Evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period

Charles C. Broaddus Department of Clinical Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74048.

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Udeni B. R. Balasuriya Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, University of Kentucky, Lexington, KY 40546.

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Jena L. R. White Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, University of Kentucky, Lexington, KY 40546.

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Peter J. Timoney Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, University of Kentucky, Lexington, KY 40546.

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Rebecca A. Funk Department of Clinical Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74048.

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G. Reed Holyoak Department of Clinical Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74048.

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Abstract

Objective—To determine whether it is safe to vaccinate pregnant or postpartum mares with a commercial modified-live virus vaccine against equine viral arteritis (EVA).

Design—Randomized controlled study.

Animals—73 mares and their foals.

Procedures—Mares were vaccinated during mid gestation, during late gestation, or 2 or 3 days after parturition with a commercial modified-live virus vaccine or were not vaccinated. Foaling outcomes were recorded, and serum, blood, milk, and nasopharyngeal samples were obtained.

Results—All mares vaccinated during mid gestation foaled without any problems; 21 of 22 mares in this group had antibody titers against EAV at the time of foaling. Of the 19 mares vaccinated during late gestation, 3 aborted; antibody titers against EAV were detected in 13 of 15 mares from which serum was obtained at the time of foaling. All postparturient vaccinates were seronegative at foaling; all of them seroconverted after vaccination. No adverse effects were detected in any of their foals.

Conclusions and Clinical Relevance—When faced with a substantial risk of natural exposure to EAV, it would appear to be safe to vaccinate healthy pregnant mares up to 3 months before foaling and during the immediate postpartum period. Vaccinating mares during the last 2 months of gestation was associated with a risk of abortion; this risk must be weighed against the much greater risk of widespread abortions in unprotected populations of pregnant mares naturally infected with EAV.

Abstract

Objective—To determine whether it is safe to vaccinate pregnant or postpartum mares with a commercial modified-live virus vaccine against equine viral arteritis (EVA).

Design—Randomized controlled study.

Animals—73 mares and their foals.

Procedures—Mares were vaccinated during mid gestation, during late gestation, or 2 or 3 days after parturition with a commercial modified-live virus vaccine or were not vaccinated. Foaling outcomes were recorded, and serum, blood, milk, and nasopharyngeal samples were obtained.

Results—All mares vaccinated during mid gestation foaled without any problems; 21 of 22 mares in this group had antibody titers against EAV at the time of foaling. Of the 19 mares vaccinated during late gestation, 3 aborted; antibody titers against EAV were detected in 13 of 15 mares from which serum was obtained at the time of foaling. All postparturient vaccinates were seronegative at foaling; all of them seroconverted after vaccination. No adverse effects were detected in any of their foals.

Conclusions and Clinical Relevance—When faced with a substantial risk of natural exposure to EAV, it would appear to be safe to vaccinate healthy pregnant mares up to 3 months before foaling and during the immediate postpartum period. Vaccinating mares during the last 2 months of gestation was associated with a risk of abortion; this risk must be weighed against the much greater risk of widespread abortions in unprotected populations of pregnant mares naturally infected with EAV.

Equine viral arteritis is an infectious disease of equids caused by EAV Although most primary cases of EVA are subclinical infections and therefore unnoticed by horse owners, some outbreaks may be associated with the appearance of influenza-like clinical signs, abortions, and interstitial pneumonia in neonatal foals.1,2 The risk of abortion in pregnant mares and the potential for persistent infection in stallions have important economic ramifications for the horse breeding industry. Equine arteritis virus is an enveloped RNA virus belonging to the family Arteriviridae, which also includes another viral disease of major veterinary concern, PRRS.3,4 The EVA and PRRS viruses are readily inactivated by common disinfectants and detergents. Equine arteritis virus survives for only 2 or 3 days at 37°C, although it may survive for at least 75 days at 4°C.5

Equine viral arteritis is a disease of worldwide concern, with serologic evidence of infection being recorded in North and South America, Europe, Australia, Africa, and Asia.1,6 However, the seroprevalence of EAV infection varies greatly among countries and among equine populations within a country. The 1998 National Animal Health Monitoring System equine survey7,8 reported that only 2.0% of unvaccinated horses in the United States were seropositive for EAV, in contrast to horses imported into California (mostly European Warmbloods) that had a seropositive rate of 18.6%. The seroprevalence of EAV infection varies widely among horse breeds in the United States, with approximately 80% of Standardbreds and 5.4% of Thoroughbreds being seropositive. Most other breeds are believed to have a seroprevalence of < 2%.1,9–13 Differences in prevalence among breeds may be associated with differences in management practices. The low overall rate of seropositivity in some horse breeds has enhanced the risk of widespread transmission of EAV in those breeds during outbreaks of EVA. This was exemplified during the extensive outbreak of EVA in Thoroughbreds in Kentucky in 1984 and in the more recent multistate outbreak in Quarter Horses in the United States in 2006 and 2007.

