Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 23:9:171.
doi: 10.1186/s13071-016-1439-z.

Establishment of transient and stable transfection systems for Babesia ovata

Hassan Hakimi  1 , Junya Yamagishi  2   3 , Yuto Kegawa  1   4 , Osamu Kaneko  1 , Shin-Ichiro Kawazu  5 , Masahito Asada  6
Affiliations

Establishment of transient and stable transfection systems for Babesia ovata

Hassan Hakimi et al. Parasit Vectors. .

Abstract

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.

Keywords: Babesia ovata; Bovine babesiosis; Double cross-over homologous recombination; Stable transfection.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic diagram of the plasmids used for transient transfection and evaluation of promoter activity. a Plasmid construct to evaluate the promoter activity and a Renilla luciferase-expressing plasmid for normalization. b Evaluation of the promoter activity of actin 5’NR driving luciferase expression, over a time course of 24–96 h post transfection. c Comparison of luciferase activity in lysates of B. ovata transfected with different constructs at 48 h post transfection. A promoter-less plasmid was used as a negative control. Values from 3 independent transfections are shown. Asterisks indicate statistical significance between promoter-less plasmid (No Promoter) and other promoter candidates by Dunnett’s multiple comparison test (p < 0.001). RLU: Relative luciferase units
Fig. 2
Fig. 2
Schematic diagram of GFP-expressing plasmid construct, growth inhibition of B. ovata by WR99210 and fluorescence microscopy images of stably expressing GFP B. ovata. a Plasmid construct for stable GFP expression (pBS-EGRADE) showing the recombination sites for integration into the ef-1α locus by homologous double cross-over recombination. The restriction sites for linearization (SalI and SacII) are shown. b Growth inhibition rate of B. ovata in the absence or presence of different concentrations of WR99210. All data are expressed as mean + SEM of triplicate culture. c Live fluorescence microscopy images of GFP-expressing parasites. The pictures are taken from the pBS-EGRADE genome integrated isolate 1. The parasite nucleus was stained with Hoechst33342. Scale-bar: 10 μm
Fig. 3
Fig. 3
Confirmation of the integration of pBS-EGRADE into the ef-1α locus. a Schematic diagram and PCRs to confirm the integration of pBS-EGRADE into ef-1α locus. PCR-1, −2 and −3 are done with primer sets Boef1α-integ-F/eGFP-R-HindIII-IF, hDHFR-F-BamHI-IF/Boef1α-integ-R and Boef1α-F2/Boef1α-integ-R, respectively. b Schematic diagram and Southern blot analysis to confirm the integration of pBS-EGRADE into ef-1α locus. Five microgram of samples genomic DNA were digested with PacI and NotI, and hybridized with gfp and ef-1α 3’NR probes. GI: genome integrated; Episomal: two independent transfectants with circular plasmids; WT: wild type
Fig. 4
Fig. 4
Growth curve of different lines of B. ovata. Wild type (WT) and genome integrants (GI) were cultured continuously by sub-culturing every 3 days and parasitemia monitored daily
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Yoshinari T, Sivakumar T, Asada M, Battsetseg B, Huang X, Lan DT, et al. A PCR based survey of Babesia ovata in cattle from various Asian, African, and South American countries. J Vet Med Sci. 2013;75:211–214. doi: 10.1292/jvms.12-0329. - DOI - PubMed
    1. Sivakumar T, Tagawa M, Yoshinari T, Ybañez AP, Igarashi I, Ikehara Y, et al. PCR Detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis. J Clin Microbiol. 2012;50:2111–2113. doi: 10.1128/JCM.00220-12. - DOI - PMC - PubMed
    1. Abbas RZ, Zaman MA, Colwell DD, Gilleard J, Iqbal Z. Acaricide resistance in cattle ticks and approaches to its management: the state of play. Vet Parasitol. 2014;203(1–2):6–20. doi: 10.1016/j.vetpar.2014.03.006. - DOI - PubMed
    1. Zintl A, Mulcahy G, Skerrett HE, Taylor SM, Gray JS. Babesia divergens: a bovine blood parasite of veterinary and zoonotic importance. Clin Microbiol Rev. 2003;16:622–636. doi: 10.1128/CMR.16.4.622-636.2003. - DOI - PMC - PubMed
    1. Crabb BS, Cowman AF. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci USA. 1996;93:7289–7294. doi: 10.1073/pnas.93.14.7289. - DOI - PMC - PubMed

Publication types