Electrophoresis analysis was performed on material from a 10% polyacrylamide gel run with 10 μL of culture supernatant Selleck Quisinostat per well containing 0.55 μg μL−1 of protein. Subsequent Western blotting analysis was performed as previously described [25]. The rRmLTI antigen

expressed in P. pastoris was adjuvated with Montanide ISA 61 VG (Seppic, Paris) and doses of 2 mL containing 100 μg of the recombinant protein prepared. One-year old Holstein calves were randomly distributed into two groups of six animals each. One group was immunized with rRmLTI antigen purified and formulated as described above. The second group (negative controls) was injected with adjuvant/saline alone. Serum samples were collected and processed, and all procedures involving selleck chemical ticks were performed according to methods described previously [17]. Sera obtained immediately before the initial injection, and at different time points thereafter, from each of the six cattle in the vaccinated and control groups were pooled and stored in an ultralow freezer until ready for ELISA testing. For ELISA, microtiter plates were coated with 1 μg mL−1 of rRmLTI antigen in 20 mM sodium carbonate buffer (pH 9.6), 50 μL per well, and incubated overnight at 4 °C. Duplicate samples of pooled sera for each group and sampling date

were tested. Subsequent procedures were performed as described previously [25]. Procedures described before were followed to assess treatment effects on tick biology and vaccine efficacy

[17] and [26]. Engorged female ticks dropping off of cattle from each group were collected daily for 13 days and incubated in the laboratory to obtain eggs that were pooled until 1 gram was accumulated. The egg masses obtained per collection day from several ticks in each group were incubated to determine hatching rates. Serum samples from bovines immunized with rRmLTI were collected just prior to tick infestation and subjected to affinity chromatography on a protein A-Sepharose column to purify immunoglobulin G (IgG). The IgG eluted from the column resulted in a yield of 5 mg mL−1 of non-immune serum. Four groups, each consisting of ten adult female ticks, were set up for treatment. Each tick in the respective ADAMTS5 group was fed with the antibody mixture containing 0, 25, 50, or 100 μg of purified IgG in 20 μL by placing a capillary tube in its hypostome. The effect on egg eclosion was assessed as described previously [17]. Data on female reproductive parameters were analyzed using a t-test. Otherwise, differences between means were determined using one-way analysis of variance (ANOVA). Differences were considered significant when p < 0.05. Identity of the DNA insert in pPICZαRmLTI was confirmed by sequencing and alignment with the RmLTI clone sequence. One Mut+ clone was selected and analysis of the induced recombinant protein revealed a band of approximately 46 kDa. The calculated molecular weight for rRmLTI was 37.9 kDa.