Shedding of porcine circovirus type 2 by boars and the role of PCV-2 in semen transmission

Porcine circovirus type 2 (PCV2) is a small, circular, single-stranded DNA virus that affects pigs worldwide and is economically important to the swine industry. Within the past 3 years, North America has seen a marked increase and wide dissemination of PCV2-associated disease (PCVAD) that has resulted in devastating production losses. The rapid spread of PCVAD raised important questions about transmission of PCV2 amongst swine populations and regions. To date, transmission of PCV2 is not well understood or characterized; however, PCV2 DNA has been detected in serum, multiple tissues, feces, urine, saliva, ocular fluid, nasal discharge, colostrum and semen of infected pigs.^{(1,4,5,11,12,13)} It is believed that the main route of infection may be through the fecal-oral route.⁽¹⁰⁾ However, due to the rapid spread and the extensive use of artificial insemination by most production units, semen transmission has been suggested as a significant route of dissemination of PCV2; yet previous epidemiological investigations have found no association with the use of artificial insemination as a risk factor for PCVAD. ^(2,3,6,9,15) Our group recently conducted a series of experiments with the objectives ⁽¹⁾to determine if there were any differences in semen shedding of different PCV2 strains (PCV2a and PCV2b) in mature boars, ⁽²⁾to determine if PCV2 shed in semen is infectious, and ⁽³⁾to determine if PCR-positive PCV2 semen causes PCV2 infection or reproductive failure when used to inseminate PCV2 negative gilts.

Materials and methods

To assess differences in semen shedding of distinct PCV2 strains, 15 Landrace boars were segregated early weaned from a farm free of porcine reproductive and respiratory virus (PRRSV) and swine influenza virus (SIV) and brought to an isolation facility at Iowa State University. At 4 months of age, all boars were seronegative for anti-PCV2 antibodies and remained negative until experimental inoculation. At seven months of age, boars were randomly allocated to three different groups and trained for semen collection. Three boars served as negative controls, six boars were inoculated with PCV2a (North American like) and six boars were inoculated with PCV2b (European-like) cell propagated PCV2 infectious clone intranasally (3mls) and intramuscularly (2mls). Semen, blood swabs, and serum samples were collected prior to PCV2 inoculation and 20 times after inoculation until termination of the study at DPI 90. Boars from each treatment group were euthanized at 3 different time points during the study to determine the distribution and amount of PCV2 in tissues and associated histological lesions and detection of PCV2 antigen by IHC.⁽¹⁴⁾
A swine bioassay was used to evaluate if the PCV2 shed in semen of experimentally inoculated boars is infectious. Twelve, 4-week-old PCV2 negative pigs were divided into four groups (n = 3) and intraperitoneally inoculated with PCR negative, PCV2a-, PCV2b-PCR-positive raw semen (7mls) or cell culture propagated PCV2 infectious clone (3mls). Serum samples were collected prior to and weekly after inoculation until termination of the study on DPI 49 for the presence of anti-PCV2 antibodies by ELISA7 and for PCV2 DNA.⁽⁸⁾

For evaluation of reproductive failure, nine, 8-month-old PCV2 naive Landrace were randomly divided into 3 groups with three gilts in each group. Gilts were segregated early weaned from the same farm as the experimentally inoculated boars. Group 1 served as negative controls, and group 2 (n = 3) and group 3 (n = 3) were artificially inseminated with PCV2a- or PCV2b-PCR-positive semen from PCV2 inoculated boars. At 5 and 8 weeks post artificial insemination, pregnancy was confirmed by ultrasonography. Termination of the study was 105 days of gestation and all in utero fetuses were extracted and serum was collected for anti-PCV2 antibodies by ELISA7 and for PCV2 DNA.

Results

Results of the experimentally inoculated boars indicate that both the PCV2a and PCV2b groups became viremic, seroconverted (sample to positive ratio > 0.2), and shed low quantities (103 to 105/ml semen) of PCV2 DNA in raw semen as determined by quantitative real-time PCR. Serum viremia (detection of PCV2 DNA in serum) occurred prior to semen shedding under experimental parameters of this study; however, detection of PCV2 DNA in semen occurred post viremia. Peak shedding incidence of PCV2 DNA in semen occurred 2– 4 weeks post inoculation. Length of shedding varied amongst individual boars in both PCV2a and PCV2b groups, and one boar within the PCV2b group was still shedding PCV2 DNA in semen at termination of the study. In contrast to serum viremia, PCV2 DNA was detectable in blood swab samples up to 90 days post inoculation.

