Abstract
Babesia bigemina and Babesia bovis, are the two major causes of bovine babesiosis, a global neglected disease in need of improved methods of control. Here, we describe a shared method for the stable transfection of these two parasites using electroporation and blasticidin/blasticidin deaminase as a selectable marker. Stably transfected B. bigemina and B. bovis were obtained using a common transfection plasmid targeting the enhanced green fluorescent protein-BSD (egfp-bsd) fusion gene into the elongation factor-1α (ef-1α) locus of B. bigemina and B. bovis under the control of the B. bigemina ef-1α promoter. Sequencing, Southern blotting, immunoblotting and immunofluorescence analysis of parasite-infected red blood cells, demonstrated that the egfp-bsd gene was expressed and stably integrated solely into the ef-1α locus of both, B. bigemina and B. bovis. Interestingly, heterologous B. bigemina ef-1α sequences were able to drive integration into the B. bovis genome by homologous recombination, and the stably integrated B. bigemina ef-1α-A promoter is fully functional in B. bovis. Collectively, the data provides a new tool for genetic analysis of these parasites, and suggests that the development of vaccine platform delivery systems based on transfected B. bovis and B. bigemina parasites using homologous and heterologous promoters is feasible.
Highlights
Bovine babesiosis caused by Babesia bovis and B. bigemina is an acute and persistent tick-borne disease with a high negative economic impact worldwide
First we compared the inhibitory concentrations of blasticidin on in vitro cultured B. bigemina and B. bovis
A stable transfection of B. bigemina and B. bovis using identical B. bigemina insertion and gene regulatory sequences that can be used for functional gene characterization or for the delivery of exogenous antigens by Babesia spp. parasites is described
Summary
Bovine babesiosis caused by Babesia bovis and B. bigemina is an acute and persistent tick-borne disease with a high negative economic impact worldwide. The availability of a partial B. bigemina genome (http://www.sanger.ac.uk/ resources/downloads/protozoa/babesia-bigemina.html) and the previous characterization of the B. bigemina ef-1α locus[10], indicate that the structure of this locus is essentially identical in both B. bigemina and B. bovis. Together, these observations suggests that it would be feasible to use a common strategy for gene integration in both B. bovis and B. bigemina, based on targeting one of the two identical ef-1α open reading frames (orfs) present in the ef-1α locus. The same plasmid used for transfection of B. bigemina was able to insert and express foreign sequences in the ef-1α locus of B. bovis, expanding the options available for the genetic manipulation of Babesia sp. parasites more generally
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