Abstract
Objective—To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection.
Design—Randomized controlled clinical trial.
Animals—33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves.
Procedures—20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days.
Results—After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers.
Conclusions and Clinical Relevance—Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.
Bovine viral diarrhea virus and BHV1 are pathogens of the reproductive tract that affect cattle throughout the world. These viruses can cause economic loss because of infertility, abortions, and the birth of calves with poor health.1,2 Additionally, these viruses can persist within a herd, BVDV via PI cattle and BHV1 via cattle with latent infections. In areas of the United States where BVDV is enzootic, the economic losses caused by BVDV infections during 2008 were estimated to be between $361 million and $1.4 billion.3,a Thus, effective prevention and control of these 2 viruses in the cattle population would be beneficial and requires strict biosecurity, test-and-removal programs, and effective vaccination programs. Several multivalent vaccines that are labeled for fetal protection against BVDV infection (ie, prevention of PI calves)4–6,b and aiding in the prevention of abortions caused by BHV17–10 are available for use in cattle.
Most studies4–6,11 that have been conducted to assess fetal protection against BVDV infection provided by vaccination of dams prior to BVDV exposure have involved only an instantaneous exposure to a challenge strain. Results of studies4,5 conducted to evaluate BVDV vaccine efficacy in which a single intranasal dose of BVDV was used as the challenge exposure indicated that 95% to 100% of the fetuses from vaccinated dams were protected against PI. However, prolonged exposure of vaccinated dams to cattle PI with BVDV is considered to be a more rigorous and realistic challenge method for BVDV vaccine efficacy studies in cattle, and fetal protection against BVDV was 73% to 95% in studies6,11,12 that used such a challenge method.
It is important that multivalent cattle vaccines also provide protection against abortion caused by BHV1 infection. To our knowledge, a study to evaluate the ability of an MLV vaccine to prevent abortion following exposure of pregnant cows to cattle with acute BHV1 infections has not been conducted. Furthermore, an economic assessment of the effect of dam vaccination on fetal development and future calf performance following natural exposure of dams to BVDV and BHV1 during pregnancy on a beef cow-calf operation has not been performed.
The primary objective of the study reported here was to determine whether the vaccination of heifers with 2 doses of a multivalent MLV vaccine prior to breeding would provide protection of fetuses against BVDV infection and prevent abortion caused by BHV1 infection when heifers were challenged by prolonged field exposure during pregnancy to cattle shedding BVDV or BHV1. Secondary objectives were to assess the effect of prebreeding vaccination of beef heifers on the subsequent health and growth of their calves and to provide an economic analysis of the vaccination of heifers prior to exposure to BVDV and BHV1 during pregnancy on a beef cow-calf operation.
Materials and Methods
Animals—All study procedures were approved by the Auburn University Institutional Animal Care and Use Committee. Thirty-three 10- to 13-month-old crossbred beef heifers were acquired from multiple sources in Alabama and Kentucky for the study. Prior to initiation of the study, all heifers had negative results when tested for BVDV and BHV1 in serum via virus isolation and were seronegative for BVDV types 1a, 1b, and 2 and BHV1. Three 15-month-old Angus crossbred bulls were used to breed the heifers that did not conceive via AI. Prior to use, the bulls were seronegative for BVDV type 1a and had negative results for BVDV and BHV1 in serum via virus isolation. Semen from these bulls also had negative results when tested for BVDV RNA by use of an RT-nPCR assay and BHV1 DNA by use of a qPCR assay. Calves (n = 24) subsequently born to the study heifers were maintained with their dams from birth until weaning, at which time the study ended.
Three crossbred steers (age, 1 to 3 years) PI with BVDV were commingled with the heifers to provide exposure to BVDV. Steers were PI with BVDV type 1a (n = 1), 1b (1), or 2 (1) field strains. Each steer was confirmed as PI with BVDV on the basis of positive results obtained via an antigen-capture ELISAc performed on ear notch specimens, RT-nPCR assay performed on serum samples, and virus isolation performed on serial serum samples and nasal swab specimens.
Three 9-month-old Angus crossbred bulls were inoculated with BHV1 and commingled with the heifers to provide exposure to BHV1. However, prior to initiation of the study, these bulls were seronegative for BHV1 (Colorado strain) and negative for BHV1 and BVDV in serum via virus isolation.
Experimental design—Heifers were allocated to a vaccinated (n = 22) or unvaccinated control (11) group in accordance with a generalized randomized block study design with weight used as the blocking factor. The start of the study was designated as day 0. Heifers were vaccinated (multivalent MLV vaccine or sham vaccination with Dulbecco PBS solution) on days 0 and 30. Estrus was synchronized in the heifers, and all heifers were bred via AI on day 64. Beginning on day 65 and ending on day 125, the 3 bulls used for breeding purposes were commingled with the heifers. On day 125, all heifers were transrectally palpated and examined via ultrasonography to determine pregnancy status and fetal age. Pregnant heifers were then commingled with the 3 steers PI with BVDV beginning on day 132 (heifers were between 45 and 68 days of gestation) and ending on day 188. On day 265, the three 9-month-old bulls were inoculated with BHV1. These bulls were then commingled with the pregnant heifers beginning on day 268 (heifers were between 181 and 204 days of gestation) and ending on day 282. Heifers began calving on day 347, and the last calf was born on day 370. Cows and calves were kept together until calves were weaned on day 551 (mean age of calves, 205 days), the last day of the study.
Animal housing and care—During days 0 through 60, vaccinated heifers were kept in a 2.4-hectare (6-acre) pasture that was isolated from the 1-hectare (2.5-acre) pasture in which the unvaccinated control heifers were kept. Both pastures contained a mix of tall fescue, Bahia grass, and orchard grass. During the breeding period (days 61 through 131), all heifers were kept together in a 3.6-hectare (9-acre) pasture that also contained a mix of tall fescue, Bahia grass, and orchard grass. During the BVDV and BHV1 challenge periods (days 132 through 282) and until parturition (days 347 through 370), all heifers were kept together in a 1.6-hectare (4-acre) pasture that contained tall fescue, and the heifers were also fed ryegrass haylage. Immediately after parturition, vaccinated dams and their calves were moved to a 3.4-hectare (8.3-acre) pasture and unvaccinated dams and their calves were moved to a 1-hectare (2.6-acre) pasture where they were kept until the end of the study (day 551). Both pastures contained tall fescue, and cows were also fed ryegrass haylage, soy hull pellets, and corn gluten pellets on an equivalent per-animal basis.
Throughout the study, the general health of all cattle was assessed twice daily by animal caretakers who were unaware of the treatment group allocation of the cattle. On day 491 (calf age, 121 to 144 days), bull calves were castrated and all calves were vaccinated against Clostridium chauvoei, Clostridium septicum, Clostridium novyi, Clostridium sordellii, Clostridium perfringens types C and D, and Moraxella bovis. On day 551 (mean calf age, 205 days), all calves were weaned.
