Statistical significance differences among the experimental groups concerning level of antigen-specific
antibodies, tick count and cattle body weight gain was analyzed by Student’s t test. Data were expressed as mean ± S.E.M. of each group. A p value of less than 0.05 was considered significant. Statistical analysis was mTOR inhibitor performed using GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, USA) software. The recombinant proteins BYC, GST-Hl and VTDCE were expressed in E. coli strains and purified by affinity chromatography. The purity of the three recombinant proteins was analyzed by a 14% SDS-PAGE ( Fig. 1A). All preparations showed a major protein band for rBYC, rGST-Hl, and rVTDCE in the gel, and these bands matched the predicted molecular masses for respective proteins. Dot blot analysis revealed an increased antibody recognition level of vaccinated bovine sera (collected at day 78) to the three recombinant proteins, compared to the vaccinated
bovine pre-immune sera (day 1) (Fig. 2). Compared to day 1, the level of recognition from vaccinated cattle sera on day 78 for rGST-Hl, rVTDCE and rBYC increased by more than 6, 10, and 2 times, respectively. The level of recognition remained constant at the end of the experiment (day 127) for rGST-Hl, reducing by half for rVTDCE, and returning to pre-immunization level for rBYC. Also, the level of recognition measured from vaccinated cattle sera was approximately 8, 4, and 2.5 times higher for rGST-Hl, rVTDCE, and rBYC respectively, than those recorded from animals injected with placebo on day 78. Western blot revealed that sera from one representative bovine learn more of the vaccinated group recognize all recombinant proteins (Fig. 1B). The proteins rBYC, rGST-Hl and rVTDCE were not recognized by pre-immune serum of this animal. The reduction in the number of ticks attached to bovines conferred by immunization with rBYC, rGST-Hl and rVTDCE is shown in Fig. 3 and Table 1. In the first three counts, tick number means from both groups were similar. From the fourth count on (days 36–127), means in the two groups were statistically different, except for day 57. During this period, bovines
vaccinated with recombinant proteins showed statistical reductions that ranged from 35.3 to 61.6% (Table 1) in the number of semi-engorged ticks, second as compared with the control group. Interestingly, even before the immunization period had ended it was already possible to detect a drop in tick infestation (Fig. 3, day 36). Also, there was an increase in cattle body weight in both groups between days 1 and 127, although the gain was statistically higher in the vaccinated group (Fig. 4). In the vaccinated and control cattle groups, body weight gain was 39% and 25%, respectively. Tick vaccines derived from the gut antigen Bm86 have been extensively investigated in the quest for a suitable tick control method. This antigen was shown to be partially protective against R.