Sheep

Microtitre plates (Immulon 4HBX, Thermo) were coated with recombinant MSP-1₁₉.GST (to which antibodies are predominantly of the IgG1 subclass [36 (link)]) or MSP-2.GST (to which antibodies are predominantly IgG3 [36 (link)]) and blocked with 1% (w/v) skimmed milk powder. Samples were assayed as described previously[36 (link),37 (link)] except that coating antigens, test samples and secondary antibody conjugate were each added in a total volume of 50 μl per well. Ten microliters of the antibody-containing eluate of each spot were added to individual wells of the coated and blocked microtitre plate together with 40 μl blocking buffer to give a final concentration of 1:1,000 with respect to the corresponding plasma sample. Each plate included a five-fold dilution series (1:50 to 1:156,250 final dilutions) of a standard African hyperimmune plasma pool. Bound antibodies were detected with either rabbit anti-human-IgG -HRP (Dako, Ely, UK), or sheep-anti-human IgG1 or IgG3-HRPconjugates (The Binding Site, Birmigham, UK) secondary antibodies and developed with o-phenylenediamine-H₂O₂.
A titration curve was fitted to the ODs obtained for the standard plasma dilutions by least squares minimisation using a three variable sigmoid model and the solver add-in in Excel (Microsoft), assuming an arbitrary value of 1000 Units/ml of antibody against each antigen in the standard pool. OD values for the spot extracts were converted to units/ml using this fitted curve.
Recoveries for blood spots were estimated as follows (full details in Additional file 1): serum or plasma ODs were converted to concentrations as above, the concentrations were multiplied by a recovery factor and then converted back to 'corrected' ODs – the ODs which would have been obtained if the serum or plasma had been more dilute. The value of the recovery factor was then optimized by weighted least squares minimisation comparing the actual ODs for the blood spots and the corrected OD values for serum or plasma, using the solver add-in in Excel™.

The ability of known COX-2 selective inhibitor celecoxib (1), 5-azido-pyraozles (5 and 14) and new triazole products (7–12, 16–25, 28, 29, 32, and 33) to inhibit ovine COX-1 and recombinant human COX-2 was determined using a COX inhibitor assay (Cayman Chemical, Ann Arbor, USA; item number: 700100) following the manufacturer’s protocol. Each compound was assayed in concentration range of 10⁻⁹ M to 10⁻³ M, in triplicate. PRISM5 software was used to calculate IC₅₀ values. In addition to celecoxib, both Dup-697 (potent COX-2 inhibitor) and SC-560 (potent COX-1 inhibitor) were used as internal controls during screening test compounds.

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.