Cattle and ticks
All animal use was approved by the University of Idaho Institutional Animal Care and Use Committee (IACUC, protocol #2016–27). Six age-matched male Holstein calves were acquired from the University of Idaho dairy. Calves were ≥ 2 months of age with ≥ 80 kg body weight at the start of the study. This cohort of cattle had no previous exposure to R. microplus, since the tick does not occur in the United States north of the quarantine zone along the border with Mexico. Animals were randomly assigned to either the control or vaccine group (three per group) and vaccinated according to the protocol described below. At the end of the vaccination protocol, cattle were moved to individual moated concrete block stalls for the tick challenge. For the tick challenge (see below) the La Minita strain of R. microplus was used. This tick colony originated from an outbreak tick population in Star County, Texas [25, 26], and has been maintained continuously (3–4 generations per year) at the Animal Disease Research Unit tick lab at the University of Idaho since it was acquired from Texas.
Peptides
Three peptide sequences from the predicted extracellular domains of the protein coded for by the RmAQP2 gene (GenBank accession numbers: protein ALJ75650, DNA Sequence KP406519) have been described previously [22]. Peptide 1 was modified from the previously published sequence by adding four additional amino acids to encompass a predicted B-cell epitope and possibly increase its antigenic potential (AVFQLGSVGLAAAP). The amino acid sequences of peptides 2 and 3 were as described previously (#2: ADALSQVDVNLAIVYGTNATAPVFSCFPAPGV, #3: MCGWGSAVFSFRSYNWFWV) [22]. Peptides were commercially synthesized (New England Peptide, Gardner, MA, USA) and supplied either as free peptide or conjugated to carrier molecules. For vaccination, peptides were conjugated to keyhole limpet hemocyanin (KLH) as a carrier to stimulate immune response. For use in enzyme-linked immunoassay (ELISA; see below) peptides were conjugated to bovine serum albumin (BSA). KLH-conjugated peptides were supplied lyophilized and were initially dissolved by adding 50:50 dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS), then taken to 1 mg/ml concentration with PBS for a final DMSO concentration of 20%. Unconjugated peptides were solubilized by adding 75:25 DMSO/sterile water and then taken to a 1 mg/ml concentration with PBS for a final DMSO concentration of 35%. BSA-conjugated peptides were obtained solubilized in PBS at 1 mg/ml concentration.
Vaccination protocol
Cattle were each vaccinated four times at 3-week intervals (days 0, 21, 42, and 63); see Fig. 1 for a graphical representation of the experimental timeline. The initial three vaccinations were done with the peptides conjugated to KLH; the fourth injection (day 63) was with peptides alone, not conjugated to KLH, in order to boost the peptide-specific immune response in the absence of KLH. Each injection consisted of 0.05 mg of a single conjugated peptide and 0.75 mg Quil-A (saponin) adjuvant in a volume of 0.5 ml. On the day of vaccination, each animal received three injections, each with a different peptide, at three different injection sites. Individual peptides were mixed with adjuvant by drawing repeatedly through a 21-gauge needle to mix well. All three doses of each peptide were mixed in a single tube, allowing an extra dose for loss in mixing and in the needle (1 ml 0.2 mg/ml peptide plus 1 ml 3 mg/ml Quil-A). Each injection was administered subcutaneously at a different injection site to avoid any potential antigenic competition at the draining lymph node (injection sites were left neck, right neck, right flank). The controls were injected with saponin mixed with KLH (MilliporeSigma, St. Louis, MO, USA) alone (no conjugated peptide), in the same way as the KLH conjugated peptides were prepared and injected (i.e., three injections at three different sites).
Tick challenge
One week after the final peptide boost, the cattle were challenged with larval ticks. A tick-feeding patch was adhered to the back of each calf with cattle hip tag cement as described previously [27], and each patch was infested with ≈5000 larval ticks, which is the approximate number of larvae hatching from 0.25 g of eggs. Larval ticks were prepared for challenge by weighing aliquots of 0.25 g of eggs all originating from the same colony rearing; the eggs were from a mixture of egg masses from a large number of females which had all been mixed and weighed on the same day to ensure uniformity. Because R. microplus is a one-host tick, larvae will develop through the larval, nymphal, and adult stages on the same host. Feeding patches were opened on days 5 and 9 post-application to assess attached larvae and newly molted nymphs, respectively, as they progressed through stages of feeding and development. After replete females began to detach at about day 20, the patches were opened daily, and all repletes that had dropped in the previous 24-h period were removed. Daily collections were repeated until most ticks had completed feeding; on the final day all remaining attached ticks were removed and counted.
