Transfected <i>Babesia bovis</i> Expressing a Tick GST as a Live Vector Vaccine

<div><p>The <i>Rhipicephalus microplus</i> tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including <i>Babesia bovis</i>, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live <i>B</i>. <i>bovis</i> vaccine expressing the protective tick antigen glutathione-S-transferase from <i>Haemaphysalis longicornis</i> (HlGST). The <i>B</i>. <i>bovis</i> S74-T3B parasites were electroporated with a plasmid containing the bidirectional <i>Ef-1α</i> (<i>elongation factor 1 alpha</i>) promoter of <i>B</i>. <i>bovis</i> controlling expression of two independent genes, the selectable marker <i>GFP-BSD</i> (<i>green fluorescent protein–blasticidin deaminase</i>), and <i>HlGST</i> fused to the <i>MSA-1</i> (<i>merozoite surface antigen 1</i>) signal peptide from <i>B</i>. <i>bovis</i>. Electroporation followed by blasticidin selection resulted in the emergence of a mixed <i>B</i>. <i>bovis</i> transfected line (termed HlGST) in <i>in vitro</i> cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the <i>Ef-1α</i> locus. A <i>B</i>. <i>bovis</i> clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of <i>B</i>. <i>bovis</i> designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the <i>Ef-1α</i> locus of <i>B</i>. <i>bovis</i> in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in <i>R</i>. <i>microplus</i> feeding on HlGST-Cln-immunized calves. Collectively, these data show the efficacy of a transfected HlGST-Cln <i>B</i>. <i>bovis</i> parasite to induce detectable anti-glutathione-S-transferase antibodies and a reduction in tick size and fecundity of <i>R</i>. <i>microplus</i> feeding in experimentally inoculated animals.</p>