Title:
Breed and Litter Factors Related to PRRSV Persistence and Clearance
NPB# 99-165
Investigator:
David A. Benfield
Institution:
South Dakota State University
Co-Investigators: Jane Christopher-Hennings, Larry D. Holler, and Eric A. Nelson
Date Received:
4/9/2001
I. Abstract: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has
previously been identified in boar semen and several studies have confirmed
transmission of the virus via this route. Therefore, to prevent transmission of PRRSV
through semen, identifying the duration of PRRSV shedding in semen is important.
However, even when the same PRRSV strain and dose has been given experimentally,
individual boar variability in the duration of viremia and shedding of PRRSV in semen
has been observed. This would suggest there are host factors, which determine the
duration of shedding, and that PRRSV persistence is not strictly related to viral strain
differences. To determine whether there are host genetic factors, particularly litter and
breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine
breeds were used for this study. We also determined whether PRRSV could be
detected for a longer period of time in serum, semen or peripheral blood mononuclear
cells and if PRRSV could still be detected in tissues after these antemortem specimens
were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire and
2 Landrace PRRSV naïve boars were obtained and inoculated intranasally with a wildtype PRRSV isolate (SD-23983). All boars within each breed were from the same litter
and were within 9 days of age between litters. Serum and peripheral blood
mononuclear cells were collected twice weekly from each boar and analyzed for the
presence of PRRSV by virus isolation and the polymerase chain reaction. Serum was
also used to obtain virus neutralization titers and ELISA S/P values. Semen was
collected twice weekly from 7 of 8 boars and analyzed by the polymerase chain
reaction. After a minimum of 2-3 weeks of PRRSV negative specimens, each boar was
euthanized and 21 tissues plus saliva, serum, feces and urine were collected. All
postmortem specimens were evaluated by virus isolation. Specimens, which were
PRRSV negative by virus isolation, were then evaluated by the polymerase chain
reaction. The mean number of days +/- the standard deviation for the duration of
PRRSV shedding in semen was 51 +/- 26.9; 7.5 +/- 4.9; and 28.3 +/- 17.5 for Landrace,
Yorkshire and Hampshire boars, respectively. Due to small sample sizes and large
standard deviations, the differences in duration of PRRSV shedding in semen between
breeds were not considered significant. However, there was a pattern, which suggested
Yorkshire boars were more resistant to PRRSV shedding in semen compared to
Landrace boars, requiring further investigation using a larger numbers of boars.
PRRSV was detected for the longest period of time in semen compared to serum or
peripheral blood mononuclear cells in 4 of 7 boars. Viremia could be detected for the
longest period of time in serum compared to peripheral blood mononuclear cells in 6 of
8 boars. After 2-3 weeks of PRRSV negative serum, semen or peripheral blood
mononuclear cells, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus
isolation, indicating that boars still harbor PRRSV within the tonsil even though
antemortem specimens are PRRSV negative.
