Bluetongue virus is a vector-borne virus that causes a noncontagious disease of wild and domestic ruminants including domestic sheep (Ovis aries), domestic cattle (Bos tarus), white-tailed deer (Odocoileus virgineanus), mule deer (Odocoileus hemionus), pronghorn (Antilocapra americana), elk (Cervus canadenisis), and bighorn sheep (Ovis canadensis). It is transmitted by species of Culicoides biting midges.1,2 Disease caused by BTV decreases production and may result in the death of infected animals; it can also lead to quarantines and trade restrictions.3,4 The World Organisation for Animal Health classifies disease caused by BTV as a list A disease owing to its large adverse economic impact, which is estimated at $3 billion annually.5 There are 26 recognized serotypes of BTV worldwide with serotypes 1, 2, 10, 11, 13, and 17 reported in the United States.3,6 Historically, serotype 17 (BTV-17) has caused periodic disease outbreaks in wildlife and sheep in Wyoming.1,2 Serotype 11 (BTV-11) has been reported in northern Colorado,7 and given the geographic proximity to Wyoming, it has likely been in southern Wyoming as well.
In late September 2007, a BTV outbreak in the Bighorn Basin of Wyoming affected domestic sheep and was associated with high mortality rates in sympatric pronghorn and deer in the region.2 The serotype associated with that outbreak was BTV-17. On the most severely affected domestic sheep operation, the morbidity rate was 36% (500/1,404) and case fatality rate was 35% (175/500).2 Affected sheep operations were quarantined until after the first frost, which delayed producers from sending their lambs to market and resulted in increased feed costs and further monetary losses. For sheep producers who wanted to vaccinate their flocks against BTV in the hopes of garnering protection against disease the following year, the only commercially licensed vaccine contained BTV-10; commercially licensed vaccines against BTV-17 were not available.
Serum neutralizing anti-BTV antibody titers as low as 1:20 have been shown to be protective,8 but anti-BTV antibodies are serotype specific with little or no cross protection among serotypes.9,10 The only USDA-licensed BTV vaccine available throughout the United States is an MLV vaccine against BTV-10.a An MLV vaccineb that contains antigens against BTV-10, BTV-11, and BTV-17 is currently produced and widely used in California, but it is licensed for use only in that state. Another option for US sheep producers who want to vaccinate their flocks against BTV is to use a custom-made autogenous KV vaccine. Autogenous vaccines are regulated by the USDA in accordance with Title 9, Code of Federal Regulations, Section 113.113.11 An autogenous vaccine must be ordered by a veterinarian and produced from a local virus isolate, and it can only be used in a specified region.11 It takes 3 to 4 months for an autogenous vaccine to be approved, manufactured, and undergo the prerequisite quality testing, and the vaccine can be used for only 18 to 24 months, at which time a new vaccine must be made from a more recent virus isolate. For an autogenous vaccine to be available on a regular basis, an ongoing effort would be needed by producers within a defined region to have a new vaccine made every 2 years. Autogenous vaccines against BTV are commonly used by US cervid grower organizations. Unfortunately, data regarding the efficacy of KV or MLV vaccines against US strains of BTV in sheep are lacking.
Killed virus and MLV vaccines against BTV are widely used in Africa and Europe to control disease and have contributed to the eradication of certain BTV serotypes from some countries.12 Newer recombinant and subunit vaccines against BTV are in development but are not currently licensed.12 Modified-live virus vaccines are inexpensive to produce and require only 1 inoculation for vaccination but can cause a decrease in milk production, abortion, and embryonic death and can potentially revert to virulence and be transmitted by vectors.10,13 Killed-virus vaccines contain inactivated virus and are protective and safe but are more costly to produce than are MLV vaccines and typically require a second inoculation (booster) to ensure protection for > 1 year.14,15 The immune response to BTV vaccines and natural infections may cause mild and transient hyperthermia with potential detrimental effects on semen quality and production. Results of 1 study16 indicate that administration of an attenuated BTV-2 vaccine was associated with significant decreases in semen mobility and concentration and increases in the numbers of abnormal and dead spermatozoa for 69 days after vaccination. Conversely, administration of inactivated BTV-8 vaccines did not induce hyperthermia or affect semen quality.17,18 To our knowledge, studies to investigate the effect of BTV-17 vaccines on semen quality have not been performed.
