William T. Christianson, Chang-Soo Choi, James E. Collins, Thomas W. Molitor, Robert B. Morrison and Han-Soo Joo. ABSTRACT. Two experiments were under ...
Pathogenesis of Porcine Reproductive and Respiratory Syndrome Virus Infection in Mid-gestation Sows and Fetuses William T. Christianson, Chang-Soo Choi, James E. Collins, Thomas W. Molitor, Robert B. Morrison and Han-Soo Joo
ABSTRACT
Two experiments were undertaken to evaluate whether porcine reproductive and respiratory syndrome (PRRS) virus was able to cross the placenta and infect midgestation fetuses following intranasal inoculation of sows and whether PRRS virus directly infected fetuses following in utero inoculation. In experiment 1, eight sows between 45 and 50 days of gestation were intranasally inoculated with PRRS virus (ATCC VR-2332), and four control sows were inoculated with uninfected cell culture lysate. Virus inoculated sows were viremic on postinoculation (PI) days 1, 3, 5, 7 and 9, shed virus in their feces and nasal secretions, and became leukopenic. Sixty-nine of 71 fetuses from principal sows euthanized on PI day 7, 14 or 21 were alive at necropsy and no virus was isolated from any of the fetuses. Two principal sows that farrowed 65 and 67 days PI delivered 25 live piglets and three stillborn fetuses. The PRRS virus was isolated from two live piglets in one litter. In experiment 2, laparotomies were performed on five sows between 40 and 45 days of gestation and fetuses were inoculated in utero with either PRRS virus alone, PRRS virus plus a swine serum containing PRRS antibodies, or uninfected cell culture lysate. Three sows were euthanized on PI day 4 and two sows on PI day 11. Viral replication occurred in fetuses inoculated with virus alone and was enhanced in fetuses inoculated with virus plus antibody. No virus was isolated
from control fetuses. The results indicate that sows and fetuses are susceptible to PRRS virus infection in mid-gestation, that viral replication is enhanced by the addition of serum with PRRS virus antibody, and that the virus does not readily cross the placental barrier during mid-gestation.
provenant d'une meme portee. Pour la deuxieme experience, cinq truies gestantes de 40 a 45 jours ont subi une laparotomie et leurs foetus ont ete inocules in utero soit avec le virus du SRRP seulement, soit avec le virus du SRRP et du serum porcin contenant des anticorps diriges contre ce virus, ou soit avec
lysat de culture cellulaire noninfectee. Trois truies ont ete euthanasiees au jour 4 PI et deux au jour 11 PI. La replication virale s'est produite dans les foetus inocules uniquement avec le virus du SRRP mais elle a ete encore plus importante chez les foetus ayant recu le virus et le serum immun. Le virus n'a pas ete isole des porcelets temoins. Les re'sultats indiquent que les truies et les foetus peuvent etre infectes par le virus du SRRP a la mi-gestation, que la replication virale est augmentee par 1'addition de serum contenant des anticorps diriges contre le virus du SRRP et que le virus traverse difficilement la barriere placentaire durant la mi-gestation. (Traduit par Dre Christiane Girard) un
RESUME
Une experience a ete effectuee evaluer la capacite du virus du syndrome reproducteur et respiratoire porcin (SRRP) inocule par voie intranasale 'a des truies a franchir la barriere placentaire et a infecter des foetus en mi-gestation. La seconde experience visait a verifier si le virus du SRRP pouvait infecter directement des foetus a la suite d'une inoculation in utero. Lors de la premiere experience, huit truies gestantes de 45 a 50 jours ont ete inoculees par voie intranasale avec le virus du SRRP (ATCC VR2332) alors que quatre truies ont recu un lysat de culture cellulaire non-infectee. Les truies infectees etaient viremiques aux jours 1, 3, 5, 7 et 9 post-inoculation (PI), ont INTRODUCTION excrete du virus dans leurs feces et leurs secretions nasales et sont Porcine reproductive and respiradevenues leucopeniques. Soixante- tory syndrome (PRRS) is a newly neuf des 71 foetus provenant des described viral disease of pigs. The truies infectees euthanasiees aux syndrome has been reported in most jours 7, 14 et 21 etaient vivants et le swine producing areas of North virus n'a pas ete reisole de ces America (1-3) and Europe (4-6). porcelets. Deux truies infectees ont Several descriptive names for the dismis-bas de 25 porcelets vivants et de ease have been used in the past trois porcelets mort-nes aux including; swine infertility and jours 65 et 67 PI. Le virus du SRRP respiratory syndrome (7), porcine a t isole de deux porcelets vivants epidemic abortion and respiratory pour
Department of Clinical and Population Sciences (Christianson, Choi, Molitor, Morrison, Joo) and Department of Veterinary Diagnostic Medicine (Collins), College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108. Supported by funds from the Minnesota Agricultural Experiment Station (MN 62-059, MN 65-022) and the National Pork Producers Council. Submitted December 22, 1992.