Transmission of EAV among horses is principally via the respiratory or venereal routes.1,14–18 Outbreaks have frequently resulted from breeding a naïve mare with EAV-infective semen; the virus is subsequently disseminated to other naïve horses on the premises via the respiratory route. Naturally infected stallions may become persistent carriers of EAV. Establishment and maintenance of the carrier state is a testosterone-dependent event, with the virus localized to the accessory sex glands.18–20 Persistently infected stallions may be short-term (< 3 months), intermediate (3 to 7 months), or long-term (7 months to several years) carriers.15,19 Carrier stallions can play a major role in widespread dissemination of the infection and are the primary natural reservoir of the virus.6

Subsequent to aerosol infection in a horse, EAV spreads to the lungs and bronchial lymph nodes and then enters the circulation to be disseminated throughout the body within 2 days.21 Following infection, virus can be isolated from the nasopharynx for 2 to 14 days, from the buffy coat for 2 to 21 days, and from serum or plasma for 7 to 9 days. An inability to isolate virus from serum or plasma after 7 to 9 days is associated with the appearance of antibodies at that time.17 Equine arteritis virus has not been isolated from an infected horse more than 28 days after infection, except in the semen of carrier stallions.18,22,23

The hallmark histologic finding associated with EAV infection is arteritis, and the vascular injury likely results from direct virus-mediated injury to the endothelium and muscularis media of affected vessels.24–26 The resulting vasculitis is characterized by marked fibrinoid necrosis of small muscular arteries, and the increased vascular permeability leads to hemorrhage and edema around these vessels.27,28

A cornerstone of current EVA prevention and control programs in the United States involves the targeted use of a MLV vaccine, which has been commercially available since 1985. This vaccine was derived from an experimental vaccine against EVA that was developed many years earlier by serial passage of the experimentally derived highly pathogenic Bucyrus strain of EAV 131 times in primary horse kidney cells followed by 32 times in primary RK cells (ie, HK131-RK32).29 However, the original MLV vaccine was not fully attenuated in that it induced abortion in 2 late-gestation mares when injected directly into the fetus or the amniotic sac. Subsequently, a vaccine with a higher passage history (131 times in primary horse kidney cells followed by 111 times in primary RK cells [ie, HK131-RK111]) was tested and found to be safe when used in pregnant pony mares.30 The current commercially available vaccine is derived from the latter of these experimental vaccines; it has a passage history of HK131-RK111-Eq Dermis 24.31 On the basis of findings in the original experimental vaccine study, the manufacturer recommends that pregnant mares should not be vaccinated until after foaling.29

Despite the manufacturer's recommendation to refrain from use of the vaccine in pregnant mares, there have been reports of its use in pregnant mares without ensuing complications. There are also anecdotal reports of a few late-term abortions in mares that had been previously vaccinated with the MLV vaccine, but without any evidence directly linking vaccination with the abortions.32 The study reported here was conducted in an attempt to establish whether vaccinating mares during mid or late gestation with a MLV vaccine against EVA would result in abortion and to determine the safety of vaccinating mares within a few days after foaling.

Materials and Methods

Animals—A total of 73 mares were used in the study Mares were predominantly Thoroughbreds or American Quarter Horses with a small number of Arabians and 1 Gypsy Cobb mare. Mares ranged from 5 to 25 years of age, and they were pregnant or had foaled recently. None of the mares had a history of gestational or peripartum complications. All of them were confirmed negative for serum neutralizing antibodies against EAV at the beginning of the study. Owner consent was obtained for all client-owned animals used in the study. The study was approved by and conducted in accordance with the guidelines of the Oklahoma State University Institutional Animal Care and Use Committee.

Procedures—All mares were maintained at 1 of 2 breeding premises in central Oklahoma for the duration of the study. Mares in group 1 (n = 22 mid-gestation mares) were vaccinated, housed, and foaled at a commercial breeding premises in Guthrie, Okla. Mares in group 2 (n = 19 late-gestation mares) were vaccinated, housed, and foaled at the commercial breeding premises (2) or at the Center for Veterinary Health Sciences Ranch in Stillwater, Okla (17). All of the mares in group 3 (n = 28 postparturient mares) were vaccinated, housed, and foaled at the Center for Veterinary Health Sciences Ranch. The control group consisted of 4 mares (2 were housed at the commercial breeding premises, and 2 were housed at the Center for Veterinary Health Sciences Ranch) that were not vaccinated. Neither premises had a prior history of EAV infection as determined on the basis of routine serosurveillance testing.

Mares in group 1 were vaccinated between 142 and 83 days before foaling, and mares in group 2 were vaccinated between 68 and 2 days before foaling. Mares in group 3 were vaccinated within 3 days after foaling. The mares in group 3 were subdivided into 2 subgroups: 16 mares and their foals were turned out to pasture following vaccination (pasture subgroup), and 12 mares were housed indoors in individual stalls (4.6 × 7.6 m [15 × 25 feet]) with their foals for 10 days following vaccination (which enforced close physical contact [stall subgroup]), after which these mares and foals were also turned out to pasture.

Vaccination of mares—Mares were vaccinated against EVA by IM administration of a single dose of a commercially available MLV vaccine.a Mares were vaccinated in accordance with the manufacturer's instructions.

Management and collection of samples from mares and foals—Management and sample collection were based on pregnancy status at the time of vaccination.

Groups 1 and 2 (prepartum vaccinates)

Following vaccination, each mare was monitored daily to evaluate general health as well as pregnancy status. As the anticipated date of parturition approached, mares were moved into a foaling stall. Each mare was frequently observed in the foaling stall through parturition.

Blood samples (5 mL) were collected via jugular venipuncture from each mare at the time of parturition (0 hours), 12 hours later, and 30 days after parturition. Blood samples (5 mL) were similarly collected from each foal immediately after birth before they nursed the dam (if possible), 12 hours later, and 30 days after parturition. The precolostral blood samples were obtained from the foals via venipucture of the umbilical vein or jugular vein; blood samples were obtained from the foals at 12 hours and 30 days after parturition via jugular venipuncture. Samples were allowed to clot, and the serum was harvested and stored at −20°C. Milk samples (10 mL) were collected from each mare at time 0, 12 hours later, and 30 days after parturition.