Passive transfer of anti-BTV antibodies has been described for sheep and cattle vaccinated with a BTV- 8 vaccine19 and white-tailed deer that survived infection with BTV or a similar vector-borne virus, EHDV. In white-tailed deer fawns, maternal antibody titers against EHDV remain high for 5 weeks and become undetectable by 17 to 18 weeks old.20,21 Thus, in the Midwest, white-tailed deer fawns born in the spring are likely to have maternal antibodies against EHDV that will protect them from disease throughout most or all of their first vector season. The longevity of maternal antibodies against BTV in domestic livestock has not been well researched, but, assuming that it is similar to the longevity of maternal antibodies against EHDV in white-tailed deer fawns, lambs born in the spring should be protected from disease for most the their first vector season as well.
Currently, there are no effective commercially available vaccines against BTV-17 to protect domestic sheep flocks outside of California, so periodic epizootics of endemic BTV-17 in domestic sheep will continue to cause producers substantial economic losses.2 The purpose of the study reported here was to compare the humoral immune response of sheep vaccinated with the MLV vaccine against BTV-17 that is currently available only in California and that of sheep vaccinated with an autogenous KV vaccine against BTV-17.
Materials and Methods
Animals
All study protocols were reviewed and approved by the University of Wyoming Institutional Animal Care and Use Committee. The study consisted of 2 phases. The first phase was a randomized clinical trial that involved 30 yearling Rambouillet-Columbia crossbred ewes that were maintained at the Wyoming State Veterinary Laboratory in Laramie, Wyoming. The second phase was a prospective field trial that involved 344 commercial ewes and rams on 7 farms in 3 separate regions of Wyoming (Figure 1). Consent was obtained from each participating producer prior to study enrollment.
Map of Wyoming that depicts the locations where a randomized clinical trial (phase 1; black triangle) and prospective field trial (phase 2; regions A, B, and C) were conducted to compare the humoral response between sheep vaccinated with a KV vaccine and those vaccinated with an MLV vaccine against BTV-17.
Citation: Journal of the American Veterinary Medical Association 248, 9; 10.2460/javma.248.9.1043
Randomized clinical trial (phase 1)
The random number generator feature of a commercial software programc was used to allocate each of 30 yearling Rambouillet-Columbia crossbred ewes to 1 of 3 treatment groups (MLV, KV, and control groups). Each group was housed in a separate pen. The ewes in the control group had fenceline contact with the ewes in both the MLV and KV groups. On day 0, the wool from the right shoulder region was clipped from each ewe to allow for injection site monitoring, and each ewe was injected with the assigned treatment. Ewes in the MLV group were administered an MLV vaccined (2 mL, SC). Ewes in the control group were administered sterile diluent (2 mL, SC). Ewes in the KV group were administered an autogenous KV vaccinee (2 mL, SC) that was produced from a Wyoming BTV-17 isolate. The ewes in the KV group were administered a second (booster) dose of the vaccine on day 21.
For each ewe, a physical examination, which included examination of the oral mucosa and clipped injection site for signs of inflammation (swelling, heat, and redness) in response to vaccine administration, was performed daily from day −3 through day 14. Rectal temperature was monitored once daily from day −3 through day 14 and on days 21, 28, and 60. A blood sample (10 mL) was collected on days 0 (immediately prior to vaccination), 3, 7, 14, 21, 28, 35, 60, 137, and 365 for determination of serum anti–BTV-17 antibody titers. Five months after vaccination, the ewes were exposed to rams and bred. The ewes were allowed to lamb the following spring (10 months after vaccination). A blood sample (10 mL) was obtained from each lamb between 5 and 10 days after birth for detection of antibodies against BTV-17.