Vaccination—Heifers in the vaccinated group (n = 22) received 2 mL of a commercial multivalent, MLV vaccined SC in the neck region on days 0 and 30. The vaccine contained cytopathic BVDV types 1a (Singer strain) and 2 (296 strain), BHV1 (Colorado strain), parainfluenza type 3 virus, and bovine respiratory syncytial virus. Heifers in the unvaccinated control group (n = 11) received 2 mL of Dulbecco PBS solution SC in the neck region on days 0 and 30.
Estrus synchronization and breeding—To synchronize estrus, heifers were fed approximately 0.5 mg of MGAe/animal/d (the vaccinated heifer group received 11.5 mg of MGA in 50.5 kg of mixed soy hull pellets and corn gluten pellets, and the control heifer group received 6 mg of MGA in 25.4 kg of mixed soy hull pellets and corn gluten pellets) for 14 days (days 29 through 42). Each day, it was confirmed that all heifers consumed the feed containing the MGA. Each heifer was administered 25 mg of dinoprost tromethaminef IM on day 61 (19 days after the last MGA feeding), and the 2 groups of heifers were commingled in 1 pasture. On day 64 (3 days after administration of dinoprost tromethamine), each heifer was bred via AI and administered 100 μg of gonadorelin diacetate tetrahydrateg IM to induce ovulation. On day 65, 3 bulls were released into the pasture with the heifers to breed heifers that did not conceive via AI, and these bulls were commingled with the heifers for 60 days (this was in central Alabama beginning in January). On day 125, the bulls were removed from the pasture, and heifers were transrectally palpated and examined via ultrasonography to determine pregnancy status and fetal age. Subsequently, for each heifer that was determined to be pregnant on day 125, pregnancy status was monitored via transrectal palpation on study days 160, 188, 231, 268, 282, 295, 309, 323, 338, 351, and 365.
BVDV challenge exposure—During study days 132 through 188 (mid-March to mid-May), 3 steers PI with BVDV were commingled with all pregnant heifers in a 1.6-hectare pasture where they shared feed and water sources. For each steer, the BVDV titer was determined via virus isolation for serum samples and nasal swab specimens that were obtained on study days 132, 160, and 188.
BHV1 challenge exposure—On study day 265, three 9-month-old bulls that were seronegative for BHV1 were inoculated with 3 × 107 CCID50 of BHV1 (Colorado strain)h IV. These 3 bulls were then commingled with the pregnant heifers on days 268 through 282 (August) in a 1.6-hectare pasture where they shared feed and water sources. For each bull, the BHV1 titer was determined via virus isolation for serum samples and nasal swab specimens obtained on days 265 (day of BHV1 inoculation), 268 (first day of BHV1 challenge), 271, 273, 275, and 282 (last day of BHV1 challenge). Heifers and bulls were monitored daily for fever and signs of clinical disease, which included nasal and ocular discharge, abnormal respiration, cough, nasal lesions, and lethargy. Clinical signs were scored on a scale of 0 to 3; the absence of clinical signs was scored as 0, and the presence of severe clinical signs was scored as 3. Rectal temperatures from all cattle were measured and recorded on days 265, 268, 271, 272, 273, 274, 275, and 282.
Collection of samples from heifers—From each heifer, blood samples (20 mL each) were collected at the time of each vaccination (days 0 and 30) for detection of serum neutralizing antibodies against BVDV and BHV1 and virus isolation (BVDV and BHV1) from serum and WBCs. Blood samples (20 mL each) were also collected on days 132 (immediately prior to BVDV exposure), 138, 139, 140, 141, 142, and 160 (day 28 after onset of BVDV challenge exposure) for detection of BVDV from serum and WBCs via virus isolation. Serum neutralizing antibodies against BVDV were detected in samples obtained on days 132, 160, and 188. Blood samples (20 mL each) and nasal swab specimens were obtained on days 268 (immediately prior to BHV1 exposure), 271, 272, 273, 274, 275, and 282 (last day of BHV1 exposure) for detection of BHV1 via virus isolation. Serum neutralizing antibodies against BHV1 were determined for samples obtained on days 268, 282, and 295. From heifers that aborted, blood samples (20 mL each) were obtained as soon as the abortion was detected, and serum antibodies against Neospora caninum were detected via an ELISA,i antibodies against Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona were determined via serum neutralization assays.
Collection of samples from calves—From each calf, 2 blood samples (10 mL each) were collected immediately after birth for detection of BVDV RNA via an RT-nPCR assay, BHV1 DNA via a qPCR assay, both BVDV and BHV1 via virus isolation, and detection of antibodies against BVDV and BHV1 via serum neutralization assays. Ear notch specimens were obtained for detection of BVDV antigen via immunohistochemical analysis and antigen-capture ELISA. If fetal membranes were available, specimens were obtained for detection of BVDV via virus isolation and BHV1 via virus isolation and immunohistochemical analysis. Body weight was measured and recorded on the day of birth and at weaning (study day 551; mean age of calves, 205 days).
Collection of samples from aborted fetuses and dead or euthanized calves—Fetal weights were obtained on the day of abortion, and calf weight was obtained immediately after birth. Necropsies were performed on all aborted fetuses that were found and on all calves that died or were euthanized, and samples of heart blood and abomasal contents and specimens of ear, lungs, spleen, thymus, liver, kidneys, and fetal membranes (when available) were obtained. Virus isolation was used to detect BVDV and BHV1 in heart blood samples and specimens of the lungs, spleen, thymus, liver, kidneys, and fetal membranes. Serum virus neutralization assays were used to detect antibodies against BVDV and BHV1 in heart blood samples. For each fetus or calf, an RT-nPCR assay was used to detect BVDV RNA and a qPCR assay was used to detect BHV1 DNA in heart blood samples and pooled tissue samples of the lungs, spleen, thymus, liver, kidneys, and fetal membranes (when available). Ear notch specimens were tested for BVDV antigen by use of immunohistochemical analysis and antigen-capture ELISA. Liver and fetal membrane specimens were tested for BHV1 antigen by use of immunohistochemical analysis. Abomasal contents were tested for abortifacient bacteria. Kidney specimens were tested for BVDV, BHV1, and Leptospira antigens via a microscopic agglutination test.
Sample and specimen processing—Serum was removed from clotted blood samples and stored at −80°C until serum neutralization assays, the N caninum ELISAi, and the Leptospira microscopic agglutination tests were performed. To obtain WBCs, unclotted blood samples were processed as described,13 except the WBCs were resuspended in 1 mL of MEM. Serum and WBC samples and nasal swab specimens suspended in 2.5 mL of MEM were refrigerated for ≤ 72 hours before virus isolation procedures were performed. Ear notch specimens were placed in neutral-buffered 10% formalin and PBS solution. Liver and fetal membrane specimens obtained for immunohistochemical analysis were placed in neutral-buffered 10% formalin. Kidney specimens obtained for fluorescent antibody tests were frozen and stored at −23°C until the tests were performed. Other tissue specimens were homogenized individually with a mechanized stomacherj and then resuspended with 3 mL of MEM. Each homogenized tissue sample was divided into 2 samples; one was refrigerated for ≤ 72 hours before virus isolation procedures were performed, and the other was stored at −80°C until RT-nPCR and qPCR assays were performed.
