Abstract
Babesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (BspXJ), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remains unclear. Firstly, a plasmid bearing the elongation factor-1 alpha promoter and the firefly luciferase reporter gene and rap stop region were transfected into BspXJ by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program settings were evaluated to confirm the best for BspXJ transient transfection, and a series of different amounts of plasmid DNA were transfected to generate relatively high luminescence values. Finally, the activities of four promoters derived from BspXJ were evaluated using the developed transient transfection system. After evaluating of various transfection parameters, the human T cell nucleofector solution, program V-024 and 20 μg of plasmid DNA were selected as the most favorable conditions for BspXJ transient transfection. These findings provide critical information for BspXJ genetic manipulation, an essential tool to identify virulence factors and to further elucidate the basic biology of this parasite.
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Data availability
All data are available upon request.
Abbreviations
- BspXJ:
-
Babesia sp. Xinjiang
- RBCs:
-
Red blood cells
- PCR:
-
Polymerase chain reaction
References
Adamson R, Lyons K, Sharrard M, Kinnaird J, Swan D, Graham S, Shiels B, Hall R (2001) Transient transfection of Theileria annulata. Mol Biochem Parasitol 114(1):53–61. https://doi.org/10.1016/s0166-6851(01)00238-9
Antunes S, Rosa C, Couto J, Ferrolho J, Domingos A (2017) Deciphering Babesia-vector interactions. Front Cell Infect Microbiol 7:429. https://doi.org/10.3389/fcimb.2017.00429
Aouadi A, Leulmi H, Boucheikhchoukh M, Benakhla A, Raoult D, Parola P (2017) Molecular evidence of tick-borne hemoprotozoan-parasites (Theileria ovis and Babesia ovis) and bacteria in ticks and blood from small ruminants in Northern Algeria. Comp Immunol Microbiol Infect Dis 50:34–39. https://doi.org/10.1016/j.cimid.2016.11.008
Asada M, Tanaka M, Goto Y, Yokoyama N, Inoue N, Kawazu S (2012) Stable expression of green fluorescent protein and targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis with the WR99210/dhfr selection system. Mol Biochem Parasitol 181(2):162–170. https://doi.org/10.1016/j.molbiopara.2011.11.001
De Goeyse I, Jansen F, Madder M, Hayashida K, Berkvens D, Dobbelaere D, Geysen D (2015) Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva. Vet Parasitol 208(3–4):238–241. https://doi.org/10.1016/j.vetpar.2015.01.013
Guan G, Ma M, Liu A, Ren Q, Wang J, Yang J, Li A, Liu Z, Du P, Li Y, Liu Q, Zhu H, Yin H, Luo J (2012a) A recently identified ovine Babesia in China: serology and sero-epidemiology. Parasitol Int 61(4):532–537. https://doi.org/10.1016/j.parint.2012.04.004
Guan G, Ma M, Moreau E, Liu J, Lu B, Bai Q, Luo J, Jorgensen W, Chauvin A, Yin H (2009) A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum. Exp Parasitol 122(4):261–267. https://doi.org/10.1016/j.exppara.2009.05.001
Guan G, Ma M, Liu A, Du P, Ren Q, Li Y, Wang J, Liu Z, Yin H, Luo J (2012b) Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China. Vet Parasitol 187(3–4):371–378. https://doi.org/10.1016/j.vetpar.2012.02.006
Guan G, Yin H, Luo J, Lv W, Zhang Q, Ma M, Yuan G, Lu B, Wang Y, Muxtar (2001) Preliminary study on the morphology and pathogenicity of the unidentified species of Babesia in sheep. Chinese Journal of Veterinary Science and Technology 11(13):35–36. https://doi.org/CNKI:SUN:ZGSY.0.2001-11-015
Hakimi H, Yamagishi J, Kegawa Y, Kaneko O, Kawazu S, Asada M (2016) Establishment of transient and stable transfection systems for Babesia ovata. Parasit Vectors 9:171. https://doi.org/10.1186/s13071-016-1439-z
Hong SH, Kim SY, Song BG, Rho JR, Cho CR, Kim CN, Um TH, Kwak YG, Cho SH, Lee SE (2019) Detection and characterization of an emerging type of Babesia sp. similar to Babesia motasi for the first case of human babesiosis and ticks in Korea. Emerg Microbes Infect 8(1):869–878. https://doi.org/10.1080/22221751.2019.1622997
Jiang J, Zheng Y, Jiang R, Li H, Huo Q, Jiang B, Sun Y, Jia N, Wang Y, Ma L, Liu H, Chu Y, Ni X, Liu K, Song Y, Yao N, Wang H, Sun T, Cao W (2015) Epidemiological, clinical, and laboratory characteristics of 48 cases of "Babesia venatorum" infection in China: a descriptive study. Lancet Infect Dis 15(2):196–203. https://doi.org/10.1016/S1473-3099(14)71046-1
