Stable transfection system for Babesia sp. Xinjiang

Jinming Wang,Jianxun Luo,Guiquan Guan,Xiaoxing Wang,Junlong Liu,Aihong Liu,Hong Yin,Jifei Yang,Youquan Li

doi:10.1186/s13071-021-04940-x

Jinming Wang, Jianxun Luo + Show 7 more

Open Access

https://doi.org/10.1186/s13071-021-04940-x

Copy DOI

Abstract

BackgroundStable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers.MethodsWe evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites.ResultsTransfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months.ConclusionsIn our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj.Graphical abstract

Highlights

Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni
When the concentration of WR99210 increased to 150 μg/ml, the growth inhibition of Babesia sp. Xinjiang (Bxj) was as low as ~ 30%
The first description of a stable transfection system in Babesia genus is reported in B. bovis in 2009; subsequently, this technique has been developed in B. gibsoni and B. ovata [17, 22, 29]

Summary

Introduction

Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. In China, ovine babesiosis was firstly reported as early as 1982 in Sichuan Province and 1986 in Heilongjiang Province [5, 6] Since this disease was sporadically reported in Xinjiang Uygur Autonomous region, Henan, Wang et al Parasites Vectors (2021) 14:463. Xinjiang, which presented distinct morphologies from B. motasi, B. ovis, and B. crassa in a thin blood smear [7, 8]. This novel Babesia species was initially isolated from a splenectomized sheep infested with partially engorged Rhipicephalus sanguineus and Hyalomma anatolicum anatolicum ticks [9].

Methods

Results

Discussion

Conclusion