Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase

Theileria equi , an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T . equi . Herein, we report on the first development of a stable transgenic T . equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T . equi , regulatory regions of the elongation factor-1 alpha ( ef-1α ) gene were identified in T. equi . An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T . equi genome. On day 9 post-transfection, blasticidin-resistant T . equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B - locus of the ef-1α gene via PCR and Southern blot analyses . Parasitaemia dynamics between the transgenic and parental T . equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T . equi and the methodology used to generate it offer new opportunities for better understanding of T . equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.