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Can J Vet Res 1993; 57: 262-268
syndrome (8), blue-eared pig disease (9) and PRRS (10). Participants of the 1ST International Symposium on the disease (May 17-19, 1992, St. Paul, Minnesota) universally agreed to use the name PRRS when referring to this disease. Clinical reports of PRRS have focused primarily on late-term reproductive failure and preweaning mortality losses. Signs reported in sows include anorexia, early farrowings (days 107-112 of gestation), high numbers of stillborn, mummified or weakborn pigs, and blue discoloration of sows ears, udders and vulvas (5,9-12). Mid-gestation abortions or small mummies at farrowing, indications of mid-term fetal infection, are seldom associated with PRRS. Experimental reproduction of the disease has been successful in lateterm pregnant sows (8,13), but the effects of PRRS virus in mid-gestation are unknown. Microscopic lesions in fetuses or placentas after experimental late-gestation infection are uncommon; the mechanism of PRRS reproductive failure is not understood. Two main questions have arisen regarding the apparent late-gestation clinical predilection of PRRS virus. First, does the virus cross the placenta in midgestation following infection of the sow? Second, if virus crosses the placenta during mid-gestation, does it replicate in fetuses? Additionally, are other factors, such as immune competency, associated with viral replication in fetuses? Enhanced viral replication with the addition of PRRS antibodies has been demonstrated in vitro (14). As porcine fetuses do not become immune competent until approximately 70 days of gestation (15), this may be a factor in the pathogenesis of PRRS virus in fetuses. By focusing on key differences associated with gestational age, including placental structure and fetal immune competency, the investigation of mid-gestation infection could lead to a greater understanding of PRRS virus pathogenesis in pregnant sows and fetuses. The study reported here describes infection of PRRS virus in midgestation sows and fetuses. The study is divided into two experiments, the first - intranasal inoculation of sows, the second - in utero inoculation of fetuses.
MATERIALS AND METHODS VIRUS
The PRRS virus used as inoculum was ATCC VR-2332. The virus originated from a Minnesota farm that had an acute outbreak of PRRS, and the virus had previously been used to experimentally reproduce PRRS in late-gestation sows (13). The physical properties of this virus have been reported (16). Virus was propagated in the following manner: Cells (CL2621; Boehringer Ingelheim Animal Health Inc., St. Joseph, Missouri) were seeded in Eagle's minimum essential medium (MEM) supplemented with 8% fetal bovine serum (FBS) and 10 ,ug of gentamicin/mL. Medium from three-day-old monolayers was decanted and virus was absorbed for 1 h at 370C. Maintenance medium (MEM with 2% FBS) was added and the cultures were incubated until obvious cytopathic effects (CPE) were observed. Virus cultures were frozen and thawed three times and virus containing supernatants were collected after centrifugation (4500 x g, 30 min); filtered (0.45 ,um) and stored at -70°C. Virus prepared in this way had a 50% tissue culture infective dose (TCID50) of 1053/mL (17). Control inoculum was prepared with the same procedure using uninfected cell cultures. In experiment 2, PRRS virus (1044 TCID50/mL) was used alone or in combination with serum containing PRRS virus antibody. Anti-PRRS virus serum was obtained from a late term PRRS virus infected stillborn fetus (13); heated (1 h at 58°C) to inactivate virus; and diluted to maximally enhance virus replication (14). The working dilution of fetal serum (10-2) enhanced PRRS virus replication, in vitro - in swine alveolar macrophage cultures but not CL2621 cells - 50 times. ANIMALS AND EXPERIMENTAL DESIGN
Seventeen multiparous Yorkshire x Landrace sows were purchased from a farm free of reproductive diseases including PRRS. Sows were vaccinated twice a year against porcine parvovirus and Leptospira species on the farm of origin. The exact breeding dates of all sows were known; sows
included in the study were between 40 and 50 days pregnant. Before the study began, all sows tested negative for PRRS virus and antibody. Sows in groups of two were housed in an isolation room and were fed 2.75 kg of a 14% gestation ration daily. The experiments followed the guidelines of the "Guide to the Care and Use of Experimental Animals" of the Canadian Council on Animal Care. Experiment I - Eight principal and four control sows between 45 and 50 days gestation were intranasally inoculated with 4 mL of virus or uninfected cell culture lysate, respectively. Clinical signs, rectal temperatures and feed intake were recorded daily. Blood samples were collected into heparinized and serum clot vacuum tubes, by jugular venipuncture, on postinoculation (PI) days 0, 1, 3, 5, 7, 9, 11, 14 and 21. Nasal swabs (Dacron, American Scientific Products, McGaw Park, Illinois) were collected on the same days and were frozen in 1 mL of maintenance MEM. Fecal swabs, collected daily, were also frozen in 1 mL of maintenance MEM. Sows were euthanized by lethal intravenous (IV) injection of pentobarbital sodium (Anthony Products, Arcadia, California) according to the following schedule: two principals on day 7 PI, two principals and two controls on day 14 PI, two principals on day 21 PI, and the remaining two principals and two controls farrowed naturally at term. At necropsy, fetal blood and tissues were collected for virus isolation and serology, and fetal abnormalities and crown-rump lengths (CRL) were recorded. Experiment 2 - Five sows were pre-anesthetized by intramuscular (IM) injection of 1 mg/kg tiletamine/ zolazepam (Aveco, Inc., Fort Dodge, Iowa) and 0.05 mg/kg atropine sulfate (Anthony Products); anesthetized with 2-4 mg/kg IV thiopental (Abbot Labs, Chicago, Illinois); maintained with 2% Isoflurane gas (Anaquest, Madison, Wisconsin); and laparotomies were performed. The fetuses in each uterus were allotted to one of three treatment groups. The fetuses closest to the right ovarian end of the uterus received 0.5 mL of virus inoculum, the fetuses closest to the left ovarian end received 263
the virus-antibody inoculum, and the The SN test was carried out with fetuses in the middle third of the uterus CL2621 cell monolayers. Antibody received uninfected cell culture lysate. titers were expressed as the highest Fetuses of three sows were injected dilution of sera that neutralized CPE IM in the rear quarter and fetuses of five days after inoculation. two sows were inoculated by injection into the amniotic fluid. Sows, after PERIPHERAL BLOOD LEUKOCYTE recovery from anesthesia, were given PARAMETERS Total peripheral blood leukocyte daily IM injections of 100 mg medroxyprogesterone acetate (Upjohn Co., counts of sows in experiment 1 were Kalamazoo, Michigan) to assist in obtained with a Coulter S+IV maintaining pregnancy. Sows were (Hialeah, Florida) counter on hepeuthanized by IV pentobarbital sodium arinized blood collected on PI days 0, injection on PI days 4 (three sows) and 3, 7, 11 and 14. Differential leukocyte 11 (two sows). As in experiment 1, counts were made on smears within fetal blood and tissues were collected 1 h of blood collection. Leukocytes and processed for virus isolation and were further characterized by flow serology. Crown-rump lengths and any cytometry. The procedure for cell staining was adapted from Pescovitz fetal abnormalities were recorded. and others (20). Briefly, red blood VIRUS ISOLATION AND SEROLOGY cells were lysed by treatment with a Virus isolation was attempted using 0.9% NH4CL, 0.1% KHCO3 solution fetal tissues (brain, lung, heart, liver, (pH 7.3). Cells were then washed spleen and kidneys); fetal serum; pla- three times in PBS and adjusted to 2 x centa; sow blood (serum, plasma and 106 cells/mL in a buffer (PBS containunwashed ficoll density separated ing 2% normal pig serum, 0.1% NaN3 leukocyte fractions); sow fecal swabs; and 0. 1% bovine serum albumin). One and sow nasal swabs. After freezing hundred pL of the leukocyte suspenand thawing, tissues were minced, sion was mixed with 100 pL of monodiluted 1:10 in MEM with 2% FCS clonal antibodies (mAb) specific for plus gentamicin (10 ig/mL), homoge- porcine CD4 (ATCC HB147, mAb nized and centrifuged at 1500 x g for 74-12-4), porcine CD8 (ATCC HB 143, 30 min. Blood and swab samples (after mAb 76-2-11), porcine B (ATCC vortexing and removing the swabs) HB 140, mAb 76-7-4), or porcine were also centrifuged. The supernatants monocyte/macrophage (ATCC HB 142, were filtered (0.45 pm) and inoculated mAb 74-22-15) cells. Incubation was onto three-day-old CL2621 cell mono- carried out for 40 min on ice, cells layers. After one hour absorption, were then washed three times in maintenance MEM was added without buffer. A FITC-labeled, goat affinity removing the inoculum, and the cul- purified F(ab'), fragment, antimouse tures were incubated at 35°C for seven immunoglobulin G (H+L) (Cappel days. When CPE was observed, sam- Research Products, Durham, North ples were confirmed PRRS virus posi- Carolina) was used to stain the pritive by indirect fluorescent antibody mary mAb. After 40 min incubation using specific antiserum of rabbit ori- on ice, the cells were washed three gin (16). Infectivity titers were deter- times with buffer before being fixed mined for positive samples. Two pas- in 100 ,uL of PBS with 1% paraformaldehyde. Unstained cells and sages were made on negative samples. Sow and fetal sera were tested for cells stained with only the FITC-goat PRRS antibodies with either an indi- antimouse antibody were included as rect fluorescent antibody (IFA) assay controls. Flow cytometry was carried (18) or a serum neutralization (SN) out with a Coulter EPICS 751. Bit assay (19). The IFA was carried out maps were set on lymphocytes and with PRRS virus infected swine alve- monocytes - the forward and orthogolar macrophage cultures. Antibody onal light scatter signals were used to titers were expressed as the highest discriminate cells and confirm homodilution demonstrating positive, cyto- geneity of the preparations. Five thouplasmic fluorescence. Noninfected sand cells were examined for each cell controls as well as known posi- sample. Absolute cell counts were tive and negative sera were included obtained by multiplying the percenton every plate. age of fluorescent cells with the
