4) boars in an attempt to determine the local immunological factors that influence the duration and viral load of PRRSV shedding in the semen of boars. A sandwich ELISA was performed using swine specific MAbs to detect pro-inflammatory (IL-1 ~' IL-8, IL-6, TNF a (R & D Systems Inc., Mpls., MN)), Th2 (IL-10 (R & D)) and Th 1 (IL-12 (R & D), IFN-y (Biosource, Carlsbad, CA)) cytokines. Due to the observance of inhibitors in boar seminal plasma, a 100 kDa Amicon filter (Millipore, Corp., Billerica, MA ) was used to remove high molecular weight proteins, glycoproteins and lipids from the sample prior to performing the enzyme immunoassay. Both PRRSV infected and non-infected boars had levels of IL-12 ranging from 626-1442 pg/ml, IFN-y levels ranged from 177-597 pg/ml, IL-1 ~ levels ranged from 28-942 pg/ml and IL-8 levels ranged from 127-564 pg/ml. Other cytokine levels appear to be negligible or below the level of detection of the assays. IL-12 levels were significantly higher in the seminal plasma of PRRSV- infected boars when contrasting cytokine seminal plasma levels with the non-infected group (P=0.0424). INF-y seminal cytokine levels were statistically higher in the seminal plasma when contrasting the concentrations with the serum cytokine concentrations in the infected group (P

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome

Boars -- Virus diseases

Format

application/pdf

Number of Pages

123

Publisher

South Dakota State University

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