Expression and Evaluation of Recombinant Babesia bovis Antigens of Vaccine Potential Against Tick Fever in Cattle

Babesia bovis is a causative agent of bovine babesiosis and is transmitted by vector ticks, Rhipicephalus (Boophilus) spp. The disease has a high mortality rate in susceptible cattle, causing serious economic loss. At present, the only commercial vaccine is culture-based with limited availability. No effective molecular vaccine has been developed to date. Generating a vaccine with specific critical epitopes responsible for protection against B. bovis is critically important. Immunity against B. bovis requires both innate and adaptive responses, with antigen-specific CD4+ T cells essential to the latter through production of IFN-?. Fourteen B. bovis proteins were selected as putative vaccine candidates and their full-length genes cloned for recombinant protein production intended for evaluating peripheral blood mononuclear cell IFN-? secretion level from experimentally infected animals in ELISPOT. All proteins expressed in insoluble form (inclusion bodies) and could not be purified. B. bovis genes were then truncated to exclude signal peptide and transmembrane regions, then cloned and expressed using pET101/D-TOPO in Escherichia coli to obtain soluble, useable proteins. Only recombinant B. bovis MSA1, MSA2b and MSA2a1 proteins were successfully expressed in soluble form. These proteins induce invasion-blocking antibodies in immunized cattle, are hypothesized to elicit protection in susceptible animals, but were previously studied by others.

Due to failure to produce new candidates to assay, the animal experiments were not performed. Instead, sera from field-infected cattle were assayed for reactivity against the MSA proteins by indirect immunofluorescent antibody (IFA) and western blot (WB) analysis. Field sera from South Texas (#41) and the Mexican Yucatan (#6, #9 and #11) along with positive and negative controls were tested. In IFA test, cattle #6, #9 and #41 were positive while #11 was negative. In WB, #41 and #6 reacted with the recombinant MSA proteins and with control B. bovis whole parasite lysate. However, both #11 and #9 had no signal in WB, although the latter was positive in IFA. Several theories may explain this phenomenon, such as the different preparation process of the antigen in the two tests, strain differences between sera and test antigens, or the different design and nature of each test.