Abstract
The objective of the present study was to determine the effect of concurrent vaccination of boars with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) on seminal shedding of both genotypes. The boars tolerated well concurrent administration of 2 commercial PRRSV vaccines, and no adverse reactions were observed. No interference in the humoral immune response (measured as the level of anti-PRRSV antibodies) or the cell-mediated immune response (measured as the level of PRRSV-specific interferon-γ-secreting cells) was observed after concurrent administration compared with single administration of the same vaccines. Concurrent vaccination significantly reduced the load of type 1 and type 2 PRRSV in blood and semen after singular (type 1 or type 2) and dual (type 1 and type 2) PRRSV challenge, and it did not significantly affect the efficacy of each vaccine. The results demonstrate that concurrent vaccination of boars with type 1 and type 2 PRRSV reduces shedding of both genotypes in semen.
Résumé
La présente étude visait à déterminer les effets d’une vaccination simultanée de verrats avec les types 1 et 2 du virus du syndrome reproducteur et respiratoire porcin (VSRRP) sur l’excrétion séminale des deux génotypes. Les verrats ont bien toléré l’administration simultanée des deux vaccins VSRRP commerciaux, et aucune réaction adverse ne fut observée. Aucune interférence dans la réponse immune humorale (mesurée par le titre d’anticorps anti-VSRRP) ou la réponse immunitaire à médiation cellulaire (mesurée par le nombre de cellules secrétant de l’interféron γ spécifique au VSRRP) ne fut observée après l’administration simultanée comparativement à une administration unique des mêmes vaccins. L’administration simultanée a réduit significativement la charge de type 1 et de type 2 du VSRRP dans le sang et la semence après une infection défi avec un seul type (type 1 ou type 2) ou les deux (type 1 et type 2), et n’affectait pas de manière significative l’efficacité de chaque vaccin. Les résultats démontrent que la vaccination simultanée de verrats avec le type 1 et le type 2 du VSRRP diminue l’excrétion des deux génotypes dans la semence.
(Traduit par Docteur Serge Messier)
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded, positive-sense RNA virus that belongs to the genus Arterivirus, family Arteriviridae, within the order Nidovirales, which also includes equine arteritis virus, lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus (1). Two genotypes of PRRSV are prevalent, as shown by genetic analysis: type 1 (European) and type 2 (North American). The 2 genotypes share only 55% to 70% nucleotide identity (2–4). In addition, pathogenic differences between types 1 and 2 PRRSV have been described (5).
This virus has become one of the most important viral pathogens for the global swine industry, resulting in immense economic losses due to reproductive failure in breeding females and respiratory disease in growing pigs (6). It can also infect male reproductive organs (7,8), the manifestations in the boars being loss of libido and alterations in semen quality, including a decrease in sperm motility and an increase in the frequency of morphologic anomalies, including an abnormal acrosome (9). Infected boars have been found to shed PRRSV in semen for as few as 4 d and as many as 92 d after experimental infection (10). In semen the virus is transmissible to sows (11–14). Therefore, freedom of semen from PRRSV is a critical issue for commercial boars because artificial insemination (AI) is widely and routinely used in the global swine industry.
Cross-protection of boars by vaccination against heterogenotypic PRRSV is limited. Vaccination of boars with type 1 PRRSV was unable to reduce seminal shedding of type 2 PRRSV after challenge and vice versa (15,16). Although PRRSV-free semen can really only be guaranteed from a PRRSV-free herd and not from PRRSV-vaccinated herds, the importance of vaccination of boars against PRRSV is to reduce the amount of seminal shedding of PRRSV because the seminal transmissibility of PRRSV is dependent on the viral load (17). Theoretically, vaccination of boars with both type 1 and type 2 PRRSV may be necessary to reduce the seminal shedding of both genotypes efficiently. Hence, the objective of the present study was to determine the effect of concurrent vaccination of boars with type 1 and type 2 PRRSV on seminal shedding of both genotypes.
Materials and methods
Inocula
Type 1 PRRSV (SNUVR090485; pan-European subtype 1) and type 2 PRRSV (SNUVR090851; lineage 1) were used as inocula. The SNUVR090485 virus [GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) no. JN315686] was isolated from lung samples from an aborted fetus and a weaned pig in a 1000-sow herd in southwestern Kyounggi Province (18). The SNUVR090851 virus (GenBank no. JN315685) was isolated from lung samples from different newly weaned pigs and from lymph node samples from an aborted fetus in a 1000-sow herd in Chungcheung Province in 2009 (19).
Experimental design
At 8 mo of age, 45 purebred male Landrace pigs were purchased from a PRRSV-free commercial farm. All boars were negative for PRRSV according to the commercial PRRSV enzyme-linked immunosorbent assay (ELISA) HerdChek PRRS X3 Ab (IDEXX Laboratories, Westbrook, Massachusetts, USA) before delivery and on arrival. All boars were individually housed in separate experimental rooms equipped with air conditioning and high-efficiency particulate air filtration to avoid possible transmission of the pathogen between groups throughout the experiment in the research facility.
