ORIGINAL RESEARCH

MichaelJ Yaeger, DVM,PhD; Thomas Prieve, DVM;James Collins, DVM,PhD;Jane Christopher-Hennings, DVM,MS;EricNelson, MS;David Benfield, PhD

Summary: Results from an ePidemiologicinvestigationof an outbreak of PRRSin a large farrow-to-finishoperation inSouth Dakota implicated fresh semen as a potential source of infection. Subsequent experimental studies supported the

potential for PRRS virus transmission in semen. Seronegative gilts developed a fever, produced antibodies to the PRRS vi-

rus, and failed to conceive following insemination with semen from boars infected with the PRRS virus. Epidemiologic evi-

dence and results from the biological assays indicate that the PRRSvirus can be transmitted in fresh semen from acutelyinfected boars.

The economic consequences of PRRS infection can bedevastating. Infection may result in the loss of sev-eral hundred dollars per sow, and clinical signs of

reproductive failure may remain in a herd for 1-4 months.l,2Becausethe swine industry currently lacks a vaccine to pre-vent PRRSvirus,it is imperativeto understandthe meansbywhich PRRSviruscan be transmittedto prevententranceofthis disease into a herd.

Increasingly, the swine industry is using biosecurity to pre-vent this and other diseases. From a disease-control stand-

point, semen has been considered a relatively safe source foradding genetic diversity. However, epidemiologic evidencefrom Great Britain has implicated fresh semen as a possiblemodeof transmissionof PRRSvirus.3Equinearteritis virus(EAV),which is closelyrelated to PRRSvirus,can be trans-mitted in stallion semen for up to 2 years.4,5This study de-scribes epidemiologic evidence that implicates semen as apossiblesource of an outbreak of PRRSand investigateswhether the PRRSvirus can be transmittedin the semenofacutely infected boars.

Case study-epidemiologic evidenceA 200- to 225-sow farrow-to-finish operation in South Dakotaexperienced a PRRS outbreak from December 1991 to March1992. The outbreak was characterized by increased abortions,

MJY,TP: Animal Disease Research and Diagnostic Laboratory,Department of Veterinary Science, South Dakota State University,Brookings, South Dakota 57007. JC: Veterinary Diagnostic Lab,University of Minnesota, St. Paul, Minnesota 55108.

stillborn and weakborn pigs, and respiratory, signs (thump-ing) in young pigs.Biosecurity on the premises was good:

replacement animals had not been brought ontothis farm since 1977;the nearest neighboring swine operation wasapproximately 2 miles (3 km) away; andhog barns were> 100yd (90 m) from a road thatwas only a minor transport route for swine.

Fresh boar semen was suspected as a possible source of thevirus because it was the only porcine material brought ontothe premises, and because clinical signs began to appear ap-proximately 2 weeks after the semen (which was obtainedfrom representative boars at a boar stud) was used. We at-tempted to obtain serum from the boar stud to determine theserostatus of PRRSvirus antibodies; unfortunately, personnel

at the boar stud declined to cooperate due to potential legalramifications.

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Nosignificant pathogens were isolated from aborted or still-born pigs submitted from this herd to the South Dakota Ani-mal DiseaseResearchand DiagnosticLaboratory.Feedanalysiswas negative for mycotoxins. PRRSinfection was confirmedby:. positive direct-fluorescent antibody assays for

PRRSantigen in sections of fresh-frozen lung fromyoung pigs with interstitial pneumonia, as previ-

ously described;6 andseroconversion using indirect-fluorescent antibody

(IFA) assay in sows experiencing reproductiveproblems (24 of 29 sows in one farrowing group),as previously described?

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Results from this field investigation led to the initiation ofexperimentaltrials to evaluatethe potentialfor PRRSvirustransmission in semen.

Materials and methods:experimental trialsBoarsand gilts used in experimental trials were housed sepa-rately in isolation facilities. Two PRRS-seronegativeboars(Boar A and Boar B) were trained for semen collection usingthe gloved-hand technique. Semenwas collected weekly andanalyzed for volume,color, concentration, motility, and mor-phology both pre- and post-PRRS-virusinfection. Boars wereinoculated intranasally with 106Fluorescent FocusUnits/mLof the PRRSvirus (ATCC VR-2332).8-10Each boar received 2 mLof the inoculum, 1mLin each nostril. Boarswere inoculatedwith the PRRSvirus 6 days prior to the anticipated estrus oftwo PRRS-seronegativegilts (Gilt A and Gilt B). Boars weremonitored twice daily for clinical signs and assessedfor se-roconversion weekly. Semen from both boars was collectedon the day of estrus detection and on day 6 postinoculation(PI). On the day of collection, semen was mixed and 60 mLwas inseminated into each gilt. The remaining semen fromPRRSvirus-infected boars was frozen for 24 hours at -70°C,thawed, and centrifuged at 1500gat 4°Cfor 20 min. The su-pernatant was prepared and added to cultures of CL2621cellsfor virus isolation.9This procedure was repeated the follow-ing day. Gilts were assessed twice daily for clinical signs ofPRRSinfection and weekly for seroconversion using IFA andimmunoblot techniques,?,8(Immunoblot procedures detect

