Odocoileus virginianus

All experiments were performed with C. sonorensis females from the Ausman colony [31 (link)], which were reared using established protocols [32 (link)]. EHDV-2 virus stock (ID no. CC12-304) was prepared from the spleen of an infected white-tailed deer from Kansas in 2012. The virus was isolated in calf pulmonary artery endothelial (CPAE; American Type Culture Collection, Manassas, VA, USA) cells and passed twice in baby hamster kidney (BHK; American Type Culture Collection) cells before purification by centrifugation through a 25% sucrose cushion at 28,000 g for 1 h. For oral infections, adult female midges (3–4 days post eclosion) were allowed to feed for 1 h on a mixture of equal volumes of commercial defibrinated sheep blood (Lampire, Everett, PA, USA) and EHDV-2 virus suspension (10^7.2 plaque forming units (PFU)/ml in 199E cell culture medium, with a final concentration of 10^6.9 PFU/ml EHDV-2) using an artificial feeding apparatus with parafilm as a membrane. Engorged females were separated immediately and placed into cages in groups of 80 per cage.
To provide positive controls for immunohistochemical analysis, infections were also performed by intrathoracic (IT) inoculation. For IT inoculation, 3–4 day old female midges were injected as described previously [33 (link)] with 50 nl of the same EHDV-2 virus stock. After oral or IT infection, all midges were kept at 25°C and fed 10% sucrose solution ad libitum.

Our study area encompassed approximately 2,300 km² of the Southern Yellowstone Ecosystem (SYE), inclusive of Grand Teton National Park (United States Park Service), the National Elk Refuge (United States Fish and Wildlife Service), and the Bridger-Teton National Forest (United States Forest Service) north of the town of Jackson, Wyoming (Figure 1). Elevations in the study area ranged from 1,800 m in the valleys to > 3,600 m in the mountains. Plant communities included cottonwood (Populus angustifolia) riparian zones interspersed with sagebrush (Artemisia spp.) uplands at lower elevations. At intermediate elevations, aspen (P. tremuloides), Douglas-fir (Pseudotsuga menziesii), and lodgepole pine (Pinus contorta) were the dominant species. Spruce (Picea engelmannii) and fir (Abies lasiocarpa) were the primary tree species at the higher elevations [25 ]. The area was characterized by short, cool summers and long winters with frequent snowstorms. Precipitation occurred mostly as snow, and mean maximum snow depths ranged from 100 cm at lower elevations to > 245 cm at intermediate and higher elevations (2,000 m +).
The study area supported a diverse community of large mammals. Carnivores included wolves, black bears (Ursus americanus), grizzly bears (U. arctos), coyotes (Canis latrans), and red foxes (Vulpes vulpes). Ungulates included elk, mule deer, moose, bison (Bison bison), pronghorn, bighorn sheep, and a very small number of white-tailed deer (Odocoileus virginianus). Deer, elk, bighorn sheep, and pronghorn exhibited seasonal migrations [7 (link),17 ,18 (link),19 (link)].

