Establishment of transient and stable transfection systems for Babesia ovata

Background: bovine babesiosis is a tick-borne disease caused by several species of babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. babesia ovata is a benign species widespread in east asian countries and causes anemia,particularly in cattle which are co-infected with theileria orientalis. the development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. such tools have not been developed for b. ovata,and are the aim of this study. methods: in this study we transfected constructs that were designed to evaluate the ability of several b. ovata promoter candidates to drive expression of a reporter luciferase. we found that the elongation factor-1 alpha intergenic region (ef-1α ig) and the actin 5' non-coding region (nr) had highest promoter activities. to establish a stable transfection system,we generated a plasmid construct in which the ef-1α ig promoter drives gfp expression,and the actin 5' nr mediates expression of the selectable marker hdhfr. the plasmid was designed for episomal transfection,as well as to integrate by double cross-over homologous recombination into the ef-1α locus. circular or linearized plasmid was transfected by electroporation into in vitro cultured b. ovata and retention of the plasmid was facilitated by drug selection with 5 nm wr99210 initiated 48 h after transfection. results: after one-week cultivation with wr99210,gfp-expressing parasites were observed by fluorescence microscopy. integration of the plasmid construct into the ef-1α locus was confirmed by pcr,southern blot analysis,and sequencing of recombination sites. these results confirm successful development of a stable transfection system for b. ovata. conclusion: the current study provides a fundamental molecular tool to aid in molecular and cellular studies of b. ovata. © 2016 hakimi et al.