Sample analyses—At Auburn Animal Health Research, 3 individuals (MSDM, PKG, and YZ) performed all sample analyses. Because of logistic constraints, the treatment allocation of animals from which samples or specimens were obtained was known to 1 individual but remained unknown to the other 2 individuals. Diagnosticians in all other labs were unaware of treatment allocation.
Virus titration—Virus titration was performed on serum and the diluent in which nasal swab specimens were stored (nasal swab diluent) that were obtained from the 3 steers PI with BVDV and the 3 bulls with acute BHV1 infection. Virus titration procedures involved serial 10-fold dilutions (1:10 to 1:10,000,000) performed in triplicate; the statistical method of Reed and Muench14 was used to determine the quantity of BVDV or BHV1. Briefly, each dilution of serum and nasal swab specimen was assayed by adding 90 μL to 3 wells of a 96-well plate, which was subsequently seeded with approximately 2,500 MDBK cells in 50 μL of MEM. To determine the concentrations of BVDV, plates were incubated for 3 days at 38.5°C in a humidified atmosphere of 5% CO2 and air. Subsequently, an immunoperoxidase monolayer assay was used as a labeling technique to confirm the presence of BVDV in the cultured MDBK cells.15,16 To determine the concentrations of BHV1, plates that contained samples from the 3 bulls with acute BHV1 infection were incubated for 5 days at 38.5°C in a humidified atmosphere of 5% CO2 and air, and then each well was examined for the characteristic cytopathic effect of BHV1.
Virus neutralization—Virus neutralization assays were performed on serum samples as described,16 except that serum was not initially diluted. Sera were tested for neutralizing antibodies against BVDV types 1a, 1b, and 2 (the 3 subgenotypes of BVDV obtained from the PI steers) and BHV1 (Colorado strain). For the BVDV neutralization assays, after each well was inoculated with the respective serum dilution, plates were incubated for 72 hours at 38.5°C in a humidified atmosphere of 5% CO2 and air, and then an immunoperoxidase monolayer assay was performed for the detection of BVDV. For the BHV1 neutralization assay, after each well was inoculated with the respective serum dilution, plates were incubated for 120 hours at 38.5°C in a humidified atmosphere of 5% CO2 and air, and then each well was examined for the characteristic cytopathic effect of BHV1.
Virus isolation—To isolate BVDV and BHV1, serum, WBC, and homogenized fetal membrane and postmortem tissue samples as well as nasal swab diluent were passaged in monolayers of MDBK cells. The procedure was performed as described13 with some modifications. Six-well (9.6 cm2) culture plates were used for serum, WBC, and fetal membrane samples and nasal swab diluent, and 24-well (2 cm2) culture plates were used for postmortem tissue samples. Each well of each plate was seeded with MDBK cells in MEM and incubated for 24 hours. Each well was then inoculated with 768 μL of serum or fetal membrane sample diluted in 192 μL of MEM, 1,000 μL of WBC sample or nasal swab diluent, or 200 μL of postmortem tissue sample. Following a 1-hour adsorption period, fetal membrane and postmortem tissue samples were removed from the wells and the cells were washed with PBS solution to remove any tissue debris. Minimum essential medium that contained 10% (vol/vol) equine serum, sodium bicarbonate (0.75 mg/mL), l-glutamine (0.29 mg/mL), penicillin G (100 U/mL; from penicillin G sodium salt), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL) was then added to each well (3 mL in the 6-well plates and 800 μL in the 24-well plates). The media used for the nasal swab diluent, fetal membrane, and postmortem tissue samples consisted of MEM and also contained gentamicin (1 mg/mL). Plates were incubated for 96 hours and examined daily for the characteristic cytopathic effect of BHV1. When a BHV1 cytopathic effect was detected in a well, 300 μL of the sample was removed from that well and tested for BHV1 DNA via a qPCR assay. After incubation for 96 hours, the plates underwent a single freeze-thaw cycle to release intracellular BVDV. Following this procedure, lysate from each well was assayed in triplicate by the addition of 10 μL of lysate and 90 μL of MEM to a well of a 96-well (0.36 cm2) culture plate, followed by the addition of 50 μL of MEM containing MDBK cells. The plates were then incubated for 72 hours and examined daily for cytopathic effects. After incubation for 72 hours, an immunoperoxidase monolayer assay was used for the detection of BVDV.
RT-nPCR and qPCR assays—A silica gel–based membrane kitk was used to extract viral RNA or DNA from calf serum samples or fetal heart blood samples. Proteinase K and filter tubes packed with glass fibers484l were used to extract RNA or DNA from pooled tissue samples. Extracted samples were assayed for the presence of BVDV RNA by use of an RT-nPCR assay as described.17
When a serum or heart blood sample had positive results for BVDV, viral RNA within the sample was amplified in triplicate. The resulting RT-nPCR products were then purified by use of a silica gel–based membrane kitm and sequenced via automated dye terminator nucleotide sequencing by use of both the 5′ (BVD 180) and 3′ (HCV 368) primers. Consensus sequences were determined with computer softwaren and compared with the BVDV sequences obtained from the 3 PI steers used for the BVDV challenge exposure to determine the subgenotype of the infecting virus.
Extracted samples were assayed for BHV1 DNA by use of a qPCR assay as described,18 except that 3-step cycling consisted of 50 cycles. Additionally, a sample from any well in which a cytopathic effect was detected during virus isolation was evaluated by use of a qPCR assay. Samples from those wells were frozen at −80°C, thawed, and extracted with a silica gel–based membrane kit,k and then a qPCR assay was used to determine whether BHV1 was the cause of the cytopathic effect.
Immunohistochemical analysis—Ear notch, liver, and fetal membrane specimens that were fixed in neutral-buffered 10% formalin were embedded in paraffin and then transported to the University of Nebraska Veterinary Diagnostic Center. Ear notch and fetal membrane specimens were tested for BVDV antigen, and liver and fetal membrane specimens were tested for BHV1 antigen by use of immunohistochemical detection methods as described.16 Briefly, sections of formalin-fixed paraffin-embedded tissues were cut at a thickness of 4 μm and placed on a microscope slide. Slides were then deparaffinized and stained on an automated immunohistochemical stainer.o The primary antibody used was a monoclonal antibody against BHV1 glycoprotein gDp at an optimal dilution of 1:2,000. The control samples for the analysis included a slide that contained a tissue section known to have positive results when tested for BHV1 and slides that contained test samples stained with an irrelevant primary antibody. The slides were pretreatedq at 90°C for 68 minutes and then were incubated with the primary antibody for 32 minutes at ambient temperature (approx 22°C). The secondary antibody, alkaline phosphatase, and substrate used for the analysis were proprietary products.
Bacteriologic culture—Samples of abomasal contents from aborted fetuses or dead calves were submitted to the Auburn University College of Veterinary Medicine microbiology laboratory. Samples were tested for abortifacient bacteria (Histophilus spp, Brucella abortus, Listeria monocytogenes, Mycoplasma spp, Escherichia coli, Salmonella spp, and Campylobacter spp) in accordance with the standard procedures used at the laboratory.