Group 3 (postpartum vaccinates)

Rectal temperature, pulse rate, and respiratory rate were recorded for each mare and foal at the time of parturition, 12 hours after parturition, 48 hours after parturition, and on days 1, 3, 5, 7, 14, and 28 after vaccination. The type of samples collected and the sample collection schedule were the same for mare-foal pairs of both the stall and pasture subgroups. Blood samples were collected from mares at the time of vaccination and on days 14 and 28 after vaccination for serum harvest and VN testing to detect development of antibodies against EAV. Blood samples were also collected from mares into EDTA-containing tubes for separation of peripheral blood mononuclear cells for use in virus isolation; these blood samples were collected on the day of parturition and days 1, 3, 5, 7, 14, and 28 after vaccination. Blood samples for serum harvest were collected via jugular venipuncture from the foals at the time of vaccination and on days 14 and 28 after vaccination for use in determining possible exposure to vaccine virus. Nasopharyngeal swab specimens were collected from mares on the day of vaccination and days 1, 3, 5, 7, 9, 11, 14, and 21 after vaccination by use of sterile gauze swabs and placed in viral transport medium, as described elsewhere.33

Control mares

The control mares were housed and handled similarly to the mares in groups 1 and 2, except that they were not vaccinated. Blood samples were collected from the mares and their foals at the same times as for the horses of groups 1 and 2.

VN titers—Serum and milk samples were subjected to VN tests to detect antibodies against EAV. Serum VN antibody titers were determined in accordance with the method described in another study.34

Virus isolation—Virus isolation from buffy coat and nasopharyngeal swab specimens was performed in RK-13 cells in accordance with a recommended protocol.35 Briefly, blood samples collected into EDTA anticoagulant tubes were centrifuged at 500 × g for 10 minutes. Plasma and buffy coat cells were aspirated and placed in 15-mL conical centrifuge tubes. Buffy coat cells were pelleted by centrifugation at 1,500 × g for 10 minutes at 4°C. The plasma was aspirated, and the WBC pellet was resuspended in 5 mL of EMEM. The cell suspensions were frozen at −80°C until virus isolation was performed. Nasopharyngeal swab specimens in viral transport medium were vortexed and then filtered through a 0.45-μm syringe filter. Filtrates were frozen at −80°C until virus isolation could be performed. Virus isolation from peripheral blood mononuclear cells, filtrates of nasopharyngeal swab specimens, and clarified 10% fetal tissue suspensions was attempted in both high- and low-passage RK-13 cell lines in accordance with a recommended protocol.35 Briefly, serial 10-fold dilutions (10−1 to 10−3) of each sample were made in supplemented EMEM, and 1 mL of each dilution was inoculated into 2 × 25-cm2 flasks containing confluent monolayers of RK-13 cells. Flasks were incubated at 37°C for 1 hour and then were overlaid with EMEM supplemented with 0.75% carboxymethyl cellulose. Flasks were incubated at 37°C and evaluated for the appearance of viral cytopathic effect on days 3 and 4 after inoculation. If there were no detectable cytopathic effects, a second passage was performed on day 4. The RK-13 cell monolayers were fixed and stained with a 1% crystal violet solution containing 1% formaldehyde on day 5 after inoculation in the case of the first passage in cell culture and on day 4 after inoculation in the case of the second passage in cell culture. Tissue culture fluid was harvested and stored at −80°C until used for viral RNA extraction.

Viral RNA extraction and real-time RT-PCR assay— Viral RNA was directly isolated from samples of tissue culture fluid by use of a commercial kit.b Briefly, tissue culture fluid samples were placed in microcentrifuge tubes and centrifuged at 13,800 × g for 2 minutes; then, 140 μL of supernatant was removed and used for nucleic acid extraction in accordance with the manufacturer's instructions. Viral nucleic acid was eluted in 60 μL of nuclease-free water and stored at −80°C.

A 1-tube real-time RT-PCR assayc was performed with RT-PCR master mixd in a real-time PCR system.e The primers and probes used were identical to those previously described.36,37 Every sample was tested in duplicate. Briefly, 25 μL of RT-PCR mixture for each reaction contained 12.5 μL of 2× master mix without uracil-N-glycosylase, 40× RTf and RNase inhibitor mix,g 900nM forward and reverse primers (0.45 μL), 250nM probe (0.625 μL), 5.35 μL of nuclease-free water, and 5 μL of test sample RNA. Thermocycling conditions were used for the standard mode as per manufacturer's recommendation (30 minutes at 48°C, 10 minutes at 95°C, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute). Each RT-PCR assay included a negative control sample without RNA (contained the reaction mix with 5 μL of water [no template]) and positive control samples.

Postmortem examination—Gross and histologic examination of any aborted fetuses was performed at an animal disease diagnostic laboratory.h Tissues were fixed in neutral-buffered 10% formalin and embedded in paraffin, and sections were stained with H&E. Specimens of fetal membranes and fetal lung, heart, and liver were collected for attempted virus isolation and viral nucleic acid extraction and stored at −80°C. Tissues were tested for evidence of infection with EHV-1, EHV-4, and Leptospira spp. Fluorescent antibody staining and examination of fetal tissues for EHV-1 and EHV-4 were performed on approximately 10-μm-thick tissue sections that were stained with caprine origin anti-EHV-1 and anti-EHV-4 polyclonal antiserum conjugated to fluorescein isothiocyanatei as described in standard protocols and procedures. Sections were counterstained with Evans blue dye, which caused cells with positive results to stain fluorescent green and cells with negative results to stain brick red. Aerobic bacteriologic culture of fetal stomach contents, fetal lungs, or fetal membranes was performed on blood agar, MacConkey agar, and phenol-ethyl-alcohol agar.38 Heart blood samples were also obtained from each fetus for serologic examination.