Prospective field trial (phase 2)
Seven commercial sheep farms were selected to participate in a prospective field trial to evaluate the humoral response of sheep to the autogenous KV vaccine in a natural farm setting. The 7 farms were selectively chosen to be representative of small (< 50 sheep; farms 1, 3, 4, and 7) and medium-sized (50 to 200 sheep; farms 2, 5, and 6) flocks that are confined to the farm throughout the year. The farms were also selected because they were representative of the different sheep management practices within Wyoming and the producers were willing to participate in the study. Two farms were enrolled in region A (Bighorn Basin, Wyo), 3 farms were enrolled in region B (central Fremont County, Wyo), and 2 farms were enrolled in region C (Goshen County, Wyo; Figure 1).
Two hundred forty-eight ewes and rams that were representative of the age, sex, and breed distribution of sheep in Wyoming were caught in a pseudo-random manner in pens. Only production-age sheep (2 to 7 years old) that were seronegative for antibodies against BTV-17 were eligible for study inclusion as vaccinates. To minimize farm visits and animal handling, a blood sample (10 mL) was collected from each sheep immediately prior to the administration of the first of 2 doses of the autogenous KV vaccinee (2 mL, SC; day 0). The second dose was administered to the sheep by the producer or farm veterinarian 21 days later. At least half of the sheep in each flock remained unvaccinated to serve as control animals. Controls and vaccinates were commingled to ensure that each group had the same pasture and vector exposure. Because a KV vaccine was used, there was no risk of vector transmission of the vaccine virus. Producers were asked to monitor sheep for evidence of illness after vaccination. Another blood sample (10 mL) was collected from each vaccinate and an approximately equal number of controls 12 months after the initial vaccination.
Blood collection and processing
All blood samples were collected by jugular venipuncture with an 18-gauge 1-inch blood collection needle into evacuated blood collection tubes that did not contain any additives. Following collection, blood samples were chilled to 4°C and then centrifuged at 2,060 × g for 15 minutes. Serum was harvested from each sample and stored in 1-mL aliquots at −80°C until analyzed.
Serologic testing
Each serum sample was assigned a sequential sample identification number and was submitted for analysis without any information that could be used to identify treatment group assignment. Thus, personnel analyzing the samples were unaware (blinded to) of the treatment group assignment for each sample. All serum samples were analyzed for the presence of antibodies against BTV with a genus-specific cELISAf that was performed in accordance with the manufacturer's instructions. The ELISA results were read at an optical density reading absorbency of 620 nm. Serum samples with an optical density ≤ 50% of the mean optical density for the negative controls were considered positive for antibodies against BTV.
Serum neutralizing antibody titers against BTV-17 were determined by a microtiter assay for serum samples obtained from the yearling ewes of the randomized pen trial (phase 1) on days −3, 3, 7, 14, 21, 27, 35, 60, 137, and 365. Briefly, 40 μL of each serum sample was diluted 1:10 in minimal essential mediumg and incubated at 57°C for 30 minutes to inactivate complement and other nonspecific antiviral factors, then serial 2-fold dilutions up to 1:2,560 were created for each sample. Each dilution was combined with an equal volume of minimal essential medium that contained 100 TCID50 of the BTV-17 strain used in the KV vaccine and incubated at 37°C for 30 minutes. Then each dilution was added to duplicate wells on a 96-well plate that contained confluent African green monkey kidney cells.h The plates were incubated at 37°C for 5 to 7 days. Wells were stained with crystal violet stain. For each sample, the anti–BTV-17 antibody titer was determined as the highest dilution in which > 50% of the viral cytopathic effect was neutralized, compared with that for negative control wells, and was expressed as the reciprocal of the endpoint serum dilution.
Serum samples from sheep of the prospective field trial (phase 2) that yielded positive results on the cELISA also underwent SN for BTV-17 as previously described at a 1:10 dilution to verify specificity of the anti-BTV antibodies. Serum samples obtained from all sheep of the prospective field trial 12 months after administration of the initial dose of the KV vaccine underwent SN for BTV- 17 at dilutions of 1:20 and 1:40. For reporting purposes for this study, a positive result for antibodies against BTV was defined as a positive result on the cELISA or an SN anti–BTV-17 antibody titer ≥ 1:20.
