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-1 alpha) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1 alpha 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-1 alpha 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.

Automated summary

This study reports the development of a stable transgenic Theileria equi line expressing enhanced green fluorescent protein/blasticidin S deaminase, with the integration of an expression cassette into the parasite's genome. The dynamics of parasitaemia between the transgenic and parental T. equi lines were comparable in vitro, providing new opportunities for better understanding of T. equi biology.