Materials and Methods

Site selection—Blood samples were collected from sheep at 3 ranches (ranches A, B, and C) in the Big Horn Basin (Figure 1) in February and March 2008 when the flocks were gathered for shearing. These ranches had experienced substantial morbidity and mortality rates from BT in 2007. When possible, blood samples were collected from replacement lambs born in the spring of 2007 as well as adult rams and ewes. The yearling lambs were considered the sentinel population for the study to give a true indication of the infection rate. These lambs were 4 to 5 months old at the time of the outbreak and would have been past the period of passive protection from maternal antibodies.^25,26

Two of these ranches (A and B) were managed as range flocks with the ewes and their lambs moved to mountain pastures from July to September, but the rams were kept at the ranch year-round. In addition, ranch A had a number of goats commingled with the sheep flock that were also tested. The third ranch in the Big Horn Basin (C) was managed as a farm flock with ewes, lambs, and rams at the ranch year-round. Ewes and lambs from ranch A were randomly selected from the whole flock by separating out alternate groups into a catch pen as the flock was moved through a gate. Sheep were similarly selected from ranch B, but the ewe and lamb flock had the sheep identified as having had BT removed.

Sheep were also tested from ranches outside of the Big Horn Basin in areas with and without histories of previous BT. These included ranch D on the plains east of the Big Horn Mountains that had a mild BT epizootic in 2005, ranch E in the black hills of northeastern Wyoming in an area with known endemic history of BT in sheep and wildlife,¹³ and ranch F in western Wyoming that had no history of bluetongue-like disease (Figure 1). These ranches were selected for comparison to ranches within the Big Horn Basin for number of sheep seropositive for BTV and viral titers.

Lambs born in the spring of 2008 were tested from 3 farm flocks kept in the Big Horn Basin all summer and from a flock in Miles City, Mont, (Figure 1) pastured in the area affected by BT in 2007. Blood samples from these lambs were collected in October and tested for serum antibodies against BTV as evidence of the virus successfully overwintering and circulating within the outbreak area in the summer of 2008.

Elevation map of the state of Wyoming and bordering counties of southern Montana. The Big Horn Basin is roughly outlined in a broken black line and contains ranches A, B, and C (not labeled) and the farms with the tested 2008 spring-born lambs (not labeled). Ranches located outside of the Basin are indicated by letters. D = East of Big Horn Mountains. E = Black Hills. F = Western Wyoming. M = Area south of Miles City, Mont, which is roughly 75 miles north of the Wyoming state line. For elevations, m = meter.

Citation: Journal of the American Veterinary Medical Association 237, 8; 10.2460/javma.237.8.955

• Download Figure
• Download figure as PowerPoint slide

View Full Size

Elevation map of the state of Wyoming and bordering counties of southern Montana. The Big Horn Basin is roughly outlined in a broken black line and contains ranches A, B, and C (not labeled) and the farms with the tested 2008 spring-born lambs (not labeled). Ranches located outside of the Basin are indicated by letters. D = East of Big Horn Mountains. E = Black Hills. F = Western Wyoming. M = Area south of Miles City, Mont, which is roughly 75 miles north of the Wyoming state line. For elevations, m = meter.

Citation: Journal of the American Veterinary Medical Association 237, 8; 10.2460/javma.237.8.955

• Download Figure
• Download figure as PowerPoint slide

Elevation map of the state of Wyoming and bordering counties of southern Montana. The Big Horn Basin is roughly outlined in a broken black line and contains ranches A, B, and C (not labeled) and the farms with the tested 2008 spring-born lambs (not labeled). Ranches located outside of the Basin are indicated by letters. D = East of Big Horn Mountains. E = Black Hills. F = Western Wyoming. M = Area south of Miles City, Mont, which is roughly 75 miles north of the Wyoming state line. For elevations, m = meter.

Citation: Journal of the American Veterinary Medical Association 237, 8; 10.2460/javma.237.8.955

• Download Figure
• Download figure as PowerPoint slide

Blood sample collection—Blood sample collection from sheep in this study was done under a protocol reviewed and approved by the University of Wyoming Institutional Animal Care and Use Committee and with the written approval and assistance of the sheep owners. Blood samples were collected by jugular venipuncture into plain and EDTA vacuum tubes.^a Information on clinical signs and history of BT in sheep was recorded from all ranchers and the attending veterinarian. All flocks were made up of white-faced breed ewes and rams except for a few black-faced breed rams.