Porcilis PRRS (lot D353A07; MSD Animal Health, Summit, New Jersey, USA) was used as the type 1 PRRSV vaccine (Vac1) and Ingelvac PRRS MLV (lot 245-659A; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) was used as the type 2 PRRSV vaccine (Vac2). Sample size was calculated assuming a 90% power (1 – β = 0.90) of detecting a difference at the 5% level of significance (α = 0.05), which was based on expected results for virus load in semen and serum as determined by real-time polymerase chain reaction (RT-PCR) (20). The boars were divided into 9 groups (5 boars per group) by means of the Excel random number generation function (Microsoft Corporation, Redmond, Washington, USA) (Table I).
Table I.
Detection of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time polymerase chain reaction in serum and semen from 5 boars per group
| Group | Virus genotype | Number of boars with positive results in serum/in semen; number of days after challenge | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||||
| 3 | 7 | 10 | 14 | 18/21/25 | 28 | 32 | 35 | 39 | 42 | 46 | 49 | 56 | ||
| Vac1-2/Ch1-2 | Type 1 | 0/0 | 1/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Type 2 | 2/0 | 1/0 | 1/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
| Vac1-2/Ch1 | Type 1 | 0/0 | 2/0 | 0/0 | 1/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Vac1-2/Ch2 | Type 2 | 2/2 | 1/0 | 1/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Vac1/Ch1 | Type 1 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Vac2/Ch2 | Type 2 | 0/0 | 1/0 | 1/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/1 | 0/0 | 0/0 | 0/0 |
| UnVac/Ch1-2 | Type 1 | 4/3 | 5/5 | 5/4 | 5/4 | 4/4 | 2/1 | 2/2 | 2/2 | 0/2 | 1/1 | 0/0 | 0/0 | 0/0 |
| Type 2 | 4/3 | 5/5 | 4/4 | 5/3 | 4/3 | 3/2 | 0/2 | 0/2 | 0/0 | 1/1 | 0/1 | 0/0 | 0/0 | |
| UnVac/Ch1 | Type 1 | 3/2 | 5/5 | 5/4 | 5/4 | 4/3 | 2/2 | 2/0 | 2/2 | 0/2 | 1/0 | 0/0 | 0/1 | 0/0 |
| UnVac/Ch2 | Type 2 | 4/3 | 5/5 | 4/4 | 5/4 | 4/4 | 2/1 | 2/2 | 2/0 | 2/2 | 0/0 | 0/1 | 0/0 | 0/0 |
| UnVac/UnCh | Type 1 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 |
| Type 2 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | 0/0 | |
Vac1-2/Ch1-2 — vaccinated with type 1 and type 2 PRRSV and then challenged with type 1 and type 2 PRRSV; Vac1-2/Ch1 — vaccinated with type 1 and type 2 PRRSV and then challenged with only type 1 PRRSV; Vac1-2/Ch2 — vaccinated with type 1 and type 2 PRRSV and then challenged with only type 2 PRRSV; Vac1/Ch1 — vaccinated with type 1 PRRSV and then challenged with type 1 PRRSV; Vac2/Ch2 — vaccinated with type 2 PRRSV and then challenged with type 2 PRRSV; UnVac/Ch1-2 — challenged with type 1 and type 2 PRRSV without prior vaccination; UnVac/Ch1 — challenged with type 1 PRRSV without prior vaccination; UnVac/Ch2 — challenged with type 2 PRRSV without prior vaccination; and UnVac/UnCh — neither vaccinated nor challenged.
The boars in the Vac1-2/Ch1-2, Vac1-2/Ch1, and Vac1-2/Ch2 groups were vaccinated intramuscularly with the 2 vaccines at the same time, the type 1 vaccine in the left side of the neck and the type 2 vaccine in the right side of the neck, each dose being 2.0 mL. The boars in the Vac1/Ch1 group were vaccinated intramuscularly with the type 1 PRRSV vaccine (left side of the neck, 2.0 mL), and the boars in the Vac2/Ch2 group were vaccinated intramuscularly with type 2 PRRSV vaccine (right side of the neck, 2.0 mL).