Diagnostic testing performed at extramural laboratories—Ear notch specimens were transported to the Alabama State Diagnostic Laboratory for detection of BVDV antigen via antigen-capture ELISA. Frozen kidney tissue specimens were transported to the South Dakota State University Animal Disease Research and Diagnostic Laboratory for detection of BVDV, BHV1, and Leptospira antigens via fluorescent antibody testing. For heifers that aborted, serum obtained immediately after the abortion was transported to the University of Nebraska Veterinary Diagnostic Center for detection of antibodies against N caninum via ELISA and to the Michigan State University Diagnostic Center for Population and Animal Health for detection of antibodies against 6 serovars of Leptospira spp (bratislava, canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona) via a microscopic agglutination test.
Statistical analysis—Data were analyzed with a statistical software package,s and values of P ≤ 0.05 were considered significant for all analyses. A multivariate repeated-measures ANOVA was used to evaluate rectal temperatures and clinical scores after inoculation with BHV1. A 2-tailed Fisher exact test was used to compare the following proportions between the vaccinated and control heifer groups: number of pregnant heifers at day 125 (61 days after AI), number of abortions, calf morbidity rate, calf mortality rate, number of calves PI with BVDV, and results of virus isolation from serum and WBCs of heifers. A nonparametric Mann-Whitney U test was used to analyze gestational period and calf birth weights, weaning weights, adjusted 205-day weaning weights, and average daily gains. A logarithmic transformation was applied to the serum antibody titers of each virus evaluated (BVDV types 1a, 1b, 2, and BHV1) to normalize the distribution of the data; data were analyzed via a multivariate repeated-measures ANOVA, and differences between treatment groups were detected over time. For each day for which antibody titers were determined, the geometric means of the reciprocal of the antibody titers were compared between the treatment groups by use of a Wilcoxon rank sum test.
Production and economic analyses—The mass of calf weaned per inseminated heifer was calculated by dividing the sum of all weaning weights by the number of heifers in each treatment group at the start of the study. The market value of each calf was determined by the use of the mean price (in Alabama) for the calf's weight range and sex19 for the week during which the calves were weaned (ie, the week ending May 22, 2010). Market revenue from offspring per inseminated heifer was calculated by totaling the market value for all calves weaned in each treatment group divided by the number of heifers in each treatment group at the start of the study. The expense of vaccination per inseminated heifer was calculated as twice the cost of a vaccine dose (commercially available in a 10-dose vial) added to labor costs of $15/h for 2 people to vaccinate 25 heifers/h. The production loss caused by BVDV and BHV1 exposure per control heifer was calculated as the loss in market revenue associated with lack of vaccination when BVDV and BHV1 are introduced during gestation, and the production cost per vaccinated heifer equaled the cost of vaccination to prevent that loss in market revenue.
Results
Pregnancy rate—Thirty of 33 heifers were pregnant as determined by transrectal palpation and ultrasonographic detection of a fetal heartbeat on day 125 (61 days after AI). Pregnancy rate (20/22 vaccinates and 10/11 controls) did not differ between the treatment groups. Two heifers were not pregnant; 1 heifer was at approximately 20 days of gestation, but a fetal heartbeat could not be detected. Those 3 heifers were excluded from the remainder of the study. Fifteen vaccinated and 8 control heifers conceived via AI, and 5 vaccinated and 2 control heifers conceived via natural breeding during a subsequent estrous cycle.
Viral challenge exposure—Bovine viral diarrhea virus was detected in serum samples and nasal swab specimens obtained from all 3 PI steers on days 132 (the first day of BVDV challenge), 160, and 188 (last day of BVDV challenge). The BVDV titers in serum ranged from 350 to 200,000 CCID50/mL, and those in nasal swab specimens ranged from 35,000 to 350,000 CCID50/mL. The PI steers had no clinical signs of BVDV infection during the challenge period. Transmission of BVDV among the commingled cattle was confirmed by seroconversion to BVDV in the control heifers.
Bovine herpesvirus 1 was detected in serum samples and nasal swab specimens of at least 2 bulls with acute BHV1 infection on days 268 (the first day of BHV1 challenge exposure), 271, 273, and 275 (7 days after the onset of BHV1 challenge exposure). On day 282 (14 days after the onset of BHV1 challenge exposure), BHV1 was detected in the nasal swab specimen of 1 bull. Viral titers in serum samples ranged from detectable only in cell culture passage but not by the described viral titration method to 110 CCID50/mL. Viral titers in nasal swab specimens ranged from detectable only in cell culture passage but not by the described viral titration method to 350,000 CCID50/mL. On day 268 (3 days after inoculation and first day of BHV1 challenge exposure), 2 bulls had rectal temperatures > 39.2°C (102.5°F; reference range, 38° to 39.2°C [100.5° to 102.5°F]), which then returned to within the reference range for the remainder of the BHV1 challenge-exposure period. At various times during the BHV1 challenge-exposure period, all 3 bulls had small amounts of purulent nasal discharge; no other clinical signs of BHV1 infection were observed. Transmission of BHV1 among the commingled cattle was confirmed by seroconversion to BHV1 in the control heifers.
Abortion rate—The abortion rate for the vaccinated group was significantly (P = 0.03) less than that for the control group. On day 230 (98 days after the onset of BVDV challenge exposure and prior to BHV1 challenge exposure), 1 vaccinated heifer that had been pregnant was determined to have aborted on the basis of results of transrectal palpation. The abortion occurred between 125 and 166 days of gestation, and the fetus was not found for diagnostic evaluation. That heifer was excluded from the BHV1 challenge exposure; however, the BVDV neutralization antibody titers and virus isolation results for that heifer were included in the analyses for the present study. Following the BHV1 challenge exposure, none of the vaccinated heifers aborted, whereas 4 control heifers aborted. The abortions were detected on days 291 (n = 1; 227 days of gestation), 297 (2; 215 and 217 days of gestation), and 335 (1; 271 days of gestation), which corresponded to 23, 29, and 67 days after the onset of BHV1 challenge exposure, respectively. All 5 heifers that aborted had negative results when tested for antibodies against N caninum and 6 serovars of Leptospira spp.
Clinical assessments—During the BHV1 challenge period (days 268 through 282), mean rectal temperature did not differ significantly (P = 0.78) between the treatment groups over time. On day 268 (the first day of BHV1 challenge exposure), the mean rectal temperature for both treatment groups was 38.9°C (102°F). On day 271 (3 days after the onset of BHV1 challenge exposure), the mean rectal temperature for both treatment groups had increased to 39.4°C (103°F) and then decreased to within the reference range for days 272 through 275 (days 4 through 7 after onset of BHV1 challenge exposure). Six of 10 heifers in the control group developed pyrexia (> 39.7°C [103.5°F]) on at least 1 day during the BHV1 challenge period, compared with 4 of 19 vaccinated heifers that developed pyrexia on at least 1 day during the BHV1 challenge period.