Statistical analysis—All statistical analyses were conducted with a commercially available statistical program.j Repeated-measures ANOVAk was used to assess the effects of parturition group and time. Simple effects of parturition group (control vs group 1 vs group 2) for a given time were calculated, and comparisons of the groups were performed with pairwise t tests.l,m Titer values were transformed with a logarithmic (base 2) function prior to calculation of the ANOVA. Means and SEs of the raw titer values were reported. Significance was set at values of P < 0.05 for all comparisons.

Results

Foaling outcomes—All 22 mares in group 1 foaled without difficulty or assistance, and each mare gave birth to a live foal. Serum IgG testing was performed on samples obtained from 20 foals at 12 hours after birth; all foals had IgG concentrations > 800 mg/dL. Although IgG concentrations were not determined for 2 foals, these foals were healthy at birth and remained healthy throughout the 30-day study period. Two foals were healthy at birth and had IgG concentrations > 800 mg/dL; however, both foals died before 30 days after birth, and regrettably neither was submitted for necropsy.

Three mares in group 2 aborted. One mare aborted 9 days after vaccination, a second mare aborted 11 days after vaccination, and a third mare aborted 38 days after vaccination. The remaining 16 mares in group 2 foaled without difficulty or assistance, and all foals, except for 1, had a serum IgG concentration > 800 mg/dL in samples obtained at 12 hours after birth. That 1 foal had a serum IgG concentration < 400 mg/dL at 12 hours after birth and was administered 1 L of plasma; this foal thrived throughout the 30-day study period. Two foals were euthanatized at approximately 2 weeks after birth (one of these foals had evidence of severe abdominal pain, and an area of devitalized intestine was found during necropsy; the other foal was weak at birth, dehydrated, and unable to stand, but no relevant findings were detected in that foal during necropsy).

All 28 mares in group 3 gave birth to healthy foals without difficulty or assistance. Twenty-five of these foals had an IgG concentration > 800 mg/dL in samples obtained 12 hours after birth. Each of the 3 other foals required a transfusion of 1 L of plasma to increase the serum IgG concentration to > 800 mg/dL. All foals in this group remained healthy throughout the 30-day study period.

All mares in the control group gave birth to healthy foals without difficulty or assistance. All foals had an IgG concentration > 800 mg/dL in samples obtained at 12 hours after birth.

Clinical findings—The mares and foals in all groups were monitored after parturition. Mares and foals were generally bright, alert, and active throughout the 28-day period after vaccination, except for the 2 aforementioned foals in group 2 that were euthanatized. Mares and foals of group 3 were closely monitored following vaccination, with frequent assessments of rectal temperature, pulse, respiration, and overall demeanor. No significant difference from the reference range was detected for mean rectal temperature, pulse rate, or respiratory rate recorded at birth, 12 hours after parturition, 48 hours after parturition, and 1, 3, 5, 7, 14, and 28 days after vaccination.

Results of VN tests—All mares were seronegative for EAV at the beginning of the study. Serologic data were available on almost all mares and foals over the study period. All of the mares in group 1, except for 1, were seropositive (titer ≥ 1:4) for EAV at foaling; titers for the seropositive mares ranged from 1:16 to ≥ 1:512. That 1 mare was seronegative at the time of foaling but seropositive (titer ≥ 1:512) 12 hours later. Samples were obtained 30 days after parturition from 18 mares, all of which were still seropositive. The mean VN titer for the mares differed significantly between the control group and group 1 as well as between the control group and group 2 at 0 hours, 12 hours, and 30 days.

All 22 foals in group 1 were seronegative (titer < 1:4) at birth, but all were seropositive 12 hours later. All of the foals available for sample collection at 30 days after parturition were still seropositive. The mean VN titer for foals differed significantly between the control group and group 1 as well as between the control group and group 2 at 12 hours, whereas there was no significant difference between the control group or groups 1 and 2 at 0 hours. At 30 days after parturition, the mean VN titer for foals of group 1 was significantly different from that of the control group, whereas the mean VN titer did not differ significantly between the control group and group 2.

Similarly, data for milk samples were available for most mares. All mares in group 1 had antibodies against EAV in the colostrum at the time of foaling, with 18 of 22 mares having a titer ≥ 1:512. Milk samples collected 12 hours after foaling had antibodies against EAV for all of the mares, although there was a 4-fold or greater decrease in antibody titer in many samples. Samples collected 30 days after parturition did not have antibodies against EAV in 7 of 22 mares and had titers between 1:4 and 1:32 in 8 of 22 mares; the remaining 7 mares were not available for collection of milk samples. Mean VN titer in milk was not significantly different among all groups at 0 hours, but was significantly different among all groups at 12 hours; at 30 days, the mean VN titer in milk for group 1 was significantly different from that of the control group, whereas the mean VN titer in milk did not differ significantly between the control group and group 2.

Fifteen of 19 mares in group 2 were available for collection of milk samples. In those 15 mares, 13 were seropositive and 2 were seronegative at the time of foaling. Fifteen foals from which samples were collected at the time of birth were seronegative. However, serum obtained before colostral suckling from 3 foals had positive results for antibodies against EAV. The 4 control mares had negative results for antibodies against EAV in serum and milk samples obtained throughout the study.

