Establishment of transient and stable transfection systems for Babesia ovata

Abstract

BackgroundBovine 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.MethodsIn 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.ResultsAfter 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.ConclusionThe current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1439-z) contains supplementary material, which is available to authorized users.

Whole-genome assembly of Babesia ovata and comparative genomics between closely related pathogens

“…By performing comparative genome analysis, we found limited diversity in ves1b and B. ovata -specific expansion of ebh genes, together with a number of B. ovata -specific genes such as an anamorsin homolog, which is potentially involved in hematopoiesis in infected hosts. We suggest that the involvement of these genes in the unique phenotypes of B. ovata should not be overlooked, even though in the future, these candidates must be examined to verify our hypothesis, taking advantage of the available gene manipulation tools for this parasite [ 56 ].…”

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

“…In recent years, progress has been made in the ability to genetically modify B. bovis , with the development of methods for transient and stable transfection with episomally-maintained plasmids [9, 11, 14, 44–46], and by targeted integration via single- [12, 47] or double-crossover recombination [15, 45]. Although each approach is very useful, each also has its own limitations.…”

Establishment of a novel tick-Babesia experimental infection model

“…Our experimental model would be a powerful tool for clarifying the kinetics of the tick stage of Babesia parasites. With the rapid progress of genome-editing strategies, transgenic organisms are currently available as attractive tools to aid in molecular and cellular studies of Babesia and H. longicornis 3444. Precise study of tick- Babesia molecular interactions using our developed model will also give us concrete knowledge to develop novel strategies for controlling babesiosis.…”