Five weeks after vaccination (vaccination being at −35 d after challenge), the boars in the Vac1-2/Ch1-2 and UnVac/Ch1-2 groups were inoculated intranasally with 1 mL of tissue culture fluid containing 105 50% tissue culture infective doses (TCID50)/mL of type 1 PRRSV (SNUVR090485, 2nd passage in alveolar macrophages) and 1 mL of tissue culture fluid containing 105 TCID50/mL of type 2 PRRSV (SNUVR090851, 2nd passage in MARC-145 cells). The boars in the Vac1-2/Ch1, Vac1/Ch1, and UnVac/Ch1 groups were inoculated intranasally with 1 mL of tissue culture fluid containing 105 TCID50/mL of type 1 PRRSV (SNUVR090485, 2nd passage in alveolar macrophages). The boars in Vac1-2/Ch2, Vac2/Ch2, and UnVac/Ch2 groups were inoculated intranasally with 1 mL of tissue culture fluid containing 105 TCID50/mL of type 2 PRRSV (SNUVR090851, 2nd passage in MARC-145 cells). The boars in the UnVac/UnCh group were used as negative controls and were not exposed to vaccine or virus. After PRRSV inoculation the physical condition of the boars was monitored daily and the rectal temperature taken daily. Blood samples were collected from each pig by jugular venipuncture at −35, −14, 0, 7, 14, 21, 28, 35, 42, 49, and 56 d after challenge and tested with the HerdChek PRRS X3 Ab ELISA. All of the methods had been approved by the Seoul National University Institutional Animal Care and Use and Ethics Committee.
Enzyme-linked immunospot (ELISPOT) assay
The numbers of PRRSV-specific interferon-γ-secreting cells (IFN-γ-SCs) were determined in peripheral blood mononuclear cells (PBMCs) as previously described (21,22) with some modifications. Briefly, 100 μL containing 5 × 105 PBMCs in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, SelectScience, Bath, England), nonessential amino acids (1 mM; Invitrogen, Carlsbad, California, USA), sodium pyruvate (1 mM), 2-mercaptoethanol (5 mM), penicillin (50 000 IU/L), and streptomycin (50 mg/L) was seeded onto plates that had been coated with IFN-γ monoclonal antibody against porcine antigen (10 μg/mL; MABTECH, Mariemont, Ohio, USA) and incubated overnight at 4°C. The cells were stimulated with challenge type 1 or type 2 PRRSV (live virus) in RPMI 1640 medium for 20 h at 3°C in 5% humidified CO2; the linear response was tested at multiplicities of infection (ratios of the number of virions added per cell during infection to the number of cells) between 0.01 and 0.1. Phytohemagglutinin (10 μg/mL; Roche Diagnostics GmbH, Mannheim, Germany) and culture medium were used as positive and negative controls, respectively. The wells were washed 5 times with phosphate-buffered saline (PBS; 0.01M, pH 7.4, 200 μL per well). Thereafter, the procedure was conducted with the commercial ELISPOT Assay Kit (MABTECH) according to the manufacturer’s instructions. The spots on the membranes were read by an automated ELISPOT Reader (AID ELISPOT Reader, AID GmbH, Strassberg, Germany). The results were expressed as the numbers of IFN-γ-SCs per million PBMCs.
Quantification of PRRSV RNA
RNA was extracted, as previously described (23), from raw semen and blood collected −35, −21, −7, 0, 3, 7, 10, 14, 18, 21, 25, 28, 32, 35, 39, 42, 46, 49, and 56 d before challenge from all the boars. Real-time PCR for the vaccine and challenge type 1 and type 2 PRRSV was used to quantify PRRSV genomic cDNA copy numbers with the RNA extracted from semen and serum as previously described (22,23).
Virus isolation
With the use of alveolar macrophages for type 1 PRRSV and MARC-145 cells for type 2 PRRSV, PRRSV was isolated, as previously described (24,25), with slight modification, from raw semen collected at −35, −7, 0, 7, 14, 28, 42, and 56 d before challenge. Dilution of semen (26), an extensive washing method (27), and increased blind passage were used to reduce the cytotoxicity of the semen and increase the sensitivity of virus isolation. Briefly, 1 mL of boar semen was centrifuged for 20 min at 625 × g. The cell fraction was resuspended in Eagle’s Minimum Essential Medium (EMEM) containing 5% fetal calf serum (FCS) and 2% antibiotics, then diluted 10 and 100 times in EMEM containing 5% FCS and 2% antibiotics. Next, 106 cells in RPMI medium were seeded in microplate wells (6-well cell culture plates) and incubated for 1 h with 480 μL of semen diluted 10 and 100 times. The inoculum was then removed and the microplates were washed 3 times with PBS before fresh medium was added and the incubation continued for 5 d. The microplates were then frozen at −70°C. After thawing, 200 μL of the supernatant was blindly passed to fresh cell monolayers. Incubation and blind passage were done 3 times. At each passage the cells were screened for the presence of PRRSV antigens by immunoperoxidase monolayer assay (IPMA) with SR-30 monoclonal antibodies (Rural Technologies, Brookings, South Dakota, USA) against the nucleocapsid protein of PRRSV. Sequencing was done on the purified reverse transcription-PCR products of amplified open reading frame 5 (28).