II. Introduction: Pig to pig contact, aerosols and semen transmission have all been
implicated in the transmission of PRRSV.13,18,19 However, since the use of artificial
insemination is increasing worldwide, there is concern that PRRSV contaminated
semen may be widely disseminated via this route. Therefore, it is important to identify
boars and boar semen, which may transmit the virus. Previous studies have identified
PRRSV in semen primarily through the use of the polymerase chain reaction (PCR). 16,14,15 This is a useful diagnostic technique since boar semen may be toxic to cells on
which the virus is grown, preventing virus isolation (VI) and swine bioassays are time
consuming and expensive.2,16 Also, detecting boars which have seroconverted to
PRRSV may not always correlate with PRRSV shedding in semen. 2 Using PCR, the
duration of PRRSV shedding in semen has been studied, demonstrating variability in
the duration of viral shedding between boars. Differences in severity of disease and
lung lesions between 3 purebred swine breeds has also been demonstrated, indicating
host factors are important in the persistence and pathogenesis of PRRSV.11
Due to these breed differences and variability among boars in shedding duration, it will
be important to understand not only virus strain effects, but also host factors influencing
the severity of PRRSV infection and persistence.7,9,10,11 Therefore, our first objective
was to determine whether host genetic factors, particularly litter and breed, are related
to the duration or persistence of PRRSV shedding in semen. Also, to detect persistently
infected boars, it has been previously documented that PRRSV remains in semen for a
longer period of time than in serum.2,12,16 Studies have also detected PRRSV in
peripheral blood mononuclear cells (PBMC) (eg. “buffy coat) and there are questions
regarding whether PBMC might be used to detect persistently infected animals (Kolb
JR, Am Assoc. of Swine Practitioners, 1998, Abstract, pgs. 413-418; Rowland RR et al.,
77th Conf. of Research Workers in Animal Diseases, 1996, Abstract 189) 6,17
Subsequently, our second objective was to determine whether PRRSV remains in
serum, semen or PBMC for the longest period of time in the boar. This information
would be useful for “test and removal” programs.8 Finally; our third objective was to
determine whether boars actually “clear” the virus after it can no longer be detected in
these antemortem specimens. If PRRSV could be detected in tissues after serum,
semen and PBMC are PRRSV negative; there could still be a risk of transmission.
These findings would assist in preventing dissemination of PRRSV through boars and
boar semen and ensuring a PRRSV-free semen supply.
III. Objectives: To determine whether host genetic factors, particularly litter and breed
are related to the persistence of PRRSV and whether an in vivo PRRS susceptibility test
might predict which animals will be persistently infected; to determine which biological
specimen (serum, semen, peripheral blood mononuclear cells or tissues) is the best
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sample in detecting a persistent adult PRRSV carrier; and to determine whether swine
actually clear PRRSV after it can no longer be detected on antemortem samples.
IV. Procedures: Animals. Eight purebred boars consisting of 2 Landrace (Boar no.
68-9, 68-10), 3 Yorkshire (Boar no. 71-4, 71-5, 71-9) and 3 Hampshire (Boar no. 291-4,
291-6, 291-7) were utilized for this study. All boars were obtained from the same
source farm and were confirmed PRRSV negative by the IDEXX HerdChek ELISA on
arrival and 2 weeks later. Originally, 9 boars were selected at farrowing, held at the
farm source until they were 6-7 months of age, trained for semen collection and then
shipped to South Dakota State University. One Landrace boar died of undetermined
causes prior to shipping. The Landrace and Hampshire boars were born 9 days prior to
the Yorkshire boars. Experimental Protocol. Boars were acclimated for 2 weeks prior
to PRRSV inoculation. During this time, serum and semen were collected 2 and 4
times, respectively from each boar, except boar 71-9, which was lame throughout the
study and semen was unable to be collected from this boar. After the 2-week
acclimation, all boars were inoculated intranasally with PRRSV isolate SD-23983.
Semen, serum and PBMC were then collected twice weekly. Samples were collected
until all results were PRRSV negative by VI and/or PCR for a period of 2-3 weeks.
Each boar was then euthanized and 21 tissues plus serum, urine, feces and saliva were
obtained. All boars were necropsied after semen, serum or PBMC were PRRSV
negative for a minimum of 2-3 weeks. Gross and histopathologic findings were
evaluated for tonsil, iliac, submandibular, mesenteric and penile lymph nodes, thymus,
spleen, testicle, epididymus, ductus deferens, prostate, bulbourethral gland, penis,
vesicular gland, lung, heart, liver, kidney, brain and small intestine (ileum) from each
boar.
V. Results: The mean number of days +/- the standard deviation for the duration of
PRRSV shedding in semen was 51 +/- 26.9; 7.5 +/- 4.9; and 28.3 +/- 17.5 for Landrace,
Yorkshire and Hampshire boars, respectively (Tables 1, 2, 3). However, due to small
sample sizes and large standard deviations, the differences in PRRSV duration were
not considered significant (P=0.1746). PRRSV was detected for the longest period of
time in semen compared to serum or PBMC in 4 of 7 boars (Tables 1, 2, 3).