Milk was obtained from 15 mares in group 2 at the time of foaling, with 11 milk samples having positive results and 4 samples having negative results for antibodies against EAV. The milk samples with negative results were collected from 4 mares that were vaccinated 2, 4, 4, and 19 days before foaling, respectively. The milk samples obtained 12 hours after parturition still had negative results for 3 of these 4 mares (mares vaccinated on 2, 4, and 4 days before foaling, respectively; the milk sample from the other mare had a titer of 1:32). All group 2 milk samples collected at 30 days after parturition, except for 1, had negative results or had low titers (≤ 1:16).

The 28 mares and 27 foals comprising group 3 from which samples were available were negative for antibodies against EAV at the time of foaling. All of the mares responded following vaccination, with detectable VN titers at days 14 and 28 after vaccination. All of the foals, except for 2, were seronegative at days 14 and 28 after parturition. Both seropositive foals were in the pasture subgroup. The remainder of the foals in the pasture subgroup and foals in the stall subgroup had negative results for antibodies at birth and 14 and 28 days after foaling.

Virus isolation and real time RT-PCR results— Results of virus isolation for nasopharyngeal swab specimens and buffy coat specimens obtained from the mares in group 3 were determined. Because there were a number of samples that were not collected, the data set was not complete for all mares. All samples with positive results for virus isolation were confirmed by use of real-time RT-PCR assay.

Twelve of 28 mares in group 3 had positive results for virus isolation of buffy coat specimens or nasopharyngeal swab specimens (or both). Equine arteritis virus was isolated from the buffy coat of 9 mares in the period immediately following vaccination, 8 of which were in the pasture subgroup (the remaining mare with the positive results was in the stall subgroup). Virus was isolated from the nasopharynx of 5 mares, including 2 from the pasture subgroup and 3 from the stall subgroup. Two of the 12 mares with positive results for EAV (1 in the stall subgroup and 1 in the pasture subgroup) had virus isolated from both the buffy coat specimens and nasopharyngeal swab specimens. Most of the virus isolations were from specimens collected within the first few days after vaccination.

Two foals seroconverted to EAV after vaccination of their respective dams; both mares had positive results for virus in the buffy coat specimens. However, EAV was not detected in nasopharyngeal swab specimens collected from either of these mares. Both of the mares were in the pasture subgroup and had positive results for virus isolation only 1 time (day 1 after vaccination).

Postmortem examination—Fetuses aborted by 3 mares in group 2 were submitted for a complete postmortem evaluation. The fetus from 1 mare was close to full term (approx 300 to 330 days of gestation). Gross examination of that fetus revealed multiple nonspecific lesions, including mild, multifocal hemorrhage in the thymus and heart; mild mesenteric lymphadenopathy; and mild, acute, multifocal hemorrhage of the allantoic sac and chorioallantois in conjunction with chorioallantoic edema. The fetal membranes weighed 5.1 kg (11.2 lb). Histologically, lesions consistent with arteritis were observed as well as stromal neutrophilic infiltration and fibrinous inflammation of the chorioallantois and amniotic sac; arteritis with neutrophilic and fibrinous perifunisitis; mild hemorrhages of the kidneys, thymus, and adrenal glands; and hyperplasia of the lymphoid follicles of the spleen and mesenteric lymph nodes. The lungs were atelectic and not aerated, and meconium was found in the lumen of the alveoli and airways. Lung tissue from the foal had positive results for EAV as determined by use of the PCR assay. The fetus was seronegative for antibodies against EAV, EHV-1, and Leptospira spp. Fluorescent antibody staining of sections of the liver yielded negative results for EHV-1 and EHV-4 antigens. Aerobic bacteriologic culture of fetal stomach contents also yielded negative results.

Necropsy of the aborted fetus and fetal membranes from a second mare that aborted revealed heavy (8.5 kg [18.7 lb]), edematous fetal membranes; a nearly full-term fetus; adrenal gland hemorrhage; and diffusely at-electic lungs. Histologic examination revealed chronic inflammatory lesions around blood vessels in the heart and in the stroma of the thickened areas of the fetal membranes. The fetus and fetal membranes had positive results for EAV as determined by use of the PCR assay. The fetus was seronegative for EAV, EHV-1, and several serovars of Leptospira interrogans (Canicola, Grippotyphosa, Icterohemorrhagiae, Pomona, Bratislava, and Hardjo). Fluorescent antibody staining of sections of the lungs and liver yielded negative results for EHV-1 and EHV-4 antigens. Aerobic bacteriologic culture of fetal stomach contents also yielded negative results.

Fetal membranes were the only tissue available for evaluation following the abortion of the third mare. The fetal membranes were heavy (8.6 kg [18.9 lb]), with marked thickening and edema of the entire allantochorion in the region of the pregnant horn and adjacent uterine body. Histologic examination confirmed stromal edema and congestion in the thickened areas of the fetal membranes. The fetal membranes had positive results for EAV as determined by use of the PCR assay. Aerobic bacteriologic culture of the fetal membranes yielded a moderate growth of Klebsiella pneumoniae, α-Streptococcus spp, Escherichia hermanii, Raltonia picketii, Acinetobacter wolfii, and Enterobacter gergoviae; all were considered bacterial contaminants. Fungal culture of the fetal membranes yielded the growth of a small number of colonies of Penicillium spp. The fetal membranes had negative results for EHV-1 and EHV-4 as determined by use of the PCR assay. Leptospiral evaluation revealed titers against several L interrogans serovars (Grippotyphosa, 1:1,600; Icterohemorrhagiae, 1:400; and Bratislava, 1:400); results were negative for several other L interrogans serovars (Canicola, Hardjo, and Pomona). The mare had a titer of 1:64 against EAV.