Statistical analysis
Continuous data (for PRRSV RNA, PRRSV serologic findings, and PRRSV-specific IFN-γ-SCs) were analyzed with 1-way analysis of variance (ANOVA) for each time point separately. If the ANOVA showed a significant effect, Tukey’s multiple-comparison test was done at each time point. Pearson’s correlation coefficient was used to assess the relationship of PRRSV RNA load between blood and semen. A P-value of less than 0.05 was considered significant.
Results
The boars in the 3 unvaccinated, challenged groups exhibited a slightly increased rectal temperature (39.5°C to 39.8°C) from 3 to 7 d after challenge. The boars in the 5 vaccinated, challenged groups and in the unvaccinated, unchallenged group were clinically normal in health and rectal temperature (38.2°C to 39.4°C) throughout the experiment (Figure 1).
Figure 1.
Mean rectal temperatures in the 9 groups of boars: those vaccinated with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) and then challenged with type 1 and type 2 PRRSV [Vac1-2/Ch1-2 (
)], challenged with only type 1 PRRSV [Vac1-2/Ch1 (
)], or challenged with only type 2 PRRSV [Vac1-2/Ch2 (
)]; those vaccinated with type 1 PRRSV and then challenged with type 1 PRRSV [Vac1/Ch1 (
)]; those vaccinated with type 2 PRRSV and then challenged with type 2 PRRSV [Vac2/Ch2 (
)]; those challenged with type 1 and type 2 PRRSV without prior vaccination [UnVac/Ch1-2 (
)]; those challenged with type 1 PRRSV without prior vaccination [UnVac/Ch1 (♦)]; those challenged with type 2 PRRSV without prior vaccination [UnVac/Ch2 (
)]; and those neither vaccinated nor challenged [UnVac/UnCh (⋄)]. Variation is expressed as the standard deviation.
Anti-PRRSV antibodies were detected 2 wk after vaccination (−21 d after challenge) in the serum samples from the 5 vaccinated, challenged groups. In the unvaccinated, challenged boars no anti-PRRSV antibodies were detected in the serum samples until challenge (0 d after challenge), and thereafter these antibodies were detected. Regardless of the commercial PRRSV vaccines used, the vaccinated, challenged boars exhibited significantly higher (P < 0.05) anti-PRRSV antibody values than the unvaccinated, challenged boars −21 to 35 d after challenge (Figure 2). There were no significant differences in anti-PRRSV antibody values between the single-vaccinated (Vac1/Ch1 and Vac2/Ch2) and dual-vaccinated (Vac1-2/Ch1-2, Vac1-2/Ch1, and Vac1-2/Ch2) boars before or after challenge. As expected, no anti-PRRSV antibodies were detected in the serum of the negative-control (UnVac/UnCh) boars throughout the experiment.
Figure 2.
Mean values for the anti-PRRSV IgG antibodies in the serum samples from the 8 challenged groups; S/P — sample/positive. Variation as in Figure 1. The asterisks indicate a significant difference at a P-value of less than 0.05.
After challenge with type 1 PRRSV the mean frequencies of type 1 PRRSV-specific IFN-γ-SCs in the Vac1-2/Ch1-2, Vac1-2/Ch1, and Vac1/Ch1 groups remained at basal levels (< 20 cells/106 PBMCs) until −21 d after challenge, when they reached an average of 47.8 ± 9.15 cells/106 PBMCs; the mean frequencies in the same 3 groups then decreased to an average of 38.8 ± 10.7 cells/106 PBMCs at 0 d after challenge and were significantly higher (P < 0.05) than those in the UnVac/Ch1-2 and UnVac/Ch1 groups at 7, 14, and 21 d after challenge (Figure 3A). There were no significant differences in the frequencies between the single-vaccinated (Vac1/Ch1) and dual-vaccinated (Vac1-2/Ch1-2 and Vac1-2/Ch1) boars before or after challenge. The mean frequencies remained at basal levels in the unvaccinated, unchallenged group throughout the experiment.
Figure 3.
Mean frequencies of type 1 PRRSV (A) — and type 2 PRRSV (B) — specific interferon-γ-secreting cells (IFN-γ-SCs) per 106 peripheral blood mononuclear cells (PBMCs) from the 8 challenged groups. Variation and asterisks as in Figures 1 and 2.
After challenge with type 2 PRRSV the mean frequencies of type 2 PRRSV-specific IFN-γ-SCs in the Vac1-2/Ch1-2, Vac1-2/Ch2, and Vac2/Ch2 groups remained at basal levels until −21 d after challenge, when they reached an average of 49 ± 12.9 cells/106 PBMCs; the mean frequencies in the same 3 groups then decreased to an average of 27.2 ± 7 cells/106 PBMCs at 0 d after challenge and were significantly higher (P < 0.05) than those in the UnVac/Ch1-2 and UnVac/Ch2 groups at 7, 14, and 21 d after challenge (Figure 3B). There were no significant differences in the frequencies between the single-vaccinated (Vac2/Ch2) and dual-vaccinated (Vac1-2/Ch1-2 and Vac1-2/Ch2) boars before or after challenge. The mean frequencies remained at basal levels in the unvaccinated, unchallenged group throughout the experiment.