Statistical analysis
For phase 1, the geometric mean anti–BTV-17 antibody titer and mean rectal temperature were compared among the 3 treatment groups (MLV, KV, and control) with an ANOVA,i and pairwise comparisons among the treatment groups at each measurement time were performed with a Student t test.c The Bonferroni method was used to adjust for multiple pairwise comparisons (ie, the cutoff for a significant P value was adjusted from < 0.05 to < 0.013). For significant comparisons, the P value reported is the higher of the pairwise or overall group analysis.
For phase 2, a χ2 testi was used to evaluate differences in the percentage of sheep seropositive for antibodies against BTV among the 3 geographic regions and differences in the percentage of seropositive sheep as determined by cELISA and SN assay. The 95% CIs for results by region or test method were determined by the method for estimating population prevalence as described.22
Results
Phase 1
None of the 30 yearling ewes developed an adverse reaction at the vaccine injection site within 60 days after administration. On day 4 (4 days after the initial vaccine injection), 1 ewe in the MLV group and 1 ewe in the KV group developed mild reddening of the labial mucosa. Three ewes in the MLV group developed a swollen red vulva on day 7 that persisted for 24 to 48 hours. The mean rectal temperature for ewes in the MLV group was significantly (P < 0.01 for all comparisons) higher than that for ewes in the KV and control groups on days 8, 9, and 10. On day 28 (7 days after ewes in the KV group received the booster dose of vaccine), the mean rectal temperature for the ewes of the KV group was 0.5°C higher than that for ewes in both the MLV and control groups (P = 0.002) but was within the reference range (38.3 to 39.9°C [101 to 104°F]) for domestic sheep. The mean rectal temperature for each treatment group remained within the reference range throughout the observation period.
All 30 ewes were seronegative for antibodies against BTV immediately prior to vaccination on day 0, and the sheep in the control group remained seronegative throughout the observation period. Positive results for antibodies against BTV were first detected on day 7 by both the cELISA and SN assay. All ewes in the MLV and KV groups had detectable SN antibody titers against BTV-17 by day 14, and those titers remained detectable for the duration of the observation period. On the basis of cELISA results, 6 of 10 ewes in the KV group and all 10 ewes in the MLV group were seropositive for antibodies against BTV on day 60 and 8 of 10 ewes in the KV group and all 10 ewes in the MLV group were seropositive on day 365. The geometric mean anti–BTV-17 antibody titer for ewes in the MLV group peaked sooner and was significantly (P < 0.01 for all comparisons) greater than that for ewes in the KV group on days 14, 21, and 27 (Figure 2). However, the geometric mean anti–BTV-17 antibody titer for the KV group was significantly (P < 0.01) greater than that for the MLV group on day 35 (14 days after administration of the booster dose of vaccine). On day 365, the geometric mean anti–BTV-17 antibody titer was 453 (range, 160 to 1,280) for the MLV group and 260 (range, 80 to 640) for the KV group.
Geometric mean anti–BTV-17 antibody titers over time for yearling Rambouillet-Columbia crossbred ewes that received an MLV vaccine (2 mL, SC; solid line; n = 10) against BTV-17 on day 0 or an autogenous KV vaccine (2 mL, SC; dashed line; n = 10) against BTV-17 on days 0 and 21 (phase 1). All ewes were seronegative for antibodies against BTV prior to vaccination. Error bars represent the SEM. *Within a day, the geometric mean antibody titer differs significantly (P < 0.01) between the 2 groups.
Citation: Journal of the American Veterinary Medical Association 248, 9; 10.2460/javma.248.9.1043
Twenty-seven of the 30 (90%) ewes became pregnant and produced 31 lambs. One lamb (a twin) died shortly after birth, so 30 lambs were tested for serum antibodies against BTV by cELISA. At 5 to 10 days of age, all lambs born to ewes in the MLV (9/9) and KV (11/11) groups were seropositive for antibodies against BTV, whereas none of the 10 lambs born to ewes in the control group were seropositive.
