Understanding the biology of Babesia bovis

Bovine babesiosis caused by the protozoan parasite Babesia bovis remains a significant cause of avoidable economic loss to the livestock industry in many countries, including Australia. The precise mechanisms by which this parasite causes severe disease in susceptible cattle are not well understood, however, it is clear that pathophysiologically important alterations to the structure and function of red blood cells (RBCs) in which the parasites reside play a critical role. In order to gain an understanding of the mechanisms that defines these processes and shed light on the basic biology of Babesia parasites, we have developed tools and technologies that are fundamental to the study of this important veterinary pathogen. Our newly developed in vitro culture and transfection tools have allowed us to genetically manipulate the parasite genome and perform specific knock-out and knock-in studies. In particular, using a forward genetics approach involving the use of the piggyBac transposable element, we have successfully generated random mutant parasite libraries that can be screened for any desired phenotype. The precise insertion events (and therefore the mutation event responsible for the altered phenotype) are subsequently identified by sequencing. We have simultaneously used a bioinformatics-driven approach to identify a set of novel exported proteins in B. bovis that we believe are likely to be involved in alteration of infected RBCs and therefore highly likely to play major roles in the pathogenesis of babesiosis. The B. bovis ‘exportome’ contains previously described exported proteins including merozoite surface antigens (MSAs), spherical body proteins (SBPs), variant erythrocyte surface antigens (VESAs) and some small open reading frame (smORFs) proteins in addition to a large number of hypothetical proteins that currently have no identity to any other protein in any other organism for which data is available. B. bovis spherical body protein 4 (SBP4) and a putative membrane protein BBOV_II004730 (4730) are two examples of proteins identified using this bioinformatics screen. SBP4 and 4730 were selected for further characterisation as they are previously uncharacterised proteins that we believe may play a role in the development and survival of the parasite. Immunofluorescence assays using specific antibodies raised against recombinant proteins have confirmed export of these proteins into the infected-RBC (pRBC). Epitope tagging of the endogenous gene in parasites has further substantiated the localisation of SBP4 in pRBC and further characterisation of knockout transgenic parasite lines is ongoing. During this study, we have employed forward and reverse genetic techniques, bioinformatics, molecular and cellular approaches to begin unravelling the characteristics of two B. bovis proteins in pRBCs. In addition, we have developed a reliable transfection system to epitope-tag or knockout genes and generate transgenic parasite lines with relative ease in the laboratory. Identification and characterisation of exported proteins in Babesia parasites will expand our overall knowledge of the parasite and functional studies of these transgenic parasite lines will assist in the longer-term, the identification of new and urgently required therapeutics and/or better vaccines to combat bovine babesiosis.