PRRSV was detected in serum by VI for the same amount of time as PBMC in 4 of 8
boars and for a longer period of time in serum than PBMC in the remaining 4 of 8 boars,
so there did not appear to be an advantage in using PBMC compared to serum to
detect persistent viremia (Tables 1, 2, 3). There was an advantage in using PCR
compared to VI on either serum or PBMC since PCR detected viremia for a longer
period of time in 6 of 8 boars (serum) and 4 of 8 boars (PBMC). Intermittent viremia
was also observed after 11 DPI using PCR (Tables 1, 2, 3).
All boars acquired antibodies to PRRSV as detected by ELISA between 11 and 14 DPI
(Tables 1, 2, 3). Virus neutralization titers were detected between 25 and 46 DPI from 7
of 8 boars, however, neutralizing antibodies were not detected from1 boar (71-5)
(Tables 1, 2, 3). It is likely this boar would have acquired neutralizing antibodies after
40 DPI if he had not been euthanized at that time. There did not appear to be any
specific correlation between the time ELISA or VN antibodies appeared and the duration
of PRRSV shedding in semen.
After 2-3 weeks of PRRSV negative semen, serum or PBMC, PRRSV was still found
within the tonsil of 3 of 8 boars as detected by VI (Table 4). This would indicate that
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replicating virus can still be present in tissues despite PRRSV negative antemortem
samples, with the potential for transmission to occur. Viral replication within the tonsil
was found only in boars euthanized at 40 (2 boars) or 47 DPI (1 boar). However,
replicating PRRSV was not observed in the remaining boars euthanized at 47 DPI (3
boars), 61 DPI (1 boar) or 88 DPI (1 boar) (Tables 4, 5, 6). Seven of 8 boars were
PRRSV positive in various tissues as detected by PCR, indicating that nucleic acid was
present. However replicating virus was not detectable in these samples by VI. PRRSV
was identified the majority of the time within lymphoid tissues of each boar compared to
reproductive or other tissues. Most boars did not have any significant gross lesions at
necropsy other than a mild, anterior-ventral pneumonia (boars 68-10, 71-4 and 291-6).
VI. Value to the Swine Industry: This study demonstrated variability in the duration of
PRRSV shedding in semen between boars as illustrated by one Yorkshire boar
shedding PRRSV in semen for only 4 DPI while a Landrace boar shed as long as 70
DPI after being given the same PRRSV strain and dose. This suggested there are host
factors, which limit the duration of shedding, or allow for prolonged shedding and that
Yorkshire boars may be more resistant to PRRSV shedding in semen compared to
Landrace boars. However, it did not appear these factors were related to litter since
variability in PRRSV shedding was observed within the same litter and because of this
variability, no statistical differences in shedding duration between breeds was observed.
In the majority of boars, PRRSV was detected in semen for the longest period of time
when compared to serum or PBMC samples. However, 3 of 7 boars were PRRSV
positive in semen for only 4 or 11 DPI, and in these boars, serum or PBMC specimens
were needed to detect PRRSV for the longest period of time. Unfortunately, obtaining
serum, semen or PBMC which are PRRSV negative may not assure that a boar is
PRRSV-free, since tissues, particularly the tonsil may still contain infectious PRRSV.
In conclusion, this study suggested there might be individual boar factors rather than
genetic, breed or litter factors contributing to the duration of PRRSV shedding in semen.