264
appropriate direct leukocyte cell counts. STATISTICAL ANALYSIS
Differences in fetal viral titers and CRL between treatments were analyzed using t-tests (21). Leukocyte population differences over time and differences in sow blood fraction viral titers over time were tested using
repeated measures analysis (22). RESULTS EXPERIMENT 1
-
SOWS
Six of eight principal sows were listless and anorectic and three of eight sows had mild fevers (39.5-39.8°C) on PI day 4. The control sows remained clinically unchanged for the duration of the study. The PRRS virus was isolated from fecal swabs on PI days 2, 4-6, 8 and 9, from nasal swabs on PI days 3, 5, 7 and 9, and from serum, plasma and peripheral blood leukocytes on PI days 1, 3, 5, 7 and 9 (Table I). The PRRS virus was also isolated from the placenta of one principal sow euthanized on PI day 7. Control sows remained virus negative over the entire study. Antibodies to PRRS virus were detected as early as PI day 9 by the IFA test (5/6 sows tested positive), the remaining sow was positive on PI day 11. Serum neutralization antibodies were first detected on PI day 14 (3/6 sows). The PRRS virus inoculated sows had a significant decrease in the number of peripheral blood leukocytes compared to controls (p < 0.01, Fig.1). The decrease in total leukocytes was most pronounced on PI day 7. A reduction in peripheral blood lymphocytes on PI days 3-11 was also significant (p < 0.01, Fig. 2). The CD4 positive lymphocyte and CD8 positive lymphocyte levels were decreased (p < 0.01 for both subsets) as was the CD4/CD8 ratio (p < 0.05, Fig. 3). Peripheral blood B lymphocyte and monocyte populations were significantly reduced by PI day 7 (p < 0.01 for both subsets) but returned to control levels by PI day 14. EXPERIMENT 1
-
FETUSES
In the principal groups euthanized PI days 7, 14 and 21, 69 of 71 fetuses
were alive and grossly normal at necropsy (Table II). The two dead fetuses were beginning to autolyze. The 20 fetuses from the PI day 14 control litters were also alive and did not have gross lesions. All four sows that completed gestation (two principals, two controls) farrowed within one day of their expected due dates. The two principal sows delivered 25 live piglets and three stillborn fetuses. The two control sows delivered 20 live piglets and one stillborn fetus. The PRRS virus was not isolated from the 71 fetuses of the PI day 7, 14 and 21 litters or any of the 41 control fetuses. The PRRS virus was isolated from two liveborn piglets of principal sow number 7 ( 104 5 and 103-25 TCID50/mL of blood). Antibody to PRRS virus was detected in the serum of two stillborn fetuses from principal sow number 8 (SN titers of 1:8 and 1:32).
Table I. PRRS virus isolation from fecal swabs, nasal swabs, and blood of mid-gestation sows infected intranasally (Experiment 1)
EXPERIMENT 2
0 0 0
No. positive/No. tested, Nasal swab Serum Plasma 0/6 0/6 0/6 1/6 0/6 2/6
Virus infectivityb PBL Plasma PBL Serum Day PI 0/6 0 1.47±0.17 1.0±0.0 1.5±0.50 3/6 1 0/6 2 1/6 1.3±0.0 6/6 2.0±0.35 1.82±0.19 6/6 4/6 2/6 0/6 3 4 2/6 2.92±0.24 2.27±0.15 2.38±0.30 6/6 6/6 6/6 3/6 2/6 5 3/6 6 1.8±0.13 2.42±0.27 2.22±0.20 6/6 6/6 4/6 6/6 7 0/6 8 1/4 1.3±0.0 1.3±0.0 1.0±0.0 2/4 2/4 1/4 2/4 2/4 9 0/4 10 0/4 0/4 0/4 11 0/4 0/4 12 0/4 13 0/4 0/4 0/4 0/4 0/4 14 0/4 aPBL = peripheral blood leukocytes bGeometric mean virus infectivity titer, TCID.,/mL in CL2621 cells; ± SD; no significant treatment effect over time (p > 0.05) between blood fractions Fecal swab 0/6
log,()
22
20+