Genomic copies of the vaccine and challenge type 1 and type 2 viruses were not detected in the serum from any boar at −35 d after challenge, but, regardless of the vaccine used, genomic copies of the vaccine strains were detected in the serum in all 5 vaccinated groups at −21 d after challenge (14 d after vaccination). Thereafter, no vaccine virus was detected in the serum from the boars in these groups.
Genomic copies of type 1 PRRSV were detected in the serum of the boars challenged with type 1 PRRSV. The boars in groups Vac1-2/Ch1-2, Vac1-2/Ch1, and Vac1/Ch1 had significantly lower numbers (P < 0.05) of genomic copies of type 1 PRRSV in their serum compared with the boars in groups UnVac/Ch1-2 and UnVac/Ch1 at 7 to 21 d after challenge (Figure 4A). Before as well as after challenge there were no significant differences in numbers of genomic copies of type 1 PRRSV between the boars vaccinated with a single strain (Vac1/Ch1) and those vaccinated with the 2 strains (Vac1-2/Ch1-2 and Vac1-2/Ch1).
Figure 4.
Mean numbers of genomic copies of type 1 (A) and type 2 (B) PRRSV RNA in serum from the 8 challenged groups. Variation and asterisks as in Figures 1 and 2.
Genomic copies of type 2 PRRSV were detected in the serum of the boars challenged with type 2 PRRSV. The boars in groups Vac1-2/Ch1-2, Vac1-2/Ch2, and Vac2/Ch2 had significantly lower numbers (P < 0.05) of genomic copies of type 2 PRRSV in their serum compared with the boars in groups UnVac/Ch1-2 and UnVac/Ch2 at 7 to 21 d after challenge (Figure 4B). Before as well as after challenge there were no significant differences in numbers of genomic copies of type 2 PRRSV between the boars vaccinated with a single strain (Vac2/Ch2) and those vaccinated with the 2 strains (Vac1-2/Ch1-2 and Vac1-2/Ch2).
The prevalence of viremia in the boars is summarized in Table I. No type 1 PRRSV was isolated from the serum of any boar challenged with type 2 PRRSV and vice versa. No type 1 or type 2 PRRSV RNA was detected in the blood of the negative-control (UnVac/UnCh) pigs throughout the experiment.
Genomic copies of the vaccine and challenge viruses were not detected in the seminal samples from any boar at −35 d after challenge, but, regardless of the vaccine used, genomic copies of the vaccine strains were detected in the semen in all 5 vaccinated groups at −21 d after challenge (14 d after vaccination). Thereafter, no vaccine virus was detected in the semen from the boars in these groups.
Genomic copies of type 1 PRRSV were detected in the semen of the boars challenged with type 1 PRRSV. The boars in groups Vac1-2/Ch1-2, Vac1-2/Ch1, and Vac1/Ch1 had significantly lower numbers (P < 0.05) of genomic copies of type 1 PRRSV in their semen compared with the boars in groups UnVac/Ch1-2 and UnVac/Ch1 at 7 and 10 d after challenge (Figure 5A). Before as well as after challenge there were no significant differences in numbers of genomic copies of type 1 PRRSV between the boars vaccinated with a single strain (Vac1/Ch1) and those vaccinated with the 2 strains (Vac1-2/Ch1-2 and Vac1-2/Ch1).
Figure 5.
Mean numbers of genomic copies of type 1 (A) and type 2 (B) PRRSV RNA in semen from the 8 challenged groups. Variation and asterisks as in Figures 1 and 2.
Genomic copies of type 2 PRRSV were detected in the semen of the boars challenged with type 2 PRRSV. The boars in groups Vac1-2/Ch1-2, Vac1-2/Ch2, and Vac2/Ch2 had significantly lower numbers (P < 0.05) of genomic copies of type 2 PRRSV in their semen compared with the boars in groups UnVac/Ch1-2 and UnVac/Ch2 at 7 to 14 d after challenge (Figure 5B). Before as well as after challenge there were no significant differences in numbers of genomic copies of type 2 PRRSV between the boars vaccinated with a single strain (Vac2/Ch2) and those vaccinated with the 2 strains (Vac1-2/Ch1-2 and Vac1-2/Ch2).
The prevalence of seminal shedding is summarized in Table I. No type 1 PRRSV was isolated from the semen of any boar challenged with type 2 PRRSV and vice versa. No type 1 or type 2 PRRSV RNA was detected in the semen of the negative-control pigs throughout the experiment.