Discussion

The economic impact of an outbreak of EVA can be substantial, as was the case following outbreaks on Thoroughbred breeding farms in Kentucky in 1984 and the multistate outbreak in Quarter Horses in 2006 and 2007. Breeding farms can be hit especially hard economically. The question frequently arises as to the best time to vaccinate broodmares against EVA to minimize or prevent the risk of widespread abortions. The primary objective of the study reported here was to provide an answer to that question.

To address this objective, mares were vaccinated during pregnancy (mid or late gestation) or immediately after parturition, which are often the time points when breeders want to vaccinate mares to protect them against the risk of natural infection with EAV. The only commercially available vaccine against EVAa in the United States was used; this vaccine is not recommended for use in pregnant mares. The vaccine insert indicates that pregnant mares should not be vaccinated until after foaling. Furthermore, pregnant mares should not be vaccinated during the last 2 months of gestation because a few instances of fetal invasion by vaccine virus have been detected after mares were vaccinated during this period. It is preferable to vaccinate mares when they are not pregnant; however, when pregnant mares are threatened by a high risk of natural exposure, vaccination may be undertaken with considerably less risk of abortion attributable to vaccination than is inherent for natural infection. Owners should be advised of the possibility of fetal infection before vaccinating pregnant mares. The results of the present study support this recommendation.

The findings of the study reported here are consistent with those of another study39 in which investigators found that the vaccine virus was associated with an isolated abortion in a mare recently vaccinated with the commercially available vaccine.a In that study,39 there was homology between the nucleotide sequence of open reading frame 5 of the isolated virus and that of the vaccine virus. The mare aborted during an extensive outbreak of EVA on a Thoroughbred breeding farm in Illinois in 1994. In contrast to this finding, results of a subsequent study31 were reported for the outcome after vaccinating 5 pregnant mares with the commercially available vaccinea between 51 and 85 days prior to foaling. Vaccination against EVA with the MLV vaccine did not result in abortion in any of the vaccinated mares. As emphasized by the authors of that study,31 this finding must be interpreted with caution in light of the small number of horses involved. It is worth mentioning that following the 2006 and 2007 multistate outbreak of EVA, there was widespread use of the same MLV vaccine in pregnant mares, with no published reports of confirmed abortions associated with vaccination.

Studies in which the vaccine strain of EAV was administered to mares include those conducted by researchers instrumental in the development of the vaccine in the 1960s and 1970s. The original vaccine inoculated directly into a fetus or amniotic sac induced abortion in 2 mares.29 A subsequent study30 that involved the IM administration of a vaccine virus with a higher passage history did not result in any abortions in 18 mares vaccinated between 30 days of gestation and nearly the end of a full-term gestation. The present study is the largest study conducted to assess the effects of vaccination with the MLV vaccine against EVA in peripartum mares. Furthermore, the passage history of the vaccine virus was much higher than that of the modified-live experimental vaccines used in earlier studies.29,30

Of the 22 mares in group 1 (mares vaccinated during mid gestation), all gave birth to live healthy foals. Twenty foals remained healthy throughout the study. The 2 foals that died before 30 days after birth appeared to have died of apparently unrelated illness, notwithstanding the fact that a postmortem examination was not performed on either foal. Furthermore, there was no evidence of congenital EAV infection in that every foal was seronegative for antibodies against EAV at birth. All of the foals were seropositive at 12 hours after birth, which confirmed effective absorption of colostral-derived antibodies from their dams. One mare in this group was seronegative for EAV at the time of foaling, but both that mare and her foal had a titer ≥ 1:512 twelve hours later, and the milk obtained from that mare at the time of foaling was positive for antibodies (titer, ≥ 1:512). This discrepant result would strongly suggest that the sample identified as that of the mare at 0 hours was probably mislabeled and that it actually represented the sample obtained from the foal at 12 hours after birth. It is important that all foals were seronegative for EAV at birth and were subsequently seropositive at 12 hours and 30 days after parturition, which indicated that vaccination of mid-gestation mares does not result in exposure of unborn fetuses to vaccine virus and that colostrum does provide passive protection against EVA in the case of newborn foals.40 The findings indicated that vaccination with the commercially available vaccinea between 83 and 142 days before the anticipated date of foaling does not compromise maintenance of pregnancy nor result in congenital infection of the fetus.

It is important to consider the neutralizing antibody concentration that is sufficient to afford protection against wild-type EAV infection. In 1 study,41 titers as low as 1:8 were fully protective, and titers of 1:4 were moderately protective, when horses were challenge exposed to a highly virulent Bucyrus strain of EAV. All mares in the study reported here had fully protective titers of ≥ 1:8 at 30 days after parturition.

In contrast to the fact that none of the vaccinated mares in group 1 aborted, 3 of 19 mares in group 2 (all mares were vaccinated during late gestation) aborted. Those 3 mares in group 2 aborted 9, 11, and 38 days after vaccination, respectively. Tissues from all 3 aborted fetuses had positive results for EAV as determined by use of virus isolation and PCR assay. In light of the fact that all foals in group 1 were seronegative at birth, it must be assumed that these group 1 foals did not have EAV infection in utero. In contrast, detection of EAV in tissues of the 3 aborted foals in group 2 was important. Considered in conjunction with other findings, it would appear that EAV was a factor in causing abortion in each of these 3 mares. Unfortunately, the aborted fetus from 1 mare was not recovered (the mare aborted unexpectedly at pasture, and the fetus was not found). This mare also had a high titer against L interrogans serovar Grippotyphosa as well as moderate titers against L interrogans serovars Icterohemorrha-giae and Bratislava. Because of the lack of any histologic confirmation of EAV infection and in light of the titers for leptospirosis, the abortion of that mare cannot unequivocally be attributed solely to EAV.

