Establishment of a stable transfection system for genetic manipulation of Babesia gibsoni

Mingming Liu,Seung-Hun Lee,Masahito Asada,Xuenan Xuan,Patrick Vudriko,Shin-Ichiro Kawazu,Tatsunori Masatani,Paul Franck Adjou Moumouni,Hassan Hakimi,Junya Yamagishi

doi:10.1186/s13071-018-2853-1

Abstract

BackgroundGenetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. In Babesia, stable transfection systems have only been established for bovine Babesia parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis.ResultsGFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection, and consistently expressed GFP for more than 3 months without drug pressure. Genome integration was confirmed by PCR, sequencing and Southern blot analysis.ConclusionsWe present the first successful establishment of a stable transfection system for B. gibsoni. This finding will facilitate functional analysis of Babesia genomes using genetic manipulation and will serve as a foundation for the development of tick-Babesia and host-Babesia infection models.

Highlights

Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites
In order to establish B. gibsoni stable transfection, we investigated whether stable transfection of Green fluorescent protein (GFP)-expressing B. gibsoni could be achieved using
Babesia gibsoni sensitivity to WR99210 WR99210 successfully inhibited the growth of B. gibsoni in vitro at a nanomolar concentration (Additional file 1: Figure S1)

Summary

Introduction

Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis. Among Babesia species, transient and stable transfection systems have been reported for B. bovis [10], B. ovata [11] and B. bigemina [12]. Similar to B. bovis [15], the development of a stable transfection system for B. gibsoni parasites requires a drug selection system and an integration target. The WR99210/human dihydrofolate reductase gene (hdhfr) selection system and double cross-over homologous recombination locus have previously been successfully used for B. bovis [16] and B. ovata [11] stable transfection

Results

Discussion

Conclusion