Clinical scores during the BHV1 challenge period did not differ significantly (P = 0.51) between the treatment groups over time. Seven of 10 control heifers had a clinical score > 0 on at least 1 day during the BHV1 challenge period, compared with 7 of 19 vaccinated heifers that had a clinical score > 0 on at least 1 day during the BHV1 challenge period. Clinical signs of BHV1 infection (small amounts of purulent nasal and ocular discharge) were observed in heifers in both treatment groups.
Virus neutralization results for heifers—All heifers were seronegative for BVDV and BHV1 prior to vaccination on day 0 (Table 1). All of the control heifers remained seronegative for BVDV and BHV1 until the respective viral challenge periods. During the BVDV challenge period (days 132 through 188), the vaccinated heifer group had significantly higher geometric mean antibody titers against BVDV types 1a and 1b, compared with those of the control heifer group. Similarly during the BHV1 challenge period (days 268 through 282), the vaccinated heifer group had significantly higher geometric mean antibody titers against BHV1, compared with those of the control heifer group.
Geometric mean serum neutralizing antibody titers against BVDV and BHV1 by study day for vaccinated and unvaccinated (control) beef crossbred heifers in a study to determine whether administration of 2 doses of a multivalent MLV vaccine to heifers prior to breeding would provide protection against fetal infection and abortion following exposure of pregnant heifers to cattle PI with BVDV and cattle with acute BHV1 infection.
| Treatment group | Virus | Study day | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 0 | 30 | 132 | 160 | 188 | 268 | 282 | 295 | ||
| Vaccinated heifers | BVDV 1a | 1 | 385 | 861 | 2,272 | 1,448 | — | — | — |
| BVDV 1b | 1 | 155 | 256 | 1,399 | 609 | — | — | — | |
| BVDV 2 | 1 | 20 | 14 | 133 | 175 | — | — | — | |
| BHV1 | 1 | 4 | — | — | — | 3 | 5 | 123 | |
| Control heifers | BVDV 1a | 1 | 1* | 1* | 147* | 446* | — | — | — |
| BVDV 1b | 1 | 1* | 1* | 21* | 128* | — | — | — | |
| BVDV 2 | 1 | 1* | 1* | 52 | 239 | — | — | — | |
| BHV1 | 1 | 1* | — | — | — | 1* | 1* | 18* | |
On study days 0 and 30,22 heifers received 2 mL of a commercial MLV vaccine that contained BVDV types 1a (Singer strain) and 2 (296 strain), BHV1 (Colorado strain), parainfluenza virus type 3, and bovine respiratory syncytial virus SC and 11 heifers (controls) received 2 mL of Dulbecco PBS solution SC. Following pregnancy examination on day 125, 3 heifers (2 from the vaccinated group and 1 from the control group) that were not pregnant were removed from the study, so for days 132, 160, and 188, there were 20 heifers in the vaccinated group and 10 heifers in the control group. A vaccinated heifer aborted prior to BHV1 challenge exposure and was removed from the study, so for days 268, 282, and 295, there were 19 heifers in the vaccinated group and 10 heifers in the control group. The BVDV challenge exposure consisted of commingling all pregnant heifers (vaccinated, 20; control, 10) with 3 steers that were PI with BVDV, with commingling beginning on day 132 and ending on day 188. The BHV1 challenge exposure consisted of commingling all pregnant heifers (vaccinated, 19; control, 10) with 3 bulls with acute BHV1 infection, with commingling beginning on day 268 and ending on day 282. Antibody titers < 1:2 were analyzed as 1.
Within study day and strain of virus, value for control heifers is significantly (P ≤ 0.05) different from the value for vaccinated heifers.
— = Not determined.
Virus isolation results for heifers—All heifers had negative results when tested for BVDV and BHV1 via virus isolation prior to the respective viral challenge exposures. During the first 2 weeks of the BVDV challenge period (days 132 through 146), BVDV was isolated from a significantly greater number of serum and WBC samples obtained from control heifers than from serum and WBC samples obtained from vaccinated heifers (Table 2). However, on day 160 (28 days after onset of BVDV challenge exposure), BVDV was not isolated from any serum or WBC sample obtained from heifers in either treatment group. During the BHV1 challenge period (days 268 through 282), the number of serum and nasal swab specimens from which BHV1 was isolated at each time did not differ significantly between the vaccinated and control heifers (Table 3).
Number of positive BVDV isolation results for serum and WBC samples obtained from the pregnant vaccinated (n = 20) or control (10) heifers in Table 1.
| Study day (day of BVDV challenge exposure) | Sample | Vaccinated heifers | Control heifers |
|---|---|---|---|
| 138 (6) | Serum | 0 | 7 |
| WBC | 1 | 9 | |
| 139 (7) | Serum | 0 | 9 |
| WBC | 3 | 9 | |
| 140 (8) | Serum | 0 | 9 |
| WBC | 1 | 10 | |
| 141 (9) | Serum | 0 | 7 |
| WBC | 0 | 10 | |
| 142 (10) | Serum | 0 | 3 |
| WBC | 0 | 9 |
Four heifers in the vaccinated group had at least 1 positive BVDV isolation result during the BVDV challenge period, and all 10 heifers in the control group had at least 1 positive BVDV isolation result during the BVDV challenge period. For each study day and sample type, the proportion of positive samples for each treatment group are significantly (P ≤ 0.05) different.
See Table 1 for remainder of key.
Number of positive BHV1 isolation results for serum samples and nasal swab specimens obtained from the pregnant vaccinated (n = 19)* or control (10) heifers in Table 1.
| Study day (day of BHV1 challenge exposure) | Sample | Vaccinated heifers | Control heifers |
|---|---|---|---|
| 271 (3) | Serum | 0 | 0 |
| Nasal swab | 2 | 1 | |
| 272 (4) | Serum | 0 | 0 |
| Nasal swab | 4 | 1 | |
| 273 (5) | Serum | 0 | 0 |
| Nasal swab | 2 | 2 | |
| 274 (6) | Serum | 0 | 0 |
| Nasal swab | 3 | 2 | |
| 275 (7) | Serum | 0 | 0 |
| Nasal swab | 4 | 3 | |
| 282 (14) | Serum | 0 | 0 |
| Nasal swab | 14 | 8 |
Abortion was detected in 1 heifer in the vaccinated group on day 230, and that heifer was excluded from the BHV1 challenge exposure. Seventeen vaccinated heifers had at least 1 positive result for BHV1 isolation during the BHV1 challenge period, and 9 control heifers had at least 1 positive result for BHV1 isolation during the BHV1 challenge period. For each study day and sample type, the proportion of positive samples did not differ significantly (P ≤ 0.05) between treatment groups.
See Table 1 for remainder of key.