Phase 2
No adverse reactions to the autogenous KV vaccine were reported by the producers of the 7 participroportion pating farms. Only 122 of the 248 sheep evaluated on day 0 were seronegative for antibodies against BTV and eligible as vaccinates for follow-up serologic testing (Table 1). During the 1-year observation period, 32 of those sheep were lost to follow-up for various reasons (routine culling, death, and predation). None of the sheep from region C (farms 6 and 7) underwent follow-up serologic testing because of the high (88% to 100%) of sheep that were seropositive for antibodies against BTV on day 0. The seroprevalence of antibodies against BTV as determined by the cELISA on day 0 varied significantly (P < 0.001) by region. The seroprevalence was highest in region C (90%; 95% CI, 84% to 96%) followed by region A (30%; 95% CI, 19% to 41%) and region B (1%; 95% CI, 0% to 3%). On day 365, the proportion of vaccinates with detectable antibodies against BTV, as determined by the SN assay (93%; 95% CI, 88% to 98%), was significantly (P < 0.01) greater than the proportion of vaccinates with detectable antibodies against BTV, as determined by the cELISA (76%; 95% CI, 67% to 85%). When the SN assay results on day 365 were used as the gold standard, the cELISA had a sensitivity of 81% (68/84). The prevalence of control sheep with antibodies against BTV on day 365 was 28% (12/43; 95% CI, 15% to 41%) for region A and 0% (0/53) for region B, which was comparable to the seroprevalence for those regions prior to vaccination on day 0.
Summary of BTV serologic test results for a prospective field trial (phase 2) conducted on 7 farms in 3 geographic regions of Wyoming in which commercial production-age (2 to 7 years old) ewes and rams were (vaccinates) or were not (controls) vaccinated with an autogenous KV vaccine (2 mL, SC) on days 0 and 21.
| No. (%) of vaccinates seropositive at 1 year | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Region | Farm | No. tested on day 0 | No. (%) seropositive on day 0 | No. of seronegative vaccinates on day 0 | No. of vaccinates lost to follow-up | cELISA | SN assay | No. of controls tested on day 365 | No. (%) of controls seropositive on day 365 |
| A | 1 | 15 | 8 (53) | 7 | 3 | 3 (75) | 4 (100) | 17 | 3 (18) |
| 2 | 46 | 10 (22) | 29 | 14 | 10 (67) | 12 (80) | 26 | 9 (35) | |
| B | 3 | 38 | 1 (3) | 19 | 0 | 15 (79) | 19 (100) | 19 | 0 (0) |
| 4 | 16 | 0 (0) | 16 | 8 | 5 (63) | 8 (100) | 17 | 0 (0) | |
| 5 | 51 | 0 (0) | 51 | 7 | 35 (80) | 41 (93) | 17 | 0 (0) | |
| C | 6 | 68 | 60 (88) | 0 | — | — | — | — | — |
| 7 | 14 | 14 (100) | 0 | — | — | — | — | — | |
| Total | 248 | 93 (38) | 122 | 32 | 68 (76) | 84 (93) | 96 | 12 (13) | |
Region A was located in the Bighorn Basin, Wyo; region B was located in central Fremont County, Wyo; and region C was located in Goshen County, Wyo. Farms were selectively chosen to be representative of the different sheep management practices in Wyoming as well as representative of small (< 50 sheep; farms 1, 3, 4, and 7) and medium-sized (50 to 200 sheep; farms 2, 5, and 6) flocks that are confined to the farm throughout the year. The autogenous KV vaccine was produced from a Wyoming BTV-17 isolate. A blood sample was collected from each of 248 pseudorandomly selected sheep immediately before the first dose of vaccine was administered on day 0. For small flocks, some of the sheep sampled were left unvaccinated. Only sheep that were initially seronegative for antibodies against BTV as determined by a cELISA were considered as vaccinates in the study. Twelve months (approx day 365) after administration of the first dose of vaccine, a second blood sample was obtained from all vaccinates remaining in the flocks and an approximately equal number of unvaccinated flock mates for serologic analysis for antibodies against BTV as determined by a cELISA (controls and vaccinates) and SN assay (vaccinates only). Vaccinates and controls were commingled throughout the observation period to ensure that each group had the same pasture and vector exposure. None of the sheep from region C (farms 6 and 7) underwent follow-up testing because a high proportion of the sheep in those flocks were seropositive for antibodies against BTV on day 0. A positive result for the cELISA was defined as an optical density ≤ 50% of the mean optical density for the negative controls. A positive result for the SN assay was defined as an anti–BTV-17 antibody titer ≥ 1:20.