Serologic detection of antibodies against BTV—Blood samples were tested in triplicate for antibodies against BTV with a previously published competitive ELISA.²⁷ Briefly, polyclonal rabbit anti-BTV serum was used to coat the wells of microtiter plates and to capture baculovirus expressed BTV viral protein 7 antigen. Mouse monoclonal antibody specific for viral protein 7 of BTV diluted 1:50 and test serum samples diluted 1:5 were allowed to compete for binding to the captured viral protein 7 antigen. This was followed by reaction with biotinylated goat anti-mouse antibody and peroxidase-conjugated streptavidin. Optical density measurement of the color change following addition of ortho-phenylene diamine allowed calculation of the percent inhibition of the mouse monoclonal antibody binding to the captured antigen. Sera were scored positive for antibodies against BTV if the optical density was ≤ 35% of the negative control. Thirty blood samples were also assayed by the Wyoming State Veterinary Laboratory in Laramie, Wyo, by use of a commercial competitive ELISA BTV kit^b and gave identical results.

Relative serum BTV-specific antibody titers were determined for blood samples from 16 seropositive sheep from ranches A, B, and D and for blood samples from 20 seropositive sheep from ranch E. Serial 2-fold dilutions were made of each serum sample and assayed in duplicate by use of the competitive ELISA. Titer units were calculated as the reciprocal of the highest serum dilution that was positive on the competitive ELISA. Mean titers were calculated for each ranch, and the differences among mean values were analyzed.

Virus serum neutralization—Virus neutralization was used to determine the serotype of BTV for ranch B. A 1:10 dilution of serum samples from 8 seropositive and 8 seronegative lambs was used to test for virus neutralization of BTV serotypes 11 and 17, and epizootic hemorrhagic disease virus serotype 2 by use of a previously described protocol.²⁸

Virus isolation—All samples were tested by virus isolation in duplicate and on 2 cell lines. Briefly, 25 μL of RBCs or buffy coat diluted in 75 μL of media was added to 100 μL of cattle pulmonary artery endothelial or Vero cells (2.5 × 10⁵ cells/mL) in 96-well plates. All cells were incubated for 7 days at 37°C with 5% to 6% CO₂. Cells were checked for cytopathic effect after 48 hours and at 7 days. The RNA was extracted from wells that had cells with a cytopathic effect, and homogenate from these wells was passed onto fresh cells.

PCR assay—Total RNA was extracted from cell cultures that had a cytopathic effect and from all original sheep RBCs with a kit^c and eluted in 30 μL of nucle-ase-free water by following the manufacturer's protocol. Extracts were tested for BTV by use of a previously published infared RT-PCR method,²⁹ and PCR products were separated by 3% agarose gel electrophoresis under UV light with nucleic acid stain.^d Blood samples were also analyzed by use of an improved RNA extraction and a real-time RT-PCR as previously described.^29,30

Statistical analysis—Confidence intervals for sero-logic testing results were calculated by use of a software program^e on the basis of a 90% confidence level and a 5% margin of error. Difference in serum BTV-specific antibody titers of sheep among ranches was determined by use of a 2-tailed Student t test. All values of P < 0.05 were considered significant.

Results

Ranch A had the highest morbidity (500/1,404 [36%]), case fatality (175/500 [35%]), and total mortality (175/1,404 [12%]) rates, as determined on the basis of ranch records. Disease, with signs of lameness, lethargy, and lack of appetite, was first noticed in the rams pastured at the ranch near irrigated land but was not recognized as being caused by BTV infection. The ewe and lamb flock returned from mountain pastures in September and began to have signs of illness within 10 to 14 days, with sheep continuing to be newly affected into early November. Signs of lameness and reluctance to stand were quickly followed by swollen lips, tongue, and face, with salivation and nasal discharge. Sick sheep were treated with an NSAID (flunixin me-glumine) and antimicrobial (oxytetracycline) and were tube fed water. For ranch A, 93% (14/15) of tested rams were seropositive for BTV, while 27% (49/182) and 40% for BTV, and 1 of 3 suckling kids was seropositive.

Ranch B is roughly 24 km (15 miles) from ranch A and also had rams becoming ill in August before the outbreak was recognized and diagnosed as BT. Lameness and depression were noted, and 4 of 56 rams died. Rams were pastured between 2 branches of a creek and near irrigated pastures. Ewes and lambs became ill after returning to the ranch in September from mountain pasture. This ewe flock was pastured approximately 16 km (10 miles) from the ram pasture and had less exposure to small creeks and irrigated fields. The rancher recorded clinical disease in 14% (233/1,679) of the ewe and lamb flock with a 3% (7/233) case fatality rate. Sick sheep were removed from the flock and treated by the sheep herders with an NSAID (flunixin meglumine), an antimicrobial (oxytetracycline), electrolytes, citrus fruit rubbed on oral lesions, and silage offered as a soft food source. For ranch B, 90% (18/20) of tested rams were seropositive for BTV, while 4% (8/201) and 5% (8/170) of tested ewes and lambs, respectively, were seropositive (Table 1). Virus serum neutralization was used to confirm virus serotype; sera from 9 of 10 sheep seropositive for BTV neutralized BTV-17 but not BTV serotype 11 or epizootic hemorrhagic disease virus serotype 2. Mean ± SEM serum BTV-specific antibody titers for the ranch B rams, ewes, and lambs were 610 ± 168, 853 ± 213, and 1,120 ± 160, respectively