All of the 4-week old pigs inoculated intraperitoneally with PCV2 positive semen became viremic and developed anti-PCV2 antibodies during the study.

Eight of nine artificial inseminated gilts became pregnant and carried pregnancy until termination of the study at 105 days of gestation. One gilt failed to become pregnant after artificial insemination on two separate estrus cycles. Weekly serum samples from inseminated gilts were negative for anti-PCV2 antibodies for the duration of the study, and serum samples from all 105-day-gestation fetuses were also seronegative. Fetal serum was negative for PCV2 DNA by quantitative PCR.

In conclusion, PCV2a and PCV2b are shed in low amounts in semen and both strains are infectious in a swine bioassay model. However, the connection between PCV2-positive semen and transmission of PCV2 via artificial insemination resulting in reproductive failure is uncertain. More research is needed to evaluate the role of urogential transmission of PCV2 and its responsibility in the spread of PCVAD and thus clear recommendation are difficult to establish for practitioners and producers in the swine industry.

Acknowledgements

We thank the National Pork Board and the Pork CheckOff for funding of PCV2 research at Iowa State University, Monsanto Choice Genetics for animals used in the studies, and IMV International for supplies

References

^1. Bolin SR, Stoffregen WC, Nayar GP, Hamel AL: 2001, Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J Vet Diagn Invest 13: 185-194.

^2. Cottrell TS, Friendship RM, Dewey CE, et al.: 1999, A study investigating epidemiological risk factors for porcine circovirus type II in Ontario. The Pig Journal 44: 10-17.

^3. Horlen KP, Schneider P, Anderson J, et al.: 2007, A cluster of farms experiencing severe porcine circovirus associated disease: Clinical features and association with the PCV2b gentotype. Journal of Swine Health and Production 15: 270-278.

^4. Krakowka S, Ellis JA, Meehan B, et al.: 2000, Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet Pathol 37: 254-263.

^5. Larochelle R, Bielanski A, Muller P, Magar R: 2000, PCR detection and evidence of shedding of porcine circovirus type 2 in boar semen. J Clin Microbiol 38: 4629-4632.

^6. Lawton DE, Morris RS, King CM: 2004, PMWS in New Zealand part 2: Epidemiological evidence for a novel agent. 18th International Pig Veterinary Society Congress 1: 128-

^7. Nawagitgul P, Harms PA, Morozov I, et al.: 2002, Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV. Clin Diagn Lab Immunol 9: 33-40.

^8. Opriessnig T, Yu S, Gallup JM, et al.: 2003, Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol 40: 521-529.

^9. Rose N, Larour G, Le DG, et al.: 2003, Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds. Prev Vet Med 61: 209-225.

^10. Segalés J, Allan GM, Domingo M: 2005, Porcine circovirus diseases. Anim Hlth Res Rev 6: 119-142.

^11. Shibata I, Okuda Y, Kitajima K, Asai T: 2006, Shedding of porcine circovirus into colostrum of sows. J Vet Med B Infect Dis Vet Public Health 53: 278-280.

^12. Shibata I, Okuda Y, Yazawa S, et al.: 2003, PCR detection of Porcine circovirus type 2 DNA in whole blood, serum, oropharyngeal swab, nasal swab, and feces from experimentally infected pigs and field cases. J Vet Med Sci 65: 405-408.

^13. Sibila M, Calsamiglia M, Segalés J, et al.: 2004, Use of a polymerase chain reaction assay and an ELISA to monitor porcine circovirus type 2 infection in pigs from farms with and without postweaning multisystemic wasting syndrome. Am J Vet Res 65: 88-92.

^14. Sorden SD, Harms PA, Nawagitgul P, et al.: 1999, Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue. J Vet Diagn Invest 11: 528-530.

^15. Wallgren P, Hasslung F, Bergstrom G, et al.: 2004, Postweaning multisystemic wasting syndrome-PMWS. the first year with the disease in Sweden. Vet Q 26: 170-187.

By Darin Madson, DVM; Tanja Opriessnig, DVM, PhD; and others, Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa.

^{Originally published in the American Association of Swine Veterinarians, 39th Annual Meeting Proceedings, March 8 – 11, 2008, San Diego, California, Pages 129 – 130.}
Published in Swine News (October, 2008 - Volume 31, Number 10)
North Carolina State University Cooperative Extension Service
College of Agriculture and Life Sciences - Swine Husbandry

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Shedding of porcine circovirus type 2 by boars and the role of PCV-2 in semen transmission