However, a larger number of boars would be necessary to confirm this. In regard to
PRRSV persistence in the boar, sampling of semen, serum or PBMC should not be
solely relied on to detect boars, which might transmit PRRSV. Persistence of PRRSV
appears to be primarily within lymphoid tissues, specifically the tonsil, rather than the
reproductive tract of the boar. Therefore, sampling of tonsillar tissue of boars may be
necessary to identify and study persistent PRRSV carriers. This study also suggested
that boars might eventually eliminate the virus so that PRRSV infection may not
necessarily result in a life-long carrier state in boars.
VII. References:
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Reproductive and Respiratory Syndrome Virus in Boar Semen by PCR. J Clin Micro
33:1730-1734.
2. Christopher-Hennings J, EA Nelson, RJ Hines et al.: 1995b, Persistence of porcine
reproductive and respiratory syndrome virus in serum and semen of adult boars. J Vet
Diagn Invest 7:456-646.
3. Christopher-Hennings J, EA Nelson, DA Benfield et al.: 1996, Detecting porcine
reproductive and respiratory syndrome virus in boar semen. Swine Health Prod 4:3739.
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4. Christopher-Hennings J, EA Nelson, JK Nelson, DA Benfield: 1997a, Effects of a
modified-live virus vaccine against porcine reproductive and respiratory syndrome in
boars. Am J Vet Res 58:40-45.
5. Christopher-Hennings J and EA Nelson: 1997b, PCR analysis for the identification
of porcine reproductive and respiratory syndrome virus in boar semen. In: PCR in
Bioanalysis. Meltzer SJ ed. Humana Press (Methods in Molecular Biology Series). pg.
81-88.
6. Christopher-Hennings J, EA Nelson, JK Nelson, et al.: 1998, Identification of porcine
reproductive and respiratory syndrome virus in semen and tissues from vasectomized
and non-vasectomized boars. Vet Path 35:260-267.
7. Dea S, CA Gagnon, H Mardassi, G Milane: 1996, Antigenic variability among North
American and European strains of porcine reproductive and respiratory syndrome virus
as defined by monoclonal antibodies to the matrix protein. J Clin Micro 34:1488-1493.
8. Dee SA, TW Molitor: 1998, Elimination of porcine reproductive and respiratory
syndrome virus using a test and removal process. Vet Rec 143:474-476.
9. Domingo E, E Baranowski, CM Ruiz-Jarabo, et al.: 1998, Quasispecies structure and
persistence of RNA Viruses. Emerging Infect Dis 4:521-527.
10. Halbur P, P Paul, X Meng, W Hagemoser, et al.: 1996, Comparative pathogenicity
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cesarean-derived, colostrum-deprived pig model. J Vet Diagn Invest 8:11-20.
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Duroc, Hampshire and Meishan pigs to infection with a high virulence strain (VR2385)
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12. Legeay O, S Bounaix, M Denis et al.: 1997, Development of a RT-PCR test
coupled with a microplate colorimetric assay for the detection of a swine Arterivirus
(PRRSV) in boar semen. J Virol Methods 68:65-80.
13. Meredith MJ: 1993, Report of the meeting of the OIE foot and mouth disease and
other epizootics commission. Paris, France pgs. 15-24.
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15. Shin J, E Bautista, Y-B Kang et al.: 1998, Quantitation of porcine reproductive and
respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested
polymerase chain reaction. J Virol Methods 72:67-79.
16. Swenson SL, HT Hill, JJ Zimmerman, et al.: 1994b, Excretion of porcine
reproductive and respiratory syndrome virus in semen after experimentally induced
infection in boars. J Am Vet Med Assoc 204:1943-1948.
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17. Voicu IL, A Silim, M Morin, MASY Elazhary: 1994, Interaction of PRRSV with swine
monocytes. Vet Rec 134:422-423.
18. Wills RW, JJ Zimmerman, KJ Yoon, et al.: 1997b, Porcine reproductive and
respiratory syndrome virus: routes of excretion. Vet Micro 57:69-81.
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porcine reproductive and respiratory syndrome (PRRS) virus in boar semen. Swine
Health Prod 1:5-9.
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