Limited testing was done on ranch C to compare infection rates between ewes and rams when both spent the summer in ranch pastures within 1 mile of each other. All rams were tested, but only a pen of nonpregnant, poor-doing, or late-to-lamb mature ewes was tested. Of these sheep, 100% (9/9) of the rams were seropositive for BTV and 86% (18/21) of the ewes were seropositive (Table 1).

For ranch D, located east of the Big Horn Mountains (an area with BT in 2005), 34% (40/119) of mature ewes were seropositive for BTV and had a mean ± SEM serum BTV-specific antibody titer of 176 ± 82 (Table 1). For ranch E, located within the Black Hills region of Wyoming, 14% (4/29) and 16% (17/109) of the rams and ewes, respectively, were seropositive for BTV⁷. Mean ± SEM serum BTV-specific antibody titers for the rams and ewes of ranch E were 123 ± 75 and 302 ± 57, respectively. On ranch F, no ewes or yearling lambs were seropositive for BTV; none of the 2008 spring-born lambs were seropositive for BTV in the fall of 2008. Testing of blood samples yielded negative results for BTV by virus isolation and for viral RNA detection by RT-PCR assay.

Discussion

Woolgrowers suffer substantial economic losses because of BTV and the resulting quarantine that prevents movement of sheep at the time lambs are usually sent to market. Ranchers had specific questions that included whether the disease was likely to recur the next summer, whether their flocks would be immune to new infection, and whether vector control programs at the premise or animal level could help to limit the spread of disease. The current knowledge of BTV is limited, in part, because it is difficult to reproduce the disease experimentally. Most of the current knowledge of BTV infection and vector spread is based on information from experimental infections with small numbers of animals and on assumptions as to the mobility of the vector. This outbreak was a chance to collect data from a large number of sheep that had been infected in a natural epizootic and to give the woolgrowers information to help manage future outbreaks.

We had hypothesized that BTV was endemic throughout Wyoming and expected to find sheep seropositive for BTV from all ranches tested. Mature sheep were expected to have a higher seropositivity as a result of multiple years of possible exposure. We found, however, that among lambs born in 2007, there was the same number of seropositive sheep as in other age groups, suggesting an introduction into a naïve population. This could also have contributed to the severity of disease, although the possibility of a more virulent form of the virus cannot be ruled out as a contributing factor and would require further studies to identify^31,32 There were no seropositive sheep from ranch F in an area that had no history of BT.

We had also hypothesized that there were a large number of subclinical infections in the affected flocks and thus most of the sheep would be seropositive for BTV and resistant to disease the following summer. However, even for ranch A, which had the highest morbidity and mortality rates, the flock was over 60% seronegative, and in fact, the number of seropositive sheep reflected the number recorded as clinically sick. Ewes and lambs on ranch B were tested from the flock with the sick sheep removed and had a seroprevalence of < 5%, also indicating there were few subclinical infections. Both of these flocks could be susceptible to infection if conditions favored BTV recurrence in the next summer.

A notable finding in this outbreak was the apparent lack of exposure of the flocks pastured in the mountains until they returned to the lower elevation valley in the fall. This is not surprising because the higher elevation pastures had cooler temperatures less favorable for the vector and rapid virus amplification. The mountains surrounding the Big Horn Basin have likely been a natural barrier to infected vectors from endemic regions of the state. The introduction of BTV into the Big Horn Basin causing the 2007 outbreak could have occurred by the movement of infected animals (domestic or wildlife), or vectors with the help of weather events or manmade transportation.

As a disease borne by a flying insect, there is controversy as to the distance the vector for BTV is likely to travel and therefore the utility of animal and ranch-level vector control programs. With this outbreak, there were significant differences in the infection rate in flocks separated by only a few miles, suggesting that effective repellents applied to the animals or implementing site level vector control strategies might be of use in a BT outbreak. Further research to identify effective repellents for application to the animal and local vector control is needed.

An unexpected finding was the near 100% infection rate in the rams. The location of the pastures and earlier exposure to the virus are a likely cause. However, ram infection rates were still significantly higher than that of ewes kept in pastures separated by < 1 mile throughout the summer. There are no published reports on differences in susceptibility to infection in males versus females and a possible explanation for the higher rates in rams is increased vector biting opportunity because of the large scrotal surface. In the study reported here, all blood samples had negative results for BTV isolation and detection of BTV RNA.