Results for fetuses and calves—All 4 of the fetuses aborted by the control heifers following the BHV1 challenge period were obtained for diagnostic evaluation. One fetus had a domed-shaped skull, but otherwise no gross abnormalities were noted in the fetuses. One calf (birth weight, 16.8 kg [37 lb]) born to a control heifer was euthanized 36 hours after birth because of failure to thrive (ie, feeble attempts to nurse despite intervention to administer 1.4 L of colostrum via an esophageal feeding tube 8 hours after birth). A calf born to another control heifer developed acute pneumonia (rectal temperature, 41.4°C [106.6°F]) at 4 months of age. That calf survived following treatment with antimicrobials and flunixin meglumine but did not grow well throughout the remainder of the study period. A calf born to a vaccinated heifer had a traumatic injury to the left eye and developed a corneal ulcer in that eye at 5 months of age. That calf was treated with antimicrobials and flunixin meglumine and made a complete recovery. Morbidity (P = 0.13) and mortality (P = 0.24) rates for calves did not differ significantly between the treatment groups.
All fetuses and calves born to heifers in the control group had positive results when tested for BVDV via immunohistochemical analysis, RT-nPCR assay, and virus isolation (Table 4). Seven of the 10 calves born to heifers in the control group were infected with BVDV type 2; the other 3 calves were infected with BVDV type 1a. Two calves born to heifers in the control group were seropositive to BVDV (most likely from ingestion of colostral antibodies) at the time of blood sample collection; BVDV was not isolated from the serum samples of those 2 calves, but it was isolated from the WBC samples.
Test results for BVDV via immunohistochemical analysis, antigen-capture ELISA, RT-nPCR assay, and virus isolation in various specimens obtained from offspring of the vaccinated (n = 19 live calves) or control (5 live calves, 1 euthanized calf [for failure to thrive at 36 hours old], and 4 aborted fetuses) heifers in Table 1.
| Sample (test) | Offspring from vaccinated heifers | Offspring from control heifers | ||
|---|---|---|---|---|
| No. BVDV positive | No. tested | No. BVDV positive* | No. tested | |
| Ear notch (immunohistochemical analysis) | 0 | 19 | 10 | 10 |
| Ear notch (antigen-capture ELISA) | 0 | 19 | 9 | 10 |
| Serum (RT-nPCR assay) | 0 | 19 | 10 | 10 |
| Pooled fetal tissue (RT-nPCR assay) | — | — | 5 | 5 |
| Serum (virus isolation) | 0 | 19 | 8 | 10† |
| WBC (virus isolation) | 0 | 19 | 6 | 6 |
| Pooled fetal tissue (virus isolation) | — | — | 5 | 5 |
All 10 offspring from the control heifers had positive results for BVDV on at least 1 test, and for each test, the proportion of offspring from the control heifers with positive BVDV results was significantly (P ≤ 0.05) greater than that for offspring from the vaccinated heifers.
The 2 calves from control heifers that had negative virus isolation results for serum were seropositive (most likely from ingestion of colostral antibodies) to BVDV at the time the serum samples were collected.
See Table 1 for remainder of key.
From the 4 aborted fetuses and 1 euthanized calf from control heifers, BVDV was consistently isolated from specimens of the lungs, spleen, and thymus and detected via immunohistochemical analysis performed on fetal membrane tissue. Bovine viral diarrhea virus was isolated from the liver and kidneys in 4 of the 5 calves and from the fetal membranes in 1 of 4 specimens obtained. One fetal kidney specimen was lost; thus, fluorescent antibody testing (BVDV, BHV1, and Leptospira spp) could not be performed on that sample. The remaining 4 kidney specimens had positive results when tested for BVDV via fluorescent antibody testing.
No fetal or calf samples had positive results for BHV1 via virus isolation, immunohistochemical analysis, fluorescent antibody testing, or qPCR assay. Cultures of abomasal contents obtained from the 4 aborted fetuses yielded negative results for abortifacient bacteria. Evaluated kidney specimens obtained from aborted or euthanized calves had negative results when tested for Leptospira spp via fluorescent antibody testing.
Production and economic analyses—The production and economic impact of vaccination of heifers with 2 doses of a MLV vaccine prior to breeding and subsequent exposure of those heifers to BVDV and BHV1 during pregnancy were summarized (Tables 5 and 6). Excluding the 5 aborted fetuses, the gestational age for calves born to heifers in the control group was significantly (P = 0.02) less than that for calves born to heifers in the vaccinated group (Table 5). Similarly, median calf birth weight, weaning weight, and adjusted 205-day weaning weight were significantly less for calves born to control heifers, compared with those for calves born to vaccinated heifers.
Median (range) gestational age at birth and assessments of weight and market value for calves born to vaccinated and control heifers in Table 1.
| Variable | Calves from vaccinated heifers* | Calves from control heifers* | P value† | ||
|---|---|---|---|---|---|
| No. of calves | Median (range) | No. of calves | Median (range) | ||
| Gestational age at birth (d) | 19 | 278 (271–286) | 6 | 276 (266–277) | 0.02 |
| Birth weight (kg) | 19 | 29.5 (22.7–36.4) | 6 | 16.8 (13.6–22.7) | <0.001 |
| Weaning weight (kg) | 19 | 172.3 (113.6–238.2) | 5 | 130.9 (92.3–160.0) | 0.04 |
| Adjusted 205-day weaning weight (kg) | 19 | 169.5 (121.4–233.2) | 5 | 128.2 (88.6–156.8) | 0.01 |
| Average daily gain (kg) | 19 | 0.70 (0.41–0.98) | 5 | 0.54 (0.36–0.68) | 0.03 |
| Market value at weaning ($) | 19 | 455 (288–601) | 5 | 380 (233–601) | 0.03 |
Excludes 1 aborted fetus in the vaccinated group and 4 aborted fetuses in the control group.
Data analyzed via a nonparametric Mann-Whitney U test.
See Table 1 for remainder of key.
Production and economic variables for heifers that were (n = 22) or were not (11; control) vaccinated against BVDV and BHV1 prior to breeding and subsequently exposed to those viruses during pregnancy.
| Variable | Vaccinated heifers | Control heifers |
|---|---|---|
| Calf mass weaned/inseminated heifer (kg) | 148.6 | 60.5 |
| Market value of offspring/inseminated heifer ($) | 390 | 164 |
| Expense of vaccination/inseminated heifer ($) | 4 | 0 |
| Production loss due to BVDV and BHV1/heifer($) | — | 226 |
| Production cost due to vaccination against BVDV and BHV1/heifer ($) | 4 | — |
See Table 1 for key.
Calves born to vaccinated dams had a significantly (P = 0.03) greater market value at weaning, compared with the market value of weaned calves born to unvaccinated dams. When production losses per unvaccinated heifer were divided by the production costs per vaccinated heifer, the results indicated that if BVDV and BHV1 were introduced at an inopportune time during gestation on a beef cow-calf operation more frequently than once every 56 years, then vaccination of heifers between weaning and approximately 30 days prior to breeding with 2 doses of an MLV vaccine containing BVDV and BHV1 would be profitable (Table 6).