The total number of detached replete (fully fed) ticks collected each day was recorded. A random sample of up to 72 ticks each day were weighed and saved in individual wells of 24-well tissue culture plates. On days when there were fewer than 72 ticks, all were weighed and saved. Saved ticks were held for egg production, and when all oviposition was complete, egg mass weights were recorded. A subsample of eggs from up to 48 of these ticks per day was set aside individually to assess hatching rate. A daily sample of 10 replete females from each calf was saved for protein determination (see below). All remaining ticks were discarded after they were counted.
Antibody titers
To assess antibody titers and to determine what titers the ticks were actually exposed to, blood samples were taken from cattle before the first vaccination (pre-bleed) and 2 weeks after each subsequent vaccination, then weekly once the tick challenge began (days 0, 14, 35, 56, 70, 77, 84, 91, and 96). Blood was collected in red-top Vacutainer tubes and allowed to clot before being centrifuged to separate serum. An ELISA was developed to track antibody titers in response to vaccination. Nunc Polysorp 96-well flat-bottom immuno-plates (Thermo Scientific, Rochester, NY, USA) were coated with BSA-conjugated peptides. Each BSA-conjugated peptide was diluted to 0.02 mg/ml in carbonate-bicarbonate coating buffer (4 mM Na2CO3, 9 mM NaHCO3, pH 9.4); 50 µl of this peptide working solution was added to each well and held overnight at 4 °C to coat the wells with approximately 1 µg/well of peptide available to bind. Plates were washed five times by hand with 1× PBS/0.05% Tween-20 to remove unbound peptide after coating, then blocked with 225 µl/well of 1× PBS/0.05% Tween-20 plus 5% BSA (blocking buffer) for 2 h.
Diluted samples (50 µl) were added to the plates in triplicate wells and allowed to incubate for 1 h at room temperature. Unbound primary antibody was removed by washing five times by hand with 1× PBS/0.05% Tween-20, and 50 µl/well of a 1:500 dilution (in blocking buffer) of goat anti-bovine IgG (H+L) antibody-horseradish peroxidase (HRP) conjugate (Life Technologies, Frederick, MD, USA) was applied for 1 h at room temperature. Unbound secondary antibody was removed by washing five times by hand with 1× PBS/0.05% Tween-20. Plates were developed with SigmaFast OPD (MilliporeSigma, St. Louis, MO, USA) and read at 450 nm on a SpectraMax 190 microplate reader (Molecular Devices, San Jose, CA, USA).
Using the ELISA described above, we determined that a 1:256 dilution was optimal for use across all the available sample time points to show the change in antibody levels during the study. To determine the peak titers, the sample point with the highest antibody response at 1:256 for each peptide was chosen, and twofold serial dilution series of the serum samples from this day were tested to find the lowest dilution where the 95% confidence intervals for the pre-bleed and the test sample did not overlap. The reciprocal of the dilution at this point was considered to be the maximum antibody titer.
Measurement of bovine IgG1 and IgG2 using ELISA
Isotyping was completed similarly to the protocol above, with the difference that the secondary antibodies were un-conjugated and an additional HRP conjugate was used for detection. Specifically, the secondary antibodies were mouse anti-bovine IgG1 or mouse anti-bovine IgG2 (Bio-Rad Laboratories, Hercules, CA, USA) and the HRP conjugate was goat anti-mouse IgG (H+L) (Life Technologies, Frederick, MD, USA).
Protein content of replete female ticks
A random sample of 10 replete females were collected from each calf on each day after the ticks began dropping for a total of 6 days (60 ticks) from each calf. After weighing, replete female ticks were placed individually in tubes with 2 ml protein lysis buffer solution containing 0.05 M Tris, 0.005 M EDTA, 1% NP-40, and protease inhibitors (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, Roche, MO, USA), and frozen at −20 °C. In preparation for the assay, tubes were thawed, the tick bodies were punctured with a 16-gauge needle, and the tissues and solution were drawn into an attached 3-ml syringe approximately 15 times to thoroughly homogenize each sample. An aliquot of 200 μl was taken from each sample and centrifuged at 8000 rpm to pellet unlysed material (cuticle, tissue fragments, etc.), and 10 μl of the supernatant was diluted in 490 μl sterile water. Triplicate samples of 25 μl of each dilution were analyzed for total protein concentration using the Pierce BCA Protein Assay according to the manufacturer’s protocol (Thermo Scientific). A 1:50 dilution of protein lysis buffer above with sterile water was used as a diluent for the BSA standard curve. Completed plates were incubated at 37 °C for 30 min and read at 562 nm on a SpectraMax 190 microplate reader (Molecular Devices, San Jose, CA, USA). The basis for comparison between populations of ticks from vaccinated versus control cattle was total soluble protein as measured by the assay as a proportion of total tick weight.