The five sows included in the experiment recovered from surgery x 1 8 + without complication. None of the 13 16 sows was viremic at the time of \ I I necropsy. Except for fetuses that were Q9) ( apparently dead at the time of surgery (-3- 14or those that died as a result of surgiT cal injury, the fetuses from all treat- aD 1 ± 2 ment groups remained alive for the C) I duration of the experiment. The only -4 gross abnormality observed was red 0 0 discoloration of the skin consistently noted in the virus and virus-antibody 8 inoculated groups but not controls. 14 4 12 8 10 2 6 0 The PRRS virus was isolated from Days post-inoculation fetuses of the virus and virus-antibody inoculated groups but not controls Fig. 1. Changes in total peripheral blood leukocytes over time in PRRS virus inoculated (0, n 6) and control (0, n 4) mid-gestation sows. Bars represent 1 SEM. There is a sig(Table III). The mean virus titer was nificant reduction of blood leukocytes in the infected sows over time (p < 0.01). significantly higher (p < 0.05) in the virus-antibody group compared to the Mid-gestation sows are susceptible virus alone group. Virus was isolated in one litter were infected by PRRS from intramuscular as well as intra- virus. However, these pigs may have to PRRS virus infection. Sows amniotic inoculated fetuses. No dif- been infected in late-gestation because intranasally exposed to PRRS virus in ference (p > 0.05) in CRL was found virus was not isolated from fetuses in experiment 1 shed recoverable the PI day 7, 14 and 21 litters and the amounts of the virus in their feces and between groups. virus (ATCC VR-2332) readily crosses nasal secretions. Virus recovery was the placenta in the last three weeks of more consistent from blood of infected sows compared to fecal or pregnancy (13). It is possible that the DISCUSSION mid-gestation inoculated sows were nasal swabs; it is possible that blood The results of this study indicate that persistently infected, with subsequent contamination of fecal and nasal PRRS virus replicates in mid-gestation transplacental transmission occurring swabs may have occurred. The PRRS porcine fetuses, but does not readily in late-gestation. Persistent infections virus did not appear to be cell associcross the placenta when sows are have been demonstrated for at least ated because viral titers were as high exposed intranasally. Of the 99 fetuses 28 days in growing pigs (data not in serum as they were in unwashed leukocyte fractions. from infected sows, two liveborn pigs shown). cn
I10+
=
=
±
265
replication may have occurred in nonvital tissues. The fetal pathogenicity of the virus strain (ATCC VR-2332) had previously been demonstrated in
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Fig. 2. Changes in total peripheral blood lymphocytes over time in PRRS v irus inoculated (a, n = 6) and control (0, n = 4) mid-gestation sows. Bars represent ± 1 SEM. There is a significant reduction of blood lymphocytes in the infected sows over time (p < 0.01
0.800 0
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0.700
60 E> 0 0.600
a
0.500
c
0.400
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0 .300-
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6 8 10 Days post-inoculation
14
12
Fig. 3. Changes in the CD4+/CD8+ peripheral blood lymphocytes ratios over time. Bars represent + 1 SEM. There is a significant reduction of the ratio in infected sows (C n= 6), versus controls (0, n = 4) over time (p < 0.05).
Changes in leukocyte numbers provided additional evidence of sow infection. Peripheral blood leukocyte populations were consistently reduced in infected sows. All leukocyte fractions measured were decreased (total cells, CD4+, CD8+, B cells and monocytes). The change in CD4/CD8 ratios observed in infected sows indicates a greater effect on the population representing T helper cells. The presence of dual CD4, CD8 positive 266
lymphocytes in the pig (n ot differentiated in this study) may Eiave affected the ratios (23). The mid-gestation fetuases infected in utero remained grossly( normal during the experimental 1period. The absence of fetal death in spite of viral replication is unexplainecd. It is possible that the experimentt was terminated before fetal dea th could be demonstrated, that a Ilonger time period is required. Altern atively, viral -
late-gestation experiments (13).