The prevalence of isolation of type 1 and type 2 PRRSV from the semen of the 9 groups is presented in Table II. All PRRSV isolated from semen was confirmed to be the same virus as in the challenge stock by sequence analysis. No type 1 PRRSV was isolated from the semen of any boar challenged with type 2 PRRSV and vice versa. No PRRSV was isolated from the semen of the negative-control boars.
Table II.
Isolation of PRRSV from the semen of 5 boars per group
| Group | Virus genotype | Number of boars with positive results; number of days after challenge | |||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| 0 | 7 | 14 | 28 | 42 | 56 | ||
| Vac1-2/Ch1-2 | Type 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Type 2 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Vac1-2/Ch1 | Type 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Vac1-2/Ch2 | Type 2 | 0 | 0 | 0 | 0 | 0 | 0 |
| Vac1/Ch1 | Type 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Vac2/Ch2 | Type 2 | 0 | 0 | 0 | 0 | 0 | 0 |
| UnVac/Ch1-2 | Type 1 | 0 | 5 | 4 | 1 | 1 | 0 |
| Type 2 | 0 | 4 | 4 | 1 | 1 | 0 | |
| UnVac/Ch1 | Type 1 | 0 | 4 | 4 | 1 | 0 | 0 |
| UnVac/Ch2 | Type 2 | 0 | 4 | 4 | 0 | 0 | 0 |
| UnVac/UnCh | Type 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Type 2 | 0 | 0 | 0 | 0 | 0 | 0 | |
Discussion
The results of this study demonstrate that concurrent vaccination of boars with type 1 and type 2 PRRSV is able to reduce shedding of both genotypes in the semen (28). Vaccinated boars shed the 2 vaccine viruses only during the first 21 d after vaccination. These results are similar to those of previous studies (29,30). Seminal shedding of PRRSV plays a major role in the transmissibility of the virus. The transmission of PRRSV via semen to offspring by AI has been reported (31). The impact of semen that is contaminated with PRRSV can be enormous since AI is widely and routinely used in the swine industry. In Korea more than 90% of sows are bred by AI, and more than 80% of swine producers purchase semen from commercial AI centers (C. Chae. personal communication with Korea Pork Producers Association) (http://www.pigtech.co.kr). There are concerns about whether the efficacy of a particular PRRSV vaccine in boars can be affected by another PRRSV vaccine concurrently administered. No interference in the humoral and cell-mediated responses, as measured by the levels of anti-PRRSV antibodies and PRRSV-specific IFN-γ-SCs, was observed in this study after concurrent administration of 2 PRRSV vaccines when compared with single administration of the same vaccines to boars at 8 mo of age. In contrast, a previous study found interference in the induction of type 2 PRRSV-specific IFN-γ-SCs after concurrent administration of 2 PRRSV vaccines when compared with single administration of the same vaccines to 4-week-old pigs (34). We have no clear explanation for this discrepancy, but it may be due to age-dependent immune responses against PRRSV. Adult boars had higher frequencies of induction of IFN-γ-SCs in our current study than did the 4-week-old pigs in the previous study (34). Age-dependent immune responses were observed in another study: mature sows had more abundant IFN-γ-SCs than did 3-week-old piglets or finisher pigs aged 16 to 20 wk after experimental PRRSV infection (33). These results suggest that boars have a better acquired cellular immune response compared with piglets.
The importance of PRRSV vaccine is in reducing the amount of seminal shedding of PRRSV because the seminal transmissibility of PRRSV is dependent upon the viral load (17). Reduction of viral shedding in semen may be related to the cell-mediated immune responses induced by the vaccine. Despite seemingly contradictory results (34), reduction of seminal virus shedding coincided with the appearance of PRRSV-specific IFN-γ-SCs in the present study. Therefore, we believe that induction of PRRSV-specific IFN-γ-SCs by the PRRSV vaccine is one of the main factors leading to reduction of seminal virus shedding in infected boars.
To our knowledge, this is the first study that has evaluated the concurrent vaccination of boars with both genotypes of PRRSV. No interference in the efficacy of either vaccine due to concurrent administration was observed by immunologic and virologic analyses. The vaccines were administered at separate anatomic sites rather than at a single site to avoid any interference between the 2 vaccine viruses. The boars tolerated the vaccines well, and no adverse reactions were observed with concurrent administration.
Using semen from PRRSV-negative boar studs is the primary step in preventing the introduction of new strains into pig herds. Because PRRSV shedding in semen is intermittent and difficult to detect, semen testing is not totally satisfactory for monitoring. Vaccination of boars with both type 1 and type 2 PRRSV is an alternative method of reducing the shedding of PRRSV in semen when previously negative boars are unexpectedly infected with PRRSV in PRRSV-positive herds. Nevertheless, although boar vaccination is acceptable in positive herds, it is never an option in negative herds and will not guarantee PRRSV-free semen. Seminal shedding of PRRSV was evaluated in the vaccinated, challenged boars until day 56 in the present study. However, the virus can be detected until day 96 after challenge (10). Therefore, the results of this study cannot be extrapolated to the long-term, and further study is needed to evaluate the seminal shedding of PRRSV in vaccinated, challenged boars beyond day 56.
