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

Authors: ALZAN, H - Washington State University; COOKE, B - Washington State University; Suarez, Carlos

Submitted to: Ticks and Tick Borne Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/22/2019
Publication Date: 1/25/2019
Citation: Alzan, H.F., Cooke, B.M., Suarez, C.E. 2019. Transgenic babesia bovis lacking 6-Cys sexual-stage genes as the foundation for non-transmissible live vaccines against bovine babesiosis. Ticks and Tick Borne Diseases. 10(3):722-728. https://doi.org/10.1016/j.ttbdis.2019.01.006.
DOI: https://doi.org/10.1016/j.ttbdis.2019.01.006

Interpretive Summary: Babesia bovis is a tick-borne apicomplexan parasite responsible for bovine babesiosis. B. bovis has a complex life cycle including asexual development in bovine red blood cells and sexual development in the tick (usually Rhipicephalus microplus) vector. A complete understanding of the molecular mechanisms involved in sexual development is essential to analyze gene function as a first step toward generating alternative transmission blocking and/or non-transmissible attenuated live vaccines (TBV). We previously identified the highly similar and contiguous 6-Cys genes A and B as markers for tick stages of the parasites. We also hypothesize that these two genes are required for the completion of the sexual stages of the parasite in the tick vector. In this study we generated a red fluorescent B. bovis line with a mutation affecting the 6-Cys genes A and B in a single step using a transfection method. The so mutated B. bovis parasites can now be used to test the functional role of the 6-Cys genes A and B, and could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis. In addition, these new vaccine strains would incorporate a molecular marker (the rfp-bsd gene) for strain differentiation. 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.

Technical Abstract: Babesia bovis is a tick-borne apicomplexan parasite responsible for bovine babesiosis. B. bovis has a complex life cycle including asexual development in bovine red blood cells and sexual development in the tick (usually Rhipicephalus microplus) vector. Understanding the molecular mechanisms involved in sexual development is essential to analyze gene function as a first step toward generating alternative transmission blocking and/or non-transmissible attenuated live vaccines (TBV). Members of the 6-Cys gene family are widely conserved among apicomplexan parasites, where they play roles in their sexual development. The genome of B. bovis encodes at least 10 canonical 6-Cys genes. The recently defined sexual marker 6-CysA and 6-CysB of B. bovis are highly similar, strain-conserved, and differentially-expressed genes that are exclusively expressed in tick-stage parasites, but not during the red blood cell stage of parasite development in the bovine host. The high level of sequence identity between the 6-Cys A and 6-CysB proteins (52%), their similar 6-Cys domain distribution and sub-cellular localization are suggestive of redundant function. We hypothesize that disruption of both 6-CysA and 6-CysB in B. bovis will result parasites with an unaltered ability to invade and grow in red blood cells and still elicit protective immunity but the parasites would not be transmissible by ticks. Taking advantage of their contiguous genome localization, we generated a double gene-knockout system to disrupt a 3,287 bp region encompassing both 6-CysA and 6-CysB genes using a single transfection plasmid. Transfection resulted in the generation of the red-fluorescent 'A'B 6-Cys B. bovis mutated parasite line which was able to grow continuously in bovine red blood cells 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 gene manipulation approach will allow future experiments aimed at determining the tick-transmission phenotype of parasites lacking tick-stage genes. Importantly, parasites deficient in genes required for sexual reproduction could be the foundation for genetically-defined, non-transmissible live vaccines against bovine babesiosis, and can be used as experimental tools for clarifying the functional role of these two genes in Babesia parasites. In addition, 'A'B 6-Cys B. bovis parasites express a fluorescent protein marker for strain discrimination. 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.