Kim J, Cho S, Joo H, Tsuji M, Cho S, Park I, Chung G, Ju J, Cheun H, Lee H, Lee Y, Kim T (2007) First case of human babesiosis in Korea: detection and characterization of a novel type of Babesia sp. (KO1) similar to ovine babesia. J Clin Microbiol 45(6):2084–2087. https://doi.org/10.1128/JCM.01334-06
Liu M, Asada M, Cao S, Moumouni P, Vudriko P, Efstratiou A, Hakimi H, Masatani T, Sunaga F, Kawazu S, Yamagishi J, Xuan X (2017) Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter. Mol Biochem Parasitol 216:56–59. https://doi.org/10.1016/j.molbiopara.2017.07.003
Man SQ, Qiao K, Cui J, Feng M, Fu YF, Cheng XJ (2016) A case of human infection with a novel Babesia species in China. Infect Dis Poverty 5:28. https://doi.org/10.1186/s40249-016-0121-1
Padmanabhan PK, Polidoro RB, Barteneva NS, Gazzinelli RT, Burleigh BA (2014) Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi. Mol Biochem Parasitol 198(2):100–103. https://doi.org/10.1016/j.molbiopara.2015.02.001
Rosa C, Asada M, Hakimi H, Domingos A, Pimentel M, Antunes S (2019) Transient transfection of Babesia ovis using heterologous promoters. Ticks Tick Borne Dis 10(6):101279. https://doi.org/10.1016/j.ttbdis.2019.101279
Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA (2012) Babesia: A world emerging. Infect Genet Evol 12(8):1788–1809. https://doi.org/10.1016/j.meegid.2012.07.004
Silva MG, Knowles DP, Suarez CE (2016) Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in Babesia bigemina and Babesia bovis. Parasit Vectors 9(1):576. https://doi.org/10.1186/s13071-016-1859-9
Suarez CE, Bishop RP, Alzan HF, Poole WA, Cooke BM (2017) Advances in the application of genetic manipulation methods to apicomplexan parasites. Int J Parasitol 47(12):701–710. https://doi.org/10.1016/j.ijpara.2017.08.002
Suarez CE, McElwain TF (2009) Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites. Int J Parasitol 39(3):289–297. https://doi.org/10.1016/j.ijpara.2008.08.006
Uilenberg G (1995) International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet Parasitol 57(1–3):19–41. https://doi.org/10.1016/0304-4017(94)03107-8
Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, Cipriano MJ, Striepen B (2015) Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523(7561):477–480. https://doi.org/10.1038/nature14651
Wang J, Zhang S, Yang J, Liu J, Zhang D, Li Y, Luo J, Guan G, Yin H (2019) Babesia divergens in human in Gansu province. China Emerg Microbes Infect 8(1):959–961. https://doi.org/10.1080/22221751.2019.1635431
Acknowledgements
We acknowledge Carlos E. Suarez for technical support and providing pBluescript II SK ( +) plasmid.
Funding
This study was financially supported by the National Science Foundation of China (grant nos. 31972701 and 31972706), National Key Research and Development Program of China (grant no. 2017YFD0501200), the 973 Program (grant no. 2015CB150300), ASTIP (grant no. CAAS-ASTIP-2016-LVRI), NBCIS (grant no. CARS-37), and the Jiangsu Co-innovation Center Program for the Prevention and Control of Important Animal Infectious Disease and Zoonoses.
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XW and JW carried out the experiments and wrote the draft of the manuscript. JL, JY, ZL, AL, YL, YL, and HL participate plasmid construction. GG corrected the manuscript. GL, JL, and HY supervised all parts of the study. All authors have read and approved the final version of this manuscript.
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The study was approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, CAAS (Permit No. LVRIAEC-2018–001). All the procedures were conducted according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
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Xiaoxing Wang and Jinming Wang contributed equally to this work.
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Wang, X., Wang, J., Liu, J. et al. Establishment of a transient transfection system for Babesia sp. Xinjiang using homologous promoters. Parasitol Res 120, 3625–3630 (2021). https://doi.org/10.1007/s00436-021-07250-4
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DOI: https://doi.org/10.1007/s00436-021-07250-4