Acknowledgments
This research was supported by contract research funds of the Research Institute for Veterinary Science, College of Veterinary Medicine, and by the BK 21 Plus Program from College of Veterinary Medicine, Seoul National University for Creative Veterinary Science Research.
References
- 1.Snijder EJ, Kikkert M, Fang Y. Arterivirus molecular biology and pathogenesis. J Gen Virol. 2013;94:2141–2163. doi: 10.1099/vir.0.056341-0. [DOI] [PubMed] [Google Scholar]
- 2.Allende R, Lewis TL, Lu Z, et al. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J Gen Virol. 1999;80:307–315. doi: 10.1099/0022-1317-80-2-307. [DOI] [PubMed] [Google Scholar]
- 3.Nelsen CJ, Murtaugh MP, Faaberg KS. Porcine reproductive and respiratory syndrome virus comparison: Divergent evolution on two continents. J Virol. 1999;73:270–280. doi: 10.1128/jvi.73.1.270-280.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Murtaugh MP, Elam MR, Kakach LT. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch Virol. 1995;140:1451–1460. doi: 10.1007/BF01322671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Han K, Seo HW, Park C, et al. Comparative virulence of reproductive diseases caused by type 1 (European-like) and type 2 (North American-like) porcine reproductive and respiratory syndrome virus in experimentally infected pregnant gilts. J Comp Pathol. 2014;150:297–305. doi: 10.1016/j.jcpa.2013.11.205. [DOI] [PubMed] [Google Scholar]
- 6.Zimmerman JJ, Benfield DA, Dee SA, et al. Porcine reproductive and respiratory syndrome virus (porcine arterivirus) In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, editors. Diseases of Swine. 10th ed. Ames, Iowa: Wiley–Blackwell; 2012. pp. 461–486. [Google Scholar]
- 7.Han K, Seo HW, Park C, Oh Y, Kang I, Chae C. Comparative pathogenesis of type 1 (European genotype) and type 2 (North American genotype) porcine reproductive and respiratory syndrome virus in infected boar. Virol J. 2013;10:156. doi: 10.1186/1743-422X-10-156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Han K, Seo HW, Oh Y, et al. Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar. Vet Res Commun. 2013;37:155–162. doi: 10.1007/s11259-013-9558-x. [DOI] [PubMed] [Google Scholar]
- 9.Prieto C, Castro JM. Porcine reproductive and respiratory syndrome virus infection in the boar: A review. Theriogenology. 2005;63:1–16. doi: 10.1016/j.theriogenology.2004.03.018. [DOI] [PubMed] [Google Scholar]
- 10.Christopher-Hennings J, Nelson EA, Hines RJ, et al. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars. J Vet Diagn Invest. 1995;7:456–464. doi: 10.1177/104063879500700406. [DOI] [PubMed] [Google Scholar]
- 11.Prieto C, Suárez P, Simarro I, García C, Martín-Rillo S, Castro JM. Insemination of susceptible and preimmunized gilts with boar semen containing porcine reproductive and respiratory syndrome virus. Theriogenology. 1997;47:647–654. doi: 10.1016/s0093-691x(97)00023-x. [DOI] [PubMed] [Google Scholar]
- 12.Yaeger MJ, Prieve T, Collins J, Christopher-Hennings J, Nelson E, Benfield D. Evidence for the transmission of porcine reproductive and respiratory syndrome (PRRS) virus in boar semen. Swine Health Prod. 1993;1:7–9. [Google Scholar]
- 13.Gradil C, Dubuc C, Eaglesome MD. Porcine reproductive and respiratory syndrome virus: Seminal transmission. Vet Rec. 1996;138:521–522. doi: 10.1136/vr.138.21.521. [DOI] [PubMed] [Google Scholar]
- 14.Swenson SL, Hill HT, Zimmerman JJ, et al. Artificial insemination of gilts with porcine reproductive and respiratory syndrome (PRRS) virus-contaminated semen. Swine Health Prod. 1994;2:19–23. [Google Scholar]
- 15.Han K, Seo HW, Shin JH, et al. Effect of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on European and North American PRRSV shedding in semen from infected boars. Clin Vaccine Immunol. 2011;18:1600–1607. doi: 10.1128/CVI.05213-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Park C, Kim T, Choi K, et al. Two commercial type 1 porcine reproductive and respiratory syndrome virus (PRRSV)-modified live vaccines reduce seminal shedding of type 1 PRRSV but not type 2 PRRSV in infected boars. Transbound Emerg Dis. 2017;64:194–203. doi: 10.1111/tbed.12361. [DOI] [PubMed] [Google Scholar]
- 17.Benfield D, Nelson C, Steffen M, Rowland RRR. Transmission of PRRSV by artificial insemination using extended semen seeded with different concentrations of PRRSV. Proc Am Assoc Swine Vet. 2000:405–408. [Google Scholar]
