FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Sci Rep / Development of a stable transgenic Theileria equi parasite e...
Scientific reports2021; 11(1); 9107; doi: 10.1038/s41598-021-88594-w

Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase.

Abstract: Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.
Get Full Text
Publication Date: 2021-04-27 PubMed ID: 33907262PubMed Central: PMC8079379DOI: 10.1038/s41598-021-88594-wGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article presents the first successful development of a genetically modified Theileria equi parasite that expresses an enhanced green fluorescent protein and blasticidin S deaminase. This marked genetic manipulation offers potential for better understanding of the parasite’s biology and control methods for the equine piroplasmosis disease it causes.

About Theileria equi and Equine Piroplasmosis

  • This study focuses on Theileria equi, a protozoan parasite that infects red blood cells. This parasite causes equine piroplasmosis, a disease that has a significant negative impact on the global horse industry.
  • The control of protozoan parasites, such as T. equi, is considered a challenge. One potential method being explored is genetic manipulation.

Genetic Manipulation of Theileria equi

  • In order to genetically manipulate the T. equi parasite, the researchers inserted a fusion gene for enhanced green fluorescent protein and blasticidin S deaminase (eGFP/BSD) into its genome.
  • The regulatory regions of a specific gene (elongation factor-1 alpha or ef-1α) were used to control the expression of the inserted gene.
  • Through this process, the development of a transgenic, or genetically modified, T. equi line expressing eGFP/BSD became successful.

Results and Significance of the Study

  • Following this genetic modification, blasticidin-resistant T. equi emerged on the ninth day post-transfection, indicating successful integration of the eGFP/BSD gene and subsequent gene expression in the parasite.
  • The integration of the eGFP/BSD gene was further confirmed by obtaining a clonal line of T. equi and analysing it through PCR and Southern blot tests, which confirmed the presence of the gene in the designated B-locus of the ef-1α gene.
  • The in vitro parasitaemia dynamics of the transgenic and natural T. equi were comparable, indicating that the genetic modification did not affect the growth behaviour of the parasite.
  • The methodology developed here provides important tools for further studies on T. equi. The genetically modified, eGFP/BSD-expressing T. equi yields important insights into parasite biology. It may also serