Some experimental studies used this approach against
tick infestations , , , , , ,  and ; however, in most cases, this strategy resulted in a statistical significant but slightly improvement in protection level. Although tick infestation experiments using bovines in confined indoors can indicate vaccine efficacy, field trials Epigenetics inhibitor are necessary to evaluate vaccine performance under real husbandry conditions . However, most of the protocols used in experiments to evaluate bovine vaccination against ticks employ confined bovines, a more practical and cost-saving approach, compared to field experiments which demand laborious handling of cattle and the availability of a large area  and . Our research group has been studying several R. microplus molecules in order to find antigens that could be used in an anti-tick vaccine. In previous studies, immunizations of cattle with native or recombinant forms of an aspartic protease named BoophilusYolk pro-cathepsin (BYC) induced overall protections click here (measured
by the reproductive potential, including reduction in number and weight of engorging ticks and in egg weight and hatchability) around 30%  and . Also, immunization with a R. microplus cysteine endopeptidase (VTDCE), involved in vitellin digestion  and , elicited an immunoprotection of 21% in vaccinated cattle . More recently, an overall protective efficacy of 57% against R. microplus was achieved using a
recombinant Haemaphysalis longicornis GST (rGST-Hl) . In this work, we evaluated a multi-antigenic vaccine composed by BYC, VTDCE and GST-Hl recombinant proteins against R. microplus infestation in cattle. Vaccine efficiency was evaluated under field conditions, based on semi-engorged female tick numbers and weight gain differences between vaccinated and control cattle groups. rGST-Hl, rBYC, and rVTDCE were expressed and purified as previously described ,  and . Briefly, rBYC and rGST-Hl were expressed in Escherichia Parvulin coli strain AD494 (DE3) pLysS. Recombinant VTDCE was expressed in E. coli strain BL21 (DE3) Star. The insoluble forms of rBYC and rVTDCE were solubilized with 6 M guanidine hydrochloride (GuHCl) and purified using a nickel-chelating Sepharose column (GE Healthcare, Uppsala, Sweden). The soluble form of recombinant GST-Hl was purified through affinity chromatography using GSTrap FF column (GE Healthcare, Uppsala, Sweden). Protein concentrations were determined by the Bradford method  and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using bovine serum albumin as standard.