Western blots
Ovaries from fed female R. microplus (3–4 days and replete) were dissected, placed in RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 °C. To prepare protein for the western blot, samples were thawed and RNAlater was carefully pipetted off the tissues. Protein lysis buffer (0.05 M Tris, 0.005 M EDTA, 1% NP-40, cOmplete™ mini EDTA-free protease inhibitor cocktail; Roche, MO, USA) was added to the tissues according to the manufacturer’s instructions. Tubes were sonicated using a cup horn (Fisherbrand Model 705, Thermo Fisher Scientific) for 6 × 15 s at 100% power and cooled on ice between steps. The prepared samples were then analyzed using a Qubit 3 Fluorometer (Thermo Fisher Scientific) to determine protein concentration, and 25 μl of a 1:10 dilution of replete female ovary protein was prepared to run on a NuPage 4–12% Bis–Tris gel (1.0 mm × 10 wells, Thermo Fisher Scientific) by adding 12.5 μl NuPage 4× LDS loading buffer, 5 μl NuPage 10× sample reducing agent, and 7.5 μl sterile water. The mixture was vortexed and heated in a dry block at 70 °C for 10 min. A 20-gauge ½-inch needle was used to carefully remove pre-cast lane dividers between four wells of the gel to form one large well. The gel was then secured in the Mini Gel Tank (Thermo Fisher), 1× NuPage MOPS buffer containing antioxidant was added to the inner chamber, and the same buffer without antioxidant was added to the outer chamber. Ovary protein was pipetted into the large open well of the gel, and a mixture of 2.5 μl PageRuler™ Plus pre-stained and 2.5 μl MagicMark™ XP western ladder (both Thermo Fisher Scientific) was pipetted into a separate adjoining single well. Electrophoresis was completed for ~ 50 min at a constant 200 V.
The proteins were then transferred to a nitrocellulose membrane using the iBlot gel transfer stack (Thermo Fisher Scientific) and Program PO (20 V for 1 min, 23 V for 4 min, 25 V for 2 min.) The membrane was blocked with 1× Tris-buffered saline (TBS) + 0.1% Tween-20 + 5% non-fat milk for 1 h at room temperature. Equal strips were cut from the blocked membrane, serum from calves was diluted 1:1 in 1× TBS + 0.1% Tween-20 + 10% non-fat milk and incubated with the membrane strips for 2 h at room temperature, rocking side to side gently. After primary incubation was completed, the strips were washed in 1× TBS + 0.1% Tween-20 twice immediately and then three times for 5 min each. Goat anti-mouse IgG (H+L)-HRP antibody (Thermo Fisher Scientific) was diluted 1:5000 in 1× TBS + 0.1% Tween-20 + 5% non-fat milk for the ladder lane and control strip. Rabbit anti-bovine IgG (whole molecule)-HRP antibody (MilliporeSigma, St. Louis, MO, USA) was diluted 1:2000 in 1× TBS + 0.1% Tween-20 + 5% non-fat milk for the bovine serum strips. Membrane strips were incubated with the secondary antibody dilutions for 1 h at room temperature, rocking gently. After secondary incubation was completed, the strips were washed as described above. Prometheus ProSignal Pico (Genessee Scientific, San Diego, CA, USA) components were prepared 1:1 for imaging the blot. Enhanced luminol solution was added to stabilized peroxide solution, mixed, and placed on the washed strips for 2 min. Strips were drained of excess reagent, arranged in a blot development folder, and imaged using a ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).
Analysis of data
Tick data (number and weight of replete females, weight of egg masses, and egg hatching, average protein content, etc.) were collected daily and summarized as the average for that day for each calf. The daily averages were combined as a single value for each animal across the full 6 days the ticks were dropping, and the values for the three vaccinated and three control animals were compared using Student’s t-test (implemented in Microsoft Excel).