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three proteins[15,19,24 kDa],which are identifiedin PRRSvirus-infected cells but not in mock-infected cells.)ll

Postmortem examination was performed on Boar A at 5 andBoar B at 15 weeks PI, while both gilts were necropsied 7weeks after insemination. Tissuesevaluated on grossand his-tologicexamination included:brain, heart, lung, liver, kidney,spleen, adrenal, and mesenteric lymph nodes in all animals,as well as the reproductive tracts of the boars (testicles, epi-didymis, penis, prostate, vesicular and bulbourethral glands,and ductus deferens) and of the gilts (ovaries,oviduct, uterus,and cervix).

Results

BoarsOn day 3 PI,Boar A developed a fever and became anorecticand depressed. Clinical signs and fever lasted for approxi-mately 24 hours. Clinical signs were not observed in Boar B.Bothboarsseroconvertedto the PRRSvirus (Table1).

Semenvolume prior to PRRSvirus infection was 132.5mL(5.1,SD)per ejaculate with an average spermatid count of 5.3x 108spermatids/mL. The volume of the semen collected a1\terin-oculation averaged 104.5mL(4.5, SD) mL with an averageconcentration of 5.85x 108spermatids/mL. Semenvolume wassignificantly decreased PI;however, concentration, color,pro-gressivemotility, and spermatid morphologyremained withinnormal limits. Spermogram values returned to pre-infectionreference ranges within 14 days PI. The PRRSvirus was notisolated from the fresh semen of either boar used for insemi-nation or from fresh semen collected at weekly intervals for15weeks PI.

GiltsAfter insemination with fresh semen from the PRRSvirus-

inoculated boars, both gilts became depressed, anorectic, andfebrile.Weobservedclinical signsin Gilt Astarting day 3 post-insemination, which lasted 24 hours and in Gilt B starting onday 4 post-insemination, which lasted 5 days. Both gilts sub-sequently seroconvertedto PRRS-positivestatus (Table 1).Bothgilts returned to estrus after 21 days, and neither gilt wasfound pregnant at necropsy (7 weeks after insemination).

Neither gross nor histologic lesions were identified in themajor organ systems, including the reproductive systems, ofany of the animals.

Discussion

Results from an epidemiologicinvestigation of this outbreakof PRRSin South Dakota implicated fresh boar semen as asource of infection. Data from our initial studies to evaluatePRRSvirus transmission in semen suggest that the virus canbe transmitted in the semen of acutely infected boars. Recentreports describe similar findings. Investigators at Iowa State

Swine Health and Production - September and October, 1993

Universityinjectedsemen from PRRSvirus-infected boars intothe peritoneal cavity of 4- to 6-week-old pigs. These pigs dem-onstrated seroconversion to the PRRSvirus following inocu-lation with semen collected from boars on days 3, 5, 7, and 9PJ.12Both studies provide evidence that the PRRS virus is

present in semen, while our study also demonstrates the po-tential for semen transmission under field conditions.

Results presented here suggest that PRRSvirus may be trans-mitted in semen. The cytotoxicity of semen to cultured cellsmakes virus isolation difficult and IFAand Western blot tests

have failed to consistently identify the virus in semen. Ef-forts are currently underway to develop a laboratory methodcapableof identifyingthe PRRSvirus in semento ascertainthe duration of virus shedding in the semen of PRRS-infectedboars.Preliminary information from an ongoingstudy at IowaState University indicates that PRRSvirus may be present inthe semen of infected boars for up to 21 days PIl3It is im-portant that producers and practitioners be made aware thatepidemiologic evidence and results from biological studiesinvolvinga limitednumberof animalsindicatethat the PRRSvirus can be transmitted in the semen of acutely infectedboars. Studies to define how long PRRSvirus may be trans-missible in semen are still in progress.

AcknowledgmentsResearch was supported by a grant from the South DakotaPork Producers Council and by USDAHatch Project numberSDO0272-H.

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