Transgenic Babesia bovis lacking 6-Cys sexual-stage genes as the foundation for non-transmissible live vaccines against bovine babesiosis

Babesia bovis, a tick-borne apicomplexan parasite responsible for bovine babesiosis has a complex life cycle including sexual development in its Rhipicephalus microplus vector. Understanding the molecular mechanisms involved in sexual development is essential for developing future-generation transmission blocking vaccines (TBVs) and/or non-transmissible attenuated live vaccines. The widely conserved members of the 6-Cys gene family likely play roles in the development of sexual stages of B. bovis, and are candidates for developing novel TBV. The recently defined sexual markers 6-CysA and 6-CysB of B. bovis are strain-conserved and exclusively surface-expressed in tick-stage parasites. However, the high level of sequence identity among the 6-Cys A and 6-Cys B proteins (52% identity), together with similar 6-Cys domain distribution and sub-cellular localization, are suggestive of redundant function. We hypothesized that disruption of both 6-CysA and 6-CysB in B. bovis would result in unaltered ability of the parasite to invade and grow in red blood cells (RBCs), with concomitant loss of the transmission phenotype. Taking advantage of their contiguous genome localization, we generated a double gene-knockout system to disrupt a 3287 bp region encompassing both 6-CysA and 6-CysB genes using a single transfection plasmid. The resulting red-fluorescent Delta A Delta B 6-Cys B. bovis transgenic parasite line was able to grow continuously in bovine RBCs in vitro at a similar rate to wild-type parasites, demonstrating that the 6-CysA and 6-CysB genes are not required for the development of blood-stage parasites. This novel gene manipulation approach will allow future experiments aimed at determining the tick-transmission phenotype of parasites lacking tick-stage genes. Parasites deficient in genes required for sexual reproduction could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis. Developing a non-tick transmissible live vaccine based on attenuated parasites unable to express critical 6-Cys genes and including a molecular vaccine marker could help reduce the burden of bovine babesiosis globally.