Ticks were acquired from the Oklahoma State Tick Rearing Facility (OSU) (Stillwater, OK, USA). Equal numbers of each sex and species (I. scapularis and A. americanum) were obtained. For each lot of I. scapularis and A. americanum and prior to shipment to the study site, OSU screened a subsample of ticks (n = 10) for pathogens using standardized PCR assays. Ixodes scapularis were screened for B. burgdorferi and Anaplasma phagocytophilum. Amblyomma americanum were screened for the presence of Ehrlichia chaffeensis, Francisella tularensis and Rickettsia rickettsii. All PCR-screened ticks were negative for the above pathogens. Once ticks arrived at the study site, they were housed in an industry-standard desiccator with the relative humidity maintained at > 90% until enclosed in a feeding capsule for attachment to deer.
The feeding capsules utilized in this study were specifically designed for holding blood-feeding I. scapularis and A. americanum. Feeding capsules allow for the containment and localization of ticks and aid in facilitating blood-feeding [40 (link)]. The traditional stockinet sleeve method for feeding ticks on cattle [41 (link)–43 ] was determined to be inadequate for white-tailed deer. We instead developed a feeding capsule for deer application, which was in part based upon feeding capsules for ticks (referred to hereafter as tick feeding capsules) previously designed for tick-feeding on rabbits and sheep [44 ]. To make each capsule, sheets of ethylene–vinyl acetate foam were cut into three square pieces. Each square had a different outside area, allowing for flexibility (base, approx. 12 × 12 cm; middle, approx. 9 × 9 cm; top, approx. 7 × 7 cm), and had a combined depth of approximately 18 mm. The center of each square was cut away, creating an opening. The inner surface areas of the base and middle piece openings were each approximately 7 × 7 cm; the top piece had a smaller opening (approx. 1.5 × 1.5 cm) through which the ticks were to be inserted, which decreased the probability that ticks would escape through the top of the capsule (Additional file 3: Figure S2).
Deer were anesthetized using an intramuscular injection of telazol and xylazine at dosages of approximately 3 mg/kg and approximately 2.5 mg/kg, respectively. Once fully anesthetized, deer were weighed to the nearest 0.1 kg using a certified balance. Prior to blood collection and capsule attachment, large patches of fur on the neck were trimmed using electric horse clippers (Wahl®; Wahl Clipper Corp., Sterling, IL, USA). Prior to capsule attachment, 10 ml of blood was collected from the jugular vein of each deer using a 20-gauge needle. The blood from each individual deer was immediately placed into a vacutainer containing EDTA and was centrifuged for 10 min at 7000 revolutions/min. The plasma was transferred to 1.5-ml centrifuge tubes, which were then stored at − 20 °C until analysis.
Two identical tick feeding capsules were attached to opposing sides of the neck of each deer using a liberal amount of fabric glue (Tear Mender, St. Louis, MO, USA). Each capsule was held firmly in place for > 3 min to allow it to adhere to the skin and fur. For each deer, 20 I. scapularis mating pairs were placed within one capsule, and 20 A. americanum mating pairs were placed within the second capsule. Prior to tick attachment, 20 ticks (all same species and sex) were placed into a modified 5-ml syringe. Ticks were chilled in ice for approximately 5–10 min to slow movement. The 20 mating pairs were then carefully plunged into the capsules and a fine mesh lid was applied and reinforced with duct tape. Representative photos and video of the tick attachment process are presented in Fig. 2 and Additional file 4: Video S1, respectively. The capsules were further secured to deer by wrapping the neck with a veterinary bandage (3 M Company, St. Paul, MN, USA).Fig. 2

Tick capsule attachment and tick attachment. a Female ticks being plunged into capsule, b plunger being removed prior to mesh lid being secured, c completed, secured capsule being checked to ensure all corners are adhered to the neck, d closeup of completed capsule containing 20 Ixodes scapularis mating pairs

After completion of capsule and tick attachment, deer were given tolazine via intramuscular injection at a dose of 4 mg/kg to reverse the effects of the anesthetic. Deer were then housed in individual pens, observed closely until they were mobile and moving normally and monitored routinely for the remainder of the day.

Deer were assigned to groups using a random sequence generator, and groups were differentiated based on: (i) test group identity (treatment group [T], control [C]); and (ii) the length of the exposure (48 h [T48], 120 h [T120]) (Additional file 1: Table S1). While explicit guidelines for white-tailed deer are not available, federal guidelines recommend a sample size of 6–10 subjects per test group when evaluating pesticides against pests of humans and pets, such as fleas and ticks [39 ]. The size of the captive herd and the number of deer that its managers could afford to donate to this project limited the sample size that we were able to utilize, and we were unable to have an equal number of males (n = 15) and females (n = 9). It was determined that each test group could comprise eight animals (n = 24). A total of 16 deer were offered FDF, with eight deer being exposed to FDF for 48 h and eight deer being exposed to FDF for 120 h. An additional eight deer served as an untreated control group, with 50% of animals exposed to placebo for 48 h and 50% exposed to placebo for 120 h. Deer continued to be housed in the individual pens during the exposure period. Prior to tick attachment, deer within each test group were additionally assigned to subgroups, with 50% of deer to be parasitized with ticks at day 7 post-exposure to FDF, and 50% to be parasitized at day 21 post-exposure to FDF (4 animals/subgroup).