The mare that aborted while at pasture and whose fetus was not recovered aborted 38 days after vaccination, as opposed to the other 2 mares that aborted 9 and 11 days after vaccination, respectively. The closer temporal association of the abortions with vaccination in those 2 mares in addition to the histologic findings in the aborted fetuses provided a stronger case for involvement of the vaccine strain of EAV in each of those abortions. The lesions of arteritis observed in the chorioallantois and umbilical cord in association with the positive PCR findings and nondetectable or marginal titers for leptospirosis would point to the vaccine strain of EAV as the likely cause of the abortion in the mare that aborted 11 days after vaccination. Similar findings, including chronic inflammation around the blood vessels in the heart of the fetus and fetal membranes, for the mare that aborted 9 days after vaccination would also support a similar conclusion. The homology of the nucleotide sequence of open reading frames 2a to 7 of the isolated virus and the corresponding region of the vaccine virus implicated the vaccine strain of EAV as the cause of the abortions. It is worth mentioning that samples of fetal heart blood collected from the aborted fetuses of the mares that aborted 9 and 11 days after vaccination were negative for antibodies against EAV, which suggested that the immune system of those fetuses did not have time to respond to circulating vaccine virus before fetal death occurred.

Further supportive evidence of the possibility of in utero fetal infection with the vaccine virus was the finding of presuckle serum titers against EAV in a limited number of foals in group 2. In contrast to group 1, in which all of the foals were born seronegative for EAV, 3 of 18 foals in group 2 were born seropositive for EAV. The dams of these 3 foals were vaccinated 13, 36, and 68 days before foaling. The titers confirmed that in utero fetal infection with EAV had taken place. It is worth mentioning that all 3 foals were born healthy and remained healthy throughout the duration of the study. Because the fetal membranes from apparently normal births were not routinely collected and subjected to virus isolation, PCR assay, or histologic examination, it is unknown whether there may have been evidence of infection with EAV in the fetal membrane tissues of these 3 foals.

The virus isolated from each animal represented the vaccine virus rather than wild-type virus acquired during the course of the study. Although neither virus isolation nor PCR assay differentiates vaccine virus from wild-type virus, there was no other source of EAV during this study, except for the vaccination. Despite vigilant surveillance, EAV was not detected on any premises in Oklahoma during the time of the study. Neither the control mares nor other nonvaccinated horses on the breeding farms where the study was conducted seroconverted during the study.

The intent of the vaccination protocol used in mares in group 3 was to assess the responses of the mare and foal if the mare was vaccinated with the MLV vaccine within 2 to 3 days after foaling. All of the mares seroconverted as expected, and all of the foals (except for 2) remained seronegative throughout the study. The 2 foals that were seropositive were clinically normal on the basis of results of physical examination, and their heart rate, respiratory rate, and rectal temperature were not significantly different from those of their cohorts. It was thought that close contact between the mare and foal during the immediate postpartum period might increase the chance of transmission of the vaccine virus from mare to foal. Surprisingly, the 2 foals with positive results for antibodies against EAV were both part of the pasture subset. It could be argued that pastured mares and foals likely have similar patterns of physical contact to those confined in stalls. Twelve of 28 mares in group 3 that had positive results for EAV on the basis of virus isolation or PCR assay of either buffy coat specimens or nasopharyngeal swab specimens comprised mares from both the pasture subgroup (n = 9 mares) and the stall subgroup (3). Apparently, only 2 of the 12 virus-positive mares shed a sufficient amount of virus to infect their foals, as attested to by seroconversion of the foals. Neither of these 2 foals had any clinical signs of illness. The titers in the foals were the result of exposure to the vaccine virus by some undefined route, perhaps via the milk. On the basis of findings in the present study, it does not appear to be detrimental to vaccinate mares 2 to 3 days after foaling, although there is a low risk that the foal may become infected with the vaccine virus through contact with its vaccinated dam. It would be prudent to recommend against vaccinating a mare soon after foaling if there was any compromise of the foal's health. If there is a need to vaccinate a mare during the immediate postpartum period, blood should be collected from her foal prior to vaccination of the mare to document seronegative status, and the foal could then be considered potentially vaccinated on that date if detected seropositive to EAV 14 to 28 days after vaccination of the dam.

It was hypothesized that the 16 of 28 mares in group 3 that were not EAV positive on the basis of virus isolation or PCR assay of buffy coat specimens or nasopharyngeal swab specimens had negative results because of enhanced clearance of vaccine virus associated with the peripartum period. The immune system of these mares may have been more aggressive in achieving pathogen clearance during the peripartum period, as suggested by vaccination studies in humans.42,43 It is interesting that the CDC recommendation for vaccination of a pregnant woman seronegative for antibodies against tetanus, diphtheria, and pertussis is to vaccinate during the postpartum period with tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vac-cine.42 Despite the lack of any documented complications associated with prepartum vaccination, the CDC believes it prudent to make this recommendation, presumably for the same reasons the manufacturer of the commercially available vaccine against EVAa makes the same recommendation. In studies44–46 in dairy cows, it has been found that there are dramatic decreases in the percentage of peripheral blood CD4+ T cells as well as increased activity of CD8+ lymphocytes at parturition. Peripheral blood B-cell concentrations are highest immediately before parturition and lowest immediately after parturition.44 Taken together, these findings indicate that parturition affects immunity. It is hypothesized that in the study reported here, it was the effect of foaling that led to the low rate of virus isolation in the group 3 mares after vaccination.