— = Not applicable.
Discussion
In the United States, few BTV vaccines are available for use in sheep, and there are no commercially available vaccines against BTV-17 outside of California. Because the production and licensing of BTV vaccines require considerable cost and effort, it is important to know whether different types of vaccines provide similar levels of protection. Results of the present study indicated that both the MLV vaccine against BTV-17 that is licensed for use in California and an autogenous KV vaccine produced from a Wyoming BTV-17 strain induced a humoral response in sheep that lasted at least 1 year and was sufficient to ensure passive transfer of anti–BTV-17 antibodies to lambs. Although the sheep of the present study were not challenged with a virulent strain of BTV-17, results of a previous study8 suggest that an anti–BTV antibody titer > 1:20 is protective against disease. All sheep vaccinated against BTV-17 in the present study developed serum anti–BTV-17 antibody titers > 1:20; therefore, seropositive sheep were believed to be protected against disease caused by BTV-17.10
The randomized clinical trial (phase 1) was performed to compare the kinetics of the humoral response between the 2 vaccine types (MLV and KV) under controlled conditions and evaluate whether that response was sufficient to ensure the passive transfer of antibodies against BTV to the lambs born to the vaccinated ewes 10 months after initial vaccination. The prospective field trial (phase 2) was conducted to evaluate the response to vaccination on producermanaged operations. Because the MLV vaccine was not permitted for use in uncontrolled (field) conditions outside of California, only the autogenous KV vaccine was assessed during the field trial. The withinflock prevalence of vaccinates with antibodies against BTV 365 days after administration of the vaccine (range, 80% to 100%) suggested that the response to vaccination varied among farms most likely because of farm-specific differences such as producer experience and skill in vaccine administration, storage and handling of the vaccine, missed injections, equipment malfunction (fences and corral panels breaking) during animal processing, and inconsistent or inaccurate record keeping.
During phase 1, the MLV vaccine produced a more rapid humoral response than did the KV vaccine; however, the geometric mean anti–BTV-17 antibody titers 1 year after vaccination did not differ between the MLV and KV groups, which suggested that both vaccines are viable options to protect sheep against BTV. Availability, cost, and safety will be the deciding factors in determining which vaccine is most useful to producers. The cost of the autogenous KV vaccine (approx $1.20/dose) used in the present study was almost 4 times that of the MLV vaccine ($0.32/dose). Also, the KV vaccine required administration of a second dose, whereas the MLV vaccine required administration of only 1 dose. Although producers are most likely to favor the less expensive vaccine, safety and timing of vaccination must be considered. The MLV vaccine cannot be given to pregnant ewes and must be administered prior to the vector season (before May in Wyoming), which means that, in Wyoming, it must be administered in the fall or early spring. Furthermore, the virus in the MLV vaccine has the potential to be transmitted by vectors. Thus, during an outbreak, the MLV vaccine cannot be used, whereas the KV vaccine might be beneficial. Because of safety concerns and licensing restrictions, the MLV vaccine used in California is unlikely to be available for use in other states in the near future. Additionally, the cost-benefit ratio of vaccinating sheep against BTV is unknown because disease outbreaks occur only periodically. Studies are warranted to compare the expense of annual vaccination against BTV with the expense and risk associated with unpredictable disease outbreaks.
Both vaccines evaluated in the present study induced a humoral response sufficient to ensure passive transfer of antibodies against BTV to lambs born 1 year after their dams were vaccinated. In white-tailed deer fawns, maternal antibodies against BTV and EHDV generally become undetectable by 18 weeks of age.21 Assuming that maternal antibodies against BTV decay at a similar rate in lambs, then lambs born in April or May should be protected from disease until August or September, which is well into the peak vector season for the Midwest. Consequently, producers in the Midwest might choose to vaccinate only ram and ewe lambs intended as flock replacements each year.
