#07-111

Complete

Date Full Report Received

06/12/2009

Date Abstract Report Received

06/12/2009

Investigation

Institution:
Primary Investigator:

The long-term objective of this research is to provide a “PRRSV-free semen supply for artificial insemination (AI)”. Therefore, a 2-pronged approach to “on farm” methods that might be used to prevent PRRSV from infecting sows and gilts by AI included: 1) evaluating the use of a commercially available unilayer density gradient (NidaCon Intl., Sweden http://www.nidacon.com/) along with a novel insert to “purify” semen from PRRSV infected boars (Figure 1). Semen used in the purification method was obtained from 8 boars experimentally infected with a North American PRRSV strain. These boars demonstrated variability in duration of shedding of PRRSV in semen as seen in previous studies (Figure 2). The density gradient centrifugation method was then performed in a 50 ml tube (per boar ejaculate) whereby the density gradient is layered into the tube and semen is layered on top. The 50 ml tube is centrifuged and the remaining semen cell pellet that results in the bottom of the tube should consist of non-PRRSV infected semen and good quality sperm. The novel insert allows for ease of layering reagents and semen and for allowing a pipet to go through the middle of the tube to obtain the “purified” sperm pellet without contamination of PRRSV in the upper layers of the gradient. In a previous study, we had used a 2-layer gradient with the novel insert. By comparison, the unilayer gradient was easier, more efficient and quicker to use than the 2-layer gradient and completely “purified” 31 of 44 (71%) PRRSV positive semen samples as determined by the polymerase chain reaction assay (PCR). In the remaining 13 of the 44 PRRSV positive samples, the amount of PRRSV nucleic acid (RNA) detected was lowered by approximately 4.4 cycles in the PCR assay indicating a substantial reduction in the amount of RNA detected after the gradient purification (Figure 3). Since PCR detects the nucleic acid of the virus and may not necessarily be indicative of infectious virus in the sample, the semen cell pellets obtained after the gradient purification technique that were not cleared of PRRSV nucleic acid were evaluated by a “swine bioassay” to determine whether these are infectious samples. A swine bioassay is performed by using PRRSV negative piglets (4-6 weeks old) and inoculating them intraperitoneally (IP) with the suspected infectious sample (in this case, semen cell pellets obtained after the gradient purification). The piglets were then tested after injection for the presence of PRRS virus and antibodies. Swine bioassay piglets were inoculated with the cell pellets and found to become infected with PRRSV which indicated that even in samples that had high cycle thresholds (Ct) (low amount of viral RNA) as determined by PCR, infectious virus was present. The 2nd approach as an “on-farm” method to prevent PRRSV infection by AI included evaluating several compounds that could prohibit the growth of PRRSV in a laboratory cell culture assay. The compound(s) that inhibited PRRSV replication in cell culture at low doses could then be tested in the animal and evaluated for possible inhibition of PRRSV in viral contaminated semen samples. This would be similar to mixing antibiotics in semen extenders to inhibit bacterial growth. Five compounds were tested called “cysteine protease inhibitors” which act on the virus to prevent viral replication. One of the 5 compounds (chymostatin) inhibited the growth of PRRSV in cell culture at low doses (62.5 µM). This compound was then mixed with PRRSV infected semen to see whether it would prevent piglets from becoming PRRSV positive using the swine bioassay technique. This method was described previously whereby the PRRSV infected semen was mixed with the antiviral and inoculated IP into PRRSV negative piglets. Piglets were then monitored for 1 week by collecting a blood sample and evaluating whether the piglet became PRRSV “positive”. If a positive status is obtained by PCR or serology testing, then it is known that the antiviral did not prevent PRRSV replication in the pig. In this study, it was determined that in 16 piglets given various doses of the antiviral mixed with PRRSV infected semen, the antiviral did not prevent PRRSV infection in the piglets. However, a minimum antiviral dose was used to prevent any possible toxicity problems and higher doses might be needed. In summary, using a unilayer density gradient centrifugation method to “purify” PRRSV contaminated semen allowed for some “risk reduction” by eliminating PRRSV from 31 of 44 (71%) semen samples tested. In the remaining samples, a substantial reduction in the amount of viral RNA present was also noted. It has been previously demonstrated that the amount of PRRSV in a semen sample may have an effect on whether a gilt or sow becomes infected with PRRSV. Therefore, even though all of the piglets became infected with semen that had a low level of PRRSV RNA as detected by PCR by the “swine bioassay”, there may be more of a barrier (eg. uterine immune defenses) to infection if this semen was purified by the gradient and then inseminated into sows or gilts by AI. The antiviral chymostatin did inhibit PRRSV in the laboratory at low drug levels. However, further testing is needed to scale up the dose from the laboratory to an effective animal dose and evaluate if there is any effect of the drug on sperm quality parameters. Besides further testing of the drug in semen, this antiviral may be useful in preventing PRRSV replication in other swine populations. For further information, please contact Dr. Jane Christopher-Hennings, South Dakota State University, Brookings, SD 57007, 605-688-5171 or Jane.Hennings@sdstate.edu