It is also important to consider the effects of the use of vaccines during pregnancy in other species. Pregnant sows vaccinated with an MLV vaccine for PRRS virus, another member (in addition to EAV) of the family Arteriviridae, had a decreased number of pigs born alive and weaned, compared with results for pregnant sows that were not vaccinated.47 However, similar to the situation with EAV in naive pregnant mares, this decrease in productivity is sometimes an acceptable alternative to the effects of disease caused by natural exposure to the PRRS virus. The reproductive performance of sows after vaccination against PRRS depends on the stage of gestation: the largest decreases in pigs born alive and weaned were detected in sows vaccinated during the last 4 weeks of gestation,48 which again was similar to the most important reproductive dysfunction in the mares vaccinated during late gestation in the present study. In contrast to the results of these studies47,48 in pregnant sows vaccinated with an MLV vaccine against PRRS, it has been found that pregnant cows vaccinated with certain MLV respiratory vaccines did not have any detrimental effects.49 In that study,49 pregnant cows vaccinated at various stages throughout gestation with a combination vaccine containing MLV components against bovine herpesvirus-1, bovine viral diarrhea virus, parainfluenza virus-3, bovine respiratory syncytial virus, and inactivated cultures of L interrogans serovars Canicola, Grippotyphosa, Hardjo, Icterohemorrhagiae, and Pomona had abortion rates similar to those of pregnant cows and heifers administered sterile water diluent during the same stages of gestation. Therefore, effects of vaccination on reproductive performance of a pregnant animal appear to depend on the vaccine.

Serologic results of the mares and foals in group 3 indicated that the mares became seropositive after vaccination, whereas the foals did not. Several reasons may have accounted for this, including the fact that when the mares developed vaccine-induced titers following vaccination 2 days after parturition, the foals were no longer able to absorb antibodies through the gastrointestinal mucosa. Transintestinal permeability for immunoglobulins in calves progresses to complete impermeability between 12 and 24 hours after birth, and foals have been found to have a similar time frame for gastrointestinal closure.50,51 Although 12 of 28 mares in group 3 shed vaccine virus, the amount of vaccine virus shed may have been too low to exceed the threshold dose needed to infect and result in seroconversion in the foals, except for 2 of the foals.

Virus neutralization testing of milk samples revealed that the colostrum collected at foaling was antibody positive for all mares in group 1. In group 2, all mares (except for 4) had positive results for antibodies against EAV in colostrum samples collected at foaling; the 4 mares that had negative results were vaccinated 2, 4, 4, and 19 days before foaling, respectively. The milk obtained 12 hours after foaling from 3 of those mares (vaccinated 2, 4, and 4 days before foaling, respectively) was still negative, whereas the milk of the other mare had a titer of 1:32 (low positive titer), perhaps because the mare was vaccinated only 19 days before foaling. Milk samples from the other 3 mares probably had negative results because a sufficient amount of time had not elapsed between vaccination and sample collection; available serum samples obtained from these 3 mares at 0 and 12 hours also had negative results, whereas the serum samples obtained from these mares at 30 days after parturition had titers of ≥ 1:512.

Analysis of the findings of the study reported here confirmed the recommendation for vaccination of at-risk peripartum mares with the MLV vaccine against EVA. Although vaccination of pregnant mares with MLV vaccines should be undertaken with caution, it appears that the risk of adverse consequences is minimal in mares vaccinated up to 3 months before foaling. Vaccination of these mares provides the potential benefit of colostral antibody protection for the foals, which does not appear to be evident in mares vaccinated soon after foaling. It is also apparent that mares may be vaccinated during the last 2 months of gestation and they will not necessarily abort; 16 of 19 mares in group 2 gave birth to healthy foals. If mare populations are under stress or have been exposed to other infections, vaccination against EAV during the last 2 months of gestation may result in abortion. However, in most circumstances, the risk of abortion is less likely. On the basis of these findings, and in the face of a high risk of natural exposure to EAV, the risk of vaccination-related abortion is far outweighed by the substantial risk of EVA-related abortion and the potential of widespread dissemination of the virus. This conclusion should be considered in the event of future EVA outbreaks.

ABBREVIATIONS

EAV

Equine arteritis virus

EHV

Equine herpesvirus

EMEM

Eagle minimal essential medium

EVA

Equine viral arteritis

MLV

Modified-live virus

PRRS

Porcine reproductive and respiratory syndrome

RK

Rabbit kidney

RT

Reverse transcriptase

VN

Virus neutralizing

a.

Arvac, Pfizer Animal Health, Charles City, Iowa.

b.

Qiagen, Valencia, Calif.

c.

TaqMan RT-PCR assay, Applied Biosystems, Foster City, Calif.

d.

TaqMan One-Step RT-PCR Master Mix, Applied Biosystems, Foster City, Calif.

e.

7500 Fast real-time PCR System, Applied Biosystems, Foster City, Calif.

f.

MultiScribe reverse transcriptase, Applied Biosystems, Foster City, Calif.

g.

RNase inhibitor, Applied Biosystems, Foster City, Calif.

h.

Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, Okla.

i.

VRMD, Pullman, Wash.

j.

PC SAS, version 9.2, SAS Institute Inc, Cary, NC.

k.

PROC MIXED, PC SAS, version 9.2, SAS Institute Inc, Cary, NC.

l.

LSMEANS with SLICE, PC SAS, version 9.2, SAS Institute Inc, Cary, NC.

m.

LSMEANS with DIFF, PC SAS, version 9.2, SAS Institute Inc, Cary, NC.

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