- 18.Han K, Seo HW, Oh Y, Kang I, Park C, Chae C. Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs. J Comp Pathol. 2012;147:275–284. doi: 10.1016/j.jcpa.2011.12.010. [DOI] [PubMed] [Google Scholar]
- 19.Han K, Seo HW, Oh Y, Kang I, Park C, Chae C. Comparison of the virulence of European and North American genotypes of porcine reproductive and respiratory syndrome virus in experimentally infected pigs. Vet J. 2013;195:313–318. doi: 10.1016/j.tvjl.2012.06.035. [DOI] [PubMed] [Google Scholar]
- 20.Chow SC, Wang H, Shao J. Comparing means. In: Chow S-C, Wang H, Shao J, editors. Sample Size Calculations in Clinical Research. 2nd ed. New York, New York: Chapman & Hall, CRC; 2008. pp. 60–92. [Google Scholar]
- 21.Meier WA, Galeota J, Osorio FA, Husmann RJ, Schnitzlein WM, Zuckermann FA. Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination. Virology. 2003;309:18–31. doi: 10.1016/s0042-6822(03)00009-6. [DOI] [PubMed] [Google Scholar]
- 22.Diaz I, Mateu E. Use of ELISPOT and ELISA to evaluate IFN-gamma, IL-10 and IL-4 responses in conventional pigs. Vet Immunol Immunopathol. 2005;106:107–112. doi: 10.1016/j.vetimm.2005.01.005. [DOI] [PubMed] [Google Scholar]
- 23.Wasilk A, Callahan JD, Christopher-Hennings J, et al. Detection of US, Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR. J Clin Microbiol. 2004;42:4453–4461. doi: 10.1128/JCM.42.10.4453-4461.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Christopher-Hennings J, Nelson EA, Nelson JK, et al. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR. J Clin Microbiol. 1995;33:1730–1734. doi: 10.1128/jcm.33.7.1730-1734.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.van Rijn PA, Wellenberg GJ, Hakze-van der Honing R, Jacobs L, Moonen PL, Feitsma H. Detection of economically important viruses in boar semen by quantitative RealTime PCR technology. J Virol Methods. 2004;120:151–160. doi: 10.1016/j.jviromet.2004.04.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Xia JQ, Yason CV, Kibenge FSB. Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen. Can J Vet Res. 1995;59:102–109. [PMC free article] [PubMed] [Google Scholar]
- 27.Brunner D, Engels M, Schwyzer M, Wyler R. A comparison of three techniques for detecting bovine herpesvirus type 1 (BHV-1) in naturally and experimentally contaminated bovine semen. Zuchthygiene (Berlin) 1988;23:1–9. [Google Scholar]
- 28.Oleksiewicz MB, Botner A, Madsen KG, Storgaard T. Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet Microbiol. 1998;64:7–22. doi: 10.1016/S0378-1135(98)00254-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Christopher-Hennings J, Nelson EA, Nelson JK, Benfield DA. Effects of a modified-live vaccine against porcine reproductive and respiratory syndrome virus in boar. Am J Vet Res. 1997;58:40–45. [PubMed] [Google Scholar]
- 30.Nielsen TL, Nielsen J, Have P, Baekbo P, Hoff-Jørgensen R, Bøtner A. Examination of virus shedding in semen from vaccinated and from previously infected boars after experimental challenge with porcine reproductive and respiratory syndrome virus. Vet Microbiol. 1997;54:101–112. doi: 10.1016/s0378-1135(96)01272-2. [DOI] [PubMed] [Google Scholar]
- 31.Guerin B, Pozzi N. Viruses in boar semen: Detection and clinical as well as epidemiological consequences regarding disease transmission by artificial insemination. Theriogenology. 2005;63:556–572. doi: 10.1016/j.theriogenology.2004.09.030. [DOI] [PubMed] [Google Scholar]
- 32.Park C, Choi K, Jeong J, Kang I, Park SJ, Chae C. Concurrent vaccination of pigs with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) protects against type 1 PRRSV but not against type 2 PRRSV on dually challenged pigs. Res Vet Sci. 2015;103:193–200. doi: 10.1016/j.rvsc.2015.10.011. [DOI] [PubMed] [Google Scholar]
- 33.Klinge KL, Vaughn EM, Roof MB, Bautista EM, Murtaugh MP. Age-dependent resistance to Porcine reproductive and respiratory syndrome virus replication in swine. Virol J. 2009;6:177. doi: 10.1186/1743-422X-6-177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Xiao Z, Batista L, Dee S, Halbur P, Murtaugh MP. The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load. J Virol. 2004;78:5923–5933. doi: 10.1128/JVI.78.11.5923-5933.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]