Discussion
The objective of the study reported here was to determine whether 2 doses of a commercially available multivalent MLV vaccine against BVDV and BHV1 administered to beef heifers prior to breeding would provide protection against abortion and fetal infection. The viral challenge-exposure method used consisted of controlled exposure of pregnant heifers that were between 45 and 68 days of gestation to BVDV (via commingling with 3 PI steers) for 56 days followed 80 days later by exposure to BHV1 (via commingling with 3 bulls with acute BHV1 infection) for 14 days in a natural or field setting. To our knowledge, this method of viral challenge exposure with both BVDV and BHV1 has not been used previously. Results of the present study indicated that vaccination provided significant fetal protection against BVDV infection. Even though 1 vaccinated heifer aborted a fetus that was unavailable for diagnostic evaluation to determine whether it was PI with BVDV, none of the remaining 19 calves born to vaccinated heifers had positive results for BVDV despite the use of several testing methods. Conversely, all 10 offspring (6 live calves and 4 aborted fetuses) of unvaccinated control heifers had positive results for BVDV via at least 1 assay. Therefore, it appeared that calves born to vaccinated dams would not have a role in the transmission of BVDV within and between herds. Although the results of the present study are similar to results of other studies4,5,20–22,b that indicated vaccination of dams achieved complete fetal protection against BVDV infection, various determinants influenced those outcomes. Most studies4,5,21,b in which vaccination of dams resulted in complete fetal protection against BVDV infection used a challenge inoculation of a single intranasal dose of a virus that had been propagated in vitro. Also, those studies4,5,21,b generally only evaluated fetal protection against 1 strain of BVDV at a time. In studies4,21,b that challenge exposed pregnant cattle with 2 strains of BVDV, complete fetal protection was achieved for only 1 of the challenge strains. Furthermore, in a study22 in which the method of BVDV challenge exposure (commingling of pregnant cattle with cattle PI with BVDV) was the same as that in the present study, fetal protection against infection with only 1 strain of BVDV was evaluated.
Results of the present study indicated that vaccination of heifers twice with an MLV vaccine prior to breeding provided effective fetal protection against BVDV infection following rigorous viral exposure during pregnancy. These results were similar to those of another study,20 in which heifers were vaccinated 4 times prior to breeding. The fact that similar protection can be obtained by the use of fewer doses of vaccine would benefit producers because it would allow them to reduce expenses and labor. The label instructions for the vaccine used in the present study do not require that a booster vaccination be administered; however, in the present study, heifers in the vaccinated group received 2 doses of vaccine to be consistent with the common recommendation that heifers receive 2 doses of an MLV vaccine between weaning and approximately 30 days prior to breeding.
Following the BVDV challenge period, the geometric mean serum neutralizing antibody titers against BVDV types 1a, 1b, and 2 increased for both groups of heifers, which indicated that viral transmission had occurred. After vaccination, heifers achieved the highest antibody titers against BVDV type 1a; the next highest antibody titers were against BVDV type 1b, and the lowest antibody titers were against BVDV type 2, even though the vaccine administered contained only BVDV types 1a and 2.
Fetal protection against BVDV infection was achieved in the vaccinated heifers regardless of the antibody titer, despite a rigorous BVDV challenge exposure. Bovine viral diarrhea virus was not isolated from the serum of any vaccinated heifer in the present study. However, BVDV was isolated from the WBCs of 4 vaccinated heifers, which indicated that those heifers had become infected, but none of the calves from those 4 heifers was PI with BVDV. Similarly, in another study20 after challenge exposure, BVDV was isolated from a WBC sample obtained from a pregnant heifer that had been vaccinated with an MLV vaccine, and the calf born to that heifer was not PI with BVDV. Furthermore, in that study,20 BVDV was isolated from the WBCs of 10 pregnant heifers that had been vaccinated with an inactivated vaccine, and only 1 of those heifers gave birth to a PI calf. Thus, although virus isolation for BVDV in WBC samples is more sensitive than virus isolation for BVDV in serum samples, detection of BVDV in the WBCs of a pregnant dam is not a valid predictor that her fetus will be PI with BVDV.
Results of a prevalence study23 performed at a veterinary diagnostic laboratory indicated that BVDV type 1b was the most predominant subgenotype detected in submissions from the field. In the present study, most of the PI calves were infected with BVDV type 2, followed by BVDV type 1a, and those results corresponded to the fact that the unvaccinated control heifers developed higher antibody titers against BVDV types 1a and 2, compared with antibody titers against BVDV type 1b. The results of the present study are similar to results of another study20 in which, after exposure to cattle PI with BVDV types 1a, 1b, or 2, PI calves were born to all 10 unvaccinated control heifers; 4 calves each were PI with BVDV types 1a and 2, and 2 calves were PI with BVDV type 1b. We do not believe that the distribution of the subgenotypes among the PI calves in the present study was indicative of the amount of virus shed by the PI steers. The steer PI with BVDV type 1b had the same concentration of virus in its nasal secretions as did the steer PI with BVDV type 2, whereas the serum of the steer PI with BVDV type 1b contained a higher concentration of virus than that detected in the serum of the steer PI with BVDV type 2. Additionally, all 3 PI steers were observed to commingle with the heifers during feeding and shared feed bunk space, which provided ample opportunity for viral transmission via nasal secretions. For the present study, it is possible that the BVDV type 1b field strain was not as infectious as the BVDV type 1a and 2 field strains when used in a simultaneous challenge exposure. Investigators of another study24 reported that BVDV type 2 was more efficient at infecting fetuses than BVDV type 1 when the 2 strains were administered simultaneously via an intranasal route. Regardless, rigorous field exposure to BVDV types 1 and 2 was achieved in the present study.
Another objective of the present study was to evaluate protection provided by the vaccine against abortion caused by BHV1 infection. The challenge method used was an attempt to duplicate field exposure and, to our knowledge, has not been assessed in any previous study. Other studies7–10,25 conducted that required a BHV1 challenge exposure used a single inoculation of the virus. In the present study, pregnant heifers were exposed to 3 bulls with acute BHV1 infection for 14 days to maximize the opportunity for viral transmission, and the heifers were exposed to BHV1 during the same period of gestation as that in other studies7,9 conducted to evaluate vaccine efficacy against BHV1-induced abortion. The BHV1 challenge method used in the present study resulted in viral transmission as evidenced by seroconversion to BHV1 in the control heifers, the anamnestic increase in antibody titers against BHV1 in the vaccinated heifers, and the isolation of BHV1 from nasal swab specimens obtained from both vaccinated and control heifers. Following BHV1 challenge exposure in the present study, the increase in antibody titers for heifers in the control group was not as great as that reported by investigators of other studies7,10; however, the BHV1 isolation results obtained from nasal swab specimens were similar to those of another study.25
Although 4 heifers in the control group aborted after the BHV1 challenge period, BHV1 was not detected in any of the fetuses or associated fetal membranes, despite the use of multiple testing methods. Thus, the cause of those abortions was uncertain. The abortions may have been caused by BHV1 because the interval after exposure during which the abortions occurred was consistent with that during which known BHV1-induced abortions occur, and only unvaccinated control heifers aborted after the BHV1 exposure. Furthermore, no other infectious causes of abortion (except for BVDV, which infected the fetuses at least 80 days prior to the abortions) were detected, despite extensive diagnostic evaluation. However, because BHV1 was not detected in the aborted fetuses or associated fetal membranes, BHV1 cannot be declared the definitive cause of the abortions. In other studies,7–9 following IV inoculation of pregnant heifers with a single dose of BHV1, all unvaccinated control heifers aborted and BHV1 was detected in all fetuses. In another study25 in which pregnant cattle were inoculated with a single dose of BHV1 intranasally, BHV1 was detected in the fetuses from 5 of 8 unvaccinated seronegative heifers and 6 of 8 unvaccinated, seronegative multiparous cows. Therefore, the appropriateness of the BHV1 challenge method used to evaluate vaccine efficacy for protection against BHV1-induced abortion in the present study might be questioned.
A possible reason for the lack of BHV1 detection in fetal tissues tested in the present study was the relatively low concentration of virus shed by the acutely infected bulls. Although the 3 bulls were inoculated IV with 3 × 107 CCID50 of BHV1, the highest concentration of BHV1 detected in their nasal secretions was 3.5 × 105 CCID50/mL. That concentration was lower than the single-dose concentrations of BHV1 (106 CCID50/mL, IV7–9; and 108 CCID50/mL, intranasal25) used to challenge expose pregnant cattle in other studies. Fetal infection with BHV1 is the result of systemic transmission of the virus across the maternal-fetal barrier.2 In the present study, BHV1 viremia was not detected in any of the heifers following BHV1 challenge exposure. Investigators of a study26 in which an in vitro BHV1 challenge method was used reported that only a small percentage (0.07%) of a 107 CCID50/mL dose of BHV1 migrated from the maternal to the fetal side of human placenta tissue. However, in studies7,10 conducted with cattle in vivo, BHV1 viremia was detected in only 3 of 10 and 6 of 11 unvaccinated control heifers, but infection with BHV1 was detected in 10 of 10 and 10 of 11 fetuses from those heifers, respectively. Thus, results of those studies7,10 indicate that BHV1 can be transmitted to a fetus even in the absence of detectable viremia in the dam.
Another possible explanation for the lack of detection of BHV1 in fetuses in the present study is that the prior BVDV infection protected the fetuses from becoming infected with BHV1, although such a phenomenon has not been verified. In a study27 conducted to determine the distribution of BHV1 antigen in experimentally infected pregnant heifers, BHV1 antigen was detected in the mesenchyme of fetal villi and endothelial cells. Similar studies28,29 have been conducted to determine the distribution of BVDV antigen in heifers pregnant with PI fetuses, and BVDV antigen was detected primarily in the maternal crypt cells of the placenta and was also present in the fetal binuclear trophoblast cells and some capillaries. Therefore, it is possible that the BVDV infection altered the receptors on the endothelial cells of the placenta such that BHV1 could not attach to the placenta and be transmitted to the fetus. Alternatively, the BVDV infection may have compromised the pregnancy to such an extent that an additional exposure to BHV1 resulted in abortion before BHV1 could be transmitted across the placenta and into the fetus at a detectable concentration.
Importantly, results of the present study indicated that vaccination of heifers prior to breeding provided protection against the birth of calves PI with BVDV and abortion caused by BHV1 when those heifers were subsequently exposed to those 2 viruses during pregnancy, and it also resulted in a significantly longer gestational period and the birth of calves with a greater weight, compared with those of calves born to unvaccinated heifers. Also, calves born to vaccinated heifers had significantly greater weaning weights, adjusted 205-day weaning weights, average daily gains, and market values, compared with those for calves born to control heifers. The difference in the mean adjusted 205-day weaning weights between calves born to vaccinated heifers and calves born to control heifers in the present study was similar to the difference between calves PI with BVDV and calves not PI with BVDV in another field study.30 Thus, there is an economic benefit for beef producers to appropriately vaccinate cattle prior to breeding, especially if cattle should become inadvertently exposed to BVDV or BHV1 during pregnancy.
Results of the present study indicated that vaccination of beef heifers twice with a commercial multivalent MLV vaccine prior to breeding provides fetal protection against PI with BVDV following challenge with 3 strains of BVDV via field exposure to PI steers. Conversely, assessment of protection of vaccinated heifers against abortion caused by BHV1 was equivocal. Although vaccinated and unvaccinated heifers were exposed to BHV1 and a large proportion of heifers in each group shed the virus in nasal secretions, BHV1 was not detected in any calves or aborted fetuses born to vaccinated or control heifers. However, the abortion rate in vaccinated heifers was significantly less than that in control heifers following exposure to both BVDV and BHV1, and calves born to vaccinated heifers performed better than calves born to control heifers. Thus, vaccination of beef heifers with 2 doses of a multivalent MLV vaccine prior to breeding is recommended to prevent the birth of calves PI with BVDV, improve reproductive health of adult cattle, and maximize growth of subsequent offspring, all of which will result in substantial economic benefit should BVDV or BHV1 be inadvertently introduced into a beef cow-calf operation.
ABBREVIATIONS
| AI | Artificial insemination |
| BHV1 | Bovine herpesvirus 1 |
| BVDV | Bovine viral diarrhea virus |
| CCID50 | Cell culture infective dose |
| MDBK | Madin-Darby bovine kidney |
| MEM | Minimum essential medium |
| MGA | Melengestrol acetate |
| MLV | Modified-live virus |
| PI | Persistently infected |
| qPCR | Quantitative PCR |
| RT-nPCR | Reverse-transcription nested PCR |
Ridpath JF, Hessman BE, Neill JD, et al. Parameters of ear notch samples for BVDV testing; stability, size requirements and viral load (abstr), in Proceedings. 39th Annu Meet Am Assoc Bovine Pract 2006;39:38–39.
Schnackel JA, Van Campen H. Modified live type 1A bovine viral diarrhea virus (BVDV) provides fetal protection against challenge with a type 2 BVDV (abstr), in Proceedings. Detecting Controlling BVDV Infection Conf 2002;37.
IDEXX HerdChek BVDV Antigen ELISA Ear-Notch/Serum Test Kit, IDEXX Laboratories Inc, Westbrook, Me.
Express FP5, Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo.
MGA 2000, North American Nutrition Co Inc, Lewisburg, Ohio.
Lutalyse, Pfizer Animal Health, New York, NY.
Cystorelin, Merial, Iselin, NJ.
VR-864, ATCC, Manassas, Va.
IDEXX Neospora X2 Ab Test, IDEXX Laboratories Inc, Westbrook, Me.
Tekmar Stomacher, model 80, Tekmar Co, Cincinnati, Ohio.
QIAamp Viral RNA preparation kit, Qiagen, Valencia, Calif.
Roche High Pure PCR Template Preparation Kit, Roche, Indianapolis, Ind.
QIAquick PCR Purification Kit, Qiagen, Valencia, Calif.
Align X, Vector NTI Advance 11, Invitrogen, Carlsbad, Calif.
Ventana BenchMark ULTRA, Ventana Medical Systems Inc, Tucson, Ariz.
1B8-F11, VMRD Inc, Pullman, Wash.
Cell Conditioner 2, Ventana Medical Systems Inc, Tucson, Ariz.
Ventana ultraView Red, Ventana Medical Systems Inc, Tucson, Ariz.
JMP, SAS Institute Inc, Cary, NC.
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