In the present study, the prevalence of sheep with antibodies against BTV as determined by the cELISA was less than that determined by the SN assay on days 60 and 365 of phase 1 and on day 365 of phase 2, which suggested that the cELISA was less sensitive than the SN assay for detection of antibodies against BTV. This finding was in agreement with the results of another study23 in which the cELISA had a sensitivity of 98.6% and 69.5% for detection of antibodies against BTV in naturally infected sheep and sheep vaccinated with a KV vaccine, respectively. The disparity in cELISA sensitivity between naturally infected and vaccinated sheep is likely caused by the fact that naturally infected sheep generally develop a substantially higher anti–BTV antibody titer than do sheep vaccinated with a KV vaccine.23
Although the mean rectal temperature for ewes in the MLV and KV groups of phase 1 was significantly greater than that for ewes in the control group at various times after vaccine administration, it never exceeded the upper limit of the reference range for sheep. Therefore, it is unlikely that administration of either vaccine to rams will adversely affect semen quality. The effect of vaccination against BTV on semen quality of rams has been investigated,16–18 but most of that research was performed in Europe where vaccines against BTV-17 are not used. Additional research is necessary to better elucidate the effect of administration and time of administration of vaccines against BTV-17 on the semen quality of domestic rams.
As expected, the geometric mean anti–BTV-17 antibody titer for the ewes in the KV group of phase 1 increased significantly following administration of the second dose and did not differ significantly from that achieved for the ewes of the MLV group after 1 year. Comparison of the anti–BTV-17 antibody titer achieved after 1 dose of a KV vaccine with that achieved after administration of a priming and booster dose of the same vaccine is necessary to determine whether administration of only 1 dose is sufficient to induce humoral immunity that will be protective against disease for at least 1 year. If 1 dose of a KV vaccine can provide protection against disease for 1 year, it would make the KV vaccine a more economically attractive option for producers. Additionally, data regarding the immune response following administration of 1 dose of a KV vaccine against BTV-17 would provide valuable information about the protection that could be expected when the vaccine is administered during an outbreak.
During phase 2 of the present study, the prevalence of sheep with antibodies against BTV varied among regions, which suggested that not all areas will benefit equally from a vaccination protocol. In region C, where the prevalence of sheep with antibodies against BTV was high, sheep naïve to BTV may be protected by herd immunity, although sympatric wildlife infected with BTV could dilute this effect and make vaccination of naïve flock additions cost-effective. In region B, where the prevalence of sheep with antibodies against BTV was low, a BTV outbreak could have a substantial economic impact on producers and the immediate availability of a vaccine in the event that BTV-infected animals (domestic livestock or sympatric wildlife) are identified might help minimize losses. The seroprevalence of antibodies against BTV in region A was moderate (approx 29%), which suggested that BTV was endemic in the area as evidenced by the presence of seropositive sheep of various ages. However, a substantial portion of the sheep population in that region was seronegative for anti–BTV antibodies, and implementation of routine vaccination programs against BTV might be beneficial.
Results of the present study indicated that both of the BTV vaccines evaluated were safe and efficacious in inducing SN antibodies against BTV-17 that persisted for at least 1 year and provided sufficient passive immunity to protect lambs born to vaccinated ewes against disease for most, if not all, of the duration of their first vector season. Because anti–BTV antibody titers at the minimum detection limit of our study are protective against disease,8 it is likely that both vaccines will provide sheep flocks with adequate protection against disease. Consequently, we believe that pursuing the production and licensing of both vaccines for use on Wyoming sheep operations is a worthwhile endeavor.
Acknowledgments
Supported in part by the Wyoming Livestock Board.
This manuscript represents a portion of a thesis submitted by Ms. Speiser to the University of Wyoming as partial fulfillment of the requirements for a Master of Science degree.
The authors thank Drs. Jim Logan and John Duncan for technical assistance.
ABBREVIATIONS
| BTV | Bluetongue virus |
| cELISA | Competitive ELISA |
| CI | Confidence interval |
| EHDV | Epizootic hemorrhagic disease virus |
| KV | Killed virus |
| MLV | Modified-live virus |
| SN | Serum neutralization |
Footnotes
Colorado Serum Co, Denver, Colo.
BlueVac (10, 11, 17), PHL Associates Inc, Davis, Calif.
Microsoft Excel 2013, Microsoft Corp, Redmond, Wash.
BlueVac-17, PHL Associates Inc, Davis, Calif.
Autogenous vaccine produced by Newport Laboratories, Worthington, Minn.
BTV antibody test kit, cELISA, VMRD Inc, Pullman, Wash.
Minimum Essential Media, Corning Cell Treat, Manassas, Va.
Vero 76, ATTC, Manassas, Va.
SAS, version 92–9.4, SAS Institute Inc, Cary, NC.
References
1. Thorne ET, Williams ES, Spraker TR, et al. Bluetongue in freeranging pronghorn antelope (Antilocapra americana) in Wyoming: 1976 and 1984. J Wildl Dis 1988; 24:113–119.
2. Miller MM, Brown J, Cornish T, et al. Investigation of a bluetongue disease epizootic caused by bluetongue virus serotype 17 in sheep in Wyoming. J Am Vet Med Assoc 2010; 237:955–959.
3. Schwartz-Cornil I, Mertens PP, Contreras V, et al. Bluetongue virus: virology, pathogenesis and immunity. Vet Res 2008; 39:46.
4. Purse BV, Mellor PS, Rogers DJ, et al. Climate change and the recent emergence of bluetongue in Europe. Nat Rev Microbiol 2005; 3:171–181.
5. World Organisation for Health website. Available at: www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2016/. Accessed Jan 26, 2016.
6. Coetzee P, Stokstad M, Venter EH, et al. Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa. Virol J 2012; 9:198–207.
7. White DM, Blair CD, Beaty BJ. Molecular epidemiology of bluetongue virus in northern Colorado. Virus Res 2006; 118:39–45.
8. Letchworth GJ, Appleton JA. Passive protection of mice and sheep against bluetongue virus by a neutralizing monoclonal antibody. Infect Immun 1983; 39:208–212.
9. Eschbaumer M, Hoffmann B, Konig P, et al. Efficacy of three inactivated vaccines against bluetongue virus serotype 8 in sheep. Vaccine 2009; 27:4169–4175.
10. Noad R, Roy P. Bluetongue vaccines. Vaccine 2009; 27(suppl 4):D86–D89.
11. Autogenous biologics. 9 CFR 113.113.
12. Roy P, Boyce M, Noad R. Prospects for improved bluetongue vaccines. Nat Rev Microbiol 2009; 7:120–128.
13. Savini G, MacLachlan NJ, Sanchez-Vizcaino JM, et al. Vaccines against bluetongue in Europe. Comp Immunol Microbiol Infect Dis 2008; 31:101–120.
14. Bartram DJ, Heasman L, Batten CA, et al. Neutralising antibody responses in cattle and sheep following booster vaccination with two commercial inactivated bluetongue virus serotype 8 vaccines. Vet J 2011; 188:193–196.
15. Hamers C, Rehbein S, Hudelet P, et al. Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep. Vaccine 2009; 27:2789–2793.
16. Breard E, Pozzi N, Sailleau C, et al. Transient adverse effects of an attenuated bluetongue virus vaccine on the quality of ram semen. Vet Rec 2007; 160:431–435.
17. Leemans J, Raes M, Saegerman C, et al. Effect of an inactivated bluetongue serotype 8 vaccine on semen quality in rams. Vet J 2012; 193:567–569.
18. Kirschvink N, Raes M, Saegerman C. Impact of a natural bluetongue serotype 8 infection on semen quality of Belgian rams in 2007. Vet J 2009; 182:244–251.
19. Vitour D, Guillotin J, Sailleau C, et al. Colostral antibody induced interference of inactivated bluetongue serotype-8 vaccines in calves. Vet Res 2011; 42:18–25.
20. Gaydos JK, Davidson WR, Elvinger F, et al. Innate resistance to epizootic hemorrhagic disease in white-tailed deer. J Wildl Dis 2002; 38:713–719.
21. Gaydos JK, Stallknecht DE, Kavanaugh D, et al. Dynamics of maternal antibodies to hemorrhagic disease viruses (Reoviridae: Orbivirus) in white-tailed deer. J Wildl Dis 2002; 38:253–257.
22. Daniel WW. Biostatistics: a foundation for analysis in the health sciences. 9th ed. New York: John Wiley & Sons, 2009;192–206.
23. Niedbalski W. Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus. Pol J Vet Sci 2011; 14:615–619.