The difference between mid-gestation PRRS virus transplacental infection and late-gestation transplacental infection is likely due to the structure of the placenta. In experiment 1, PRRS virus was isolated from the placenta of a sow euthanized PI day 7, yet no virus was recovered from her fetuses suggesting that the virus does not cross the placenta at that stage of gestation. In experiment 2, virus was isolated from several fetuses but not from the sows suggesting, again, that the virus does not cross the placenta in mid-gestation. There is a possible anatomical explanation for this phenomenon. In the early stages of gestation, six histologically distinct layers separate fetal and maternal blood (24). As gestation advances the vascular beds are brought closer together by assuming subepithelial positions and by the invasion of the trophoblast by embryonic capillaries. As a consequence, the materno-fetal capillary interrelationship of the placenta becomes a more efficient organ for exchange as gestation progresses. The pathogenesis of PRRS virus infection in pregnant sows appears to be unique among viral swine reproductive pathogens because other porcine viruses such as pseudorabies virus and porcine parvovirus cross the placenta in early, mid, and late-gestation (25). Antibody dependent enhancement of PRRS virus replication has been demonstrated with swine alveolar macrophages in vitro by Choi and coworkers (14). In the in vitro study, virus replication was enhanced with the addition of PRRS virus antibody positive serum but was not enhanced with negative serum. Experiment 2 offers evidence that antibody dependent enhancement may also take place in vivo. Fetuses had enhanced viral replication when PRRS positive antibody was present. Late-gestation fetuses are capable of a humoral immune response so antibody dependent enhancement of PRRS virus replication could take place after lategestation transplacental infection (15).
The following conclusions can be drawn from the results of this study: 1) mid-gestation sows are susceptible to PRRS virus (ATCC VR-2332) infection when exposed to the virus by the intranasal route; 2) mid-gestation fetuses are susceptible to PRRS virus infection when they are exposed in utero; 3) viral replication in fetuses is enhanced by the addition of PRRS antibody positive serum, and; 4) PRRS virus does not readily cross the placenta during mid-gestation.
ACKNOWLEDGMENTS The authors thank Ms. K. Gustafson for her excellent assistance with sample collection and virus isolation, and Dr. I. J. Yoon and Ms. E. Bautista for assistance with cell culture and serology. We are also grateful to Dr. N. Sivula and his staff for performing the laparotomies.
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Table II. Effects of PRRS virus on fetuses of mid-gestation sows infected intranasally (Experiment 1)
Fetal virus Fetal PRRS isolation antibody Positive/no. Positive/no. Sow no.a tested Days PI Abnormal/total testedb 7 1 0/8 0/8 2 7 0/16 0/16 3 14 1/13 0/13 4 14 0/13 0/13 0/11 5 21 1/11 0/11 0/10 6 21 0/10 0/10 7 0/18 2/18 0/18 65 2/10 8 67 3/10 0/10 9 14 0/12 0/12 14 10 0/8 0/8 11 66 1/12 0/12 0/12 12 0/9 69 0/9 0/9 aSows 1-8 were intranasally inoculated with 1059 TCID50 PRRS virus; sows 9-12 were inoculated with uninfected cell lysate bSerum neutralization Fetal condition
Table III. Effects of in utero inoculation of mid-gestation fetuses with PRRS virus (Experiment 2) Virus postive/no. Treatmentb tested Virus infectivityc V 4/4 4.50 ± 0.58 VAb 4/4 5.88 ± 0.25 C 0/4 V 14 4 2/2 4.75± 1.76 VAb 3/3 6.17 ± 1.04 C 0/8 V 15 11 2/2 4.50±0.0 VAb 3/3 5.17 ± 0.29 C 0/2 V 16 4 4/4 3.38 ± 0.48 VAb 4/4 4.25 ±0.29 C 0/5 V 17 11 4/4 3.81 ±0.38 VAb 4/4 4.00 ± 0.71 C 0/6 aFetuses from sows 13-15 were inoculated by intramuscular injection; fetuses from sows 16,17 were inoculated by intra-amniotic injection bV = PRRS virus (104-1 TCID50); VAb = PRRS virus (104 TCID50) plus fetal serum with PRRS virus antibody; C = control, uninfected cell culture lysate cGeometric mean virus infectivity titer, log10 TCID50/mL in CL2621 cells; ± SD; significant difference (p < 0.05) between mean virus and virus plus antibody treatments Sow no.a 13
8.
9.
10. 11.
Days PI 4
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15.
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