Jugular Vein

Large volume serum samples were available from six (3 male and 3 female) domestic short-haired cats experimentally infected at 10 months of age with Dirofilaria immitis by subcutaneous inoculation of third-stage larvae (L₃) and confirmed to be infected by recovery of adult worms at necropsy or confirmation of histological lesions. Briefly, trickle infection of a total of 100 L₃ of D. immitis was performed by subcutaneous inguinal inoculation of each cat a total of four times, on study days 7, 14, 21, and 28, with 25 L₃ (Missouri strain) harvested shortly prior to inoculation from infected Aedes aegypti mosquitoes (Liverpool strain). Whole blood samples were collected from the jugular vein or, rarely, the cephalic vein, of each cat on days 84, 112, 140, 168, 196, and 224 directly into vacuum tubes containing either EDTA or no additive. Cats were cared for through Oklahoma State University’s Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal resources program throughout the study; all research procedures followed a detailed animal care and use protocol approved by Oklahoma State University’s Institutional Animal Care and Use Committee. Anti-coagulated whole blood was assayed for microfilaria by modified Knott’s test and by real-time PCR for Wolbachia spp. as previously described [11 ,12 (link)]. For tubes without additive, blood was allowed to clot, the serum separated by centrifugation, placed into aliquots, and stored at −80°C until further use.

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.

At the conclusion of the tick observations on day 8 post-attachment, fresh fecal samples were collected from each test deer pen. Additionally, internal tissues were collected from each deer in each treatment group. The deer were first sedated by injection of 1–2 mg/kg xylazine hydrochloride (100 mg/ml) into the large muscle bellies of the rump/rear limbs. While sedated, deer were euthanized by intravenous injection, administered via the jugular vein, of 86 mg/kg Euthasol (pentobarbital sodium, 390 mg/ml), resulting in pentobarbital sodium overdose. Death was confirmed by a combination of the following: (i) lack of heartbeat based on auscultation with a stethoscope; (ii) lack of respiration based on visual inspection of the thorax; (iii) lack of corneal reflex; and (iv) lack of response to firm toe pinch. All euthanasia was performed by the attending veterinarian exclusively.
Various tissues were collected from euthanized deer. The objective was to collect tissues similar to what would be collected by hunters when field dressing a killed deer. Thus, we focused on specific meat cuts, meat by-products and fatty tissues. Approximately 50 g of each tissue was surgically removed using disposable scalpels. Scalpels and surgical gloves were replaced between each individual tissue collection to minimize the risk of contamination. Each tissue was transferred to an individual biological specimen bag (Keefitt®), which was immediately stored at − 20 °C until analysis. In addition to collecting tissues from 16 deer in the treatment group, we collected tissues from two deer in the control group to establish a baseline and for analytical method development.
Tissues, plasma and feces were delivered to CSU for method development and analyses, and analyzed for the presence of fipronil and fipronil metabolites using validated methods of liquid chromatography/mass spectrometry (LC/MS). A list of tissue classifications, the maximum residue limits (MRL) listed by the US Environmental Protection Agency (EPA) for fipronil in cattle and the explicit tissue identifications are presented in Additional file 6: Table S2.
Critical study dates for each test deer (acclimation, exposure, post-attachment, capsule checks, tissue collection) are presented in Additional file 7: Table S3.

A temporary filter was inserted via the nonaffected femoral or jugular vein into the inferior vena cava (IVC) prior to the next procedure for patients with an extensive thrombus in the proximal vein that was evaluated as potentially life-threatening and was retrieved after the proximal DVT was removed and potentially life-threatening conditions were relieved. Consistent with local routines based on published guidelines [9 ], anticoagulant treatment was initiated immediately when DVT was identified with the use of subcutaneous low molecular weight heparin (LMWH) at a bolus dose of 100 units/kg twice daily. PTA and/or stent placement was encouraged for lesions that caused 50% or greater diameter narrowing of the iliac and/or common femoral vein, robust collateral filling, and/or a mean pressure gradient of more than 2 mmHg. At the end of LMWH, oral rivaroxaban was directly commenced at a dosage of 15 mg twice a day over the subsequent 21 days and 20 mg once a day thereafter for at least 6 months. In addition, the use of compression stockings (ankle pressure was approximately 30–40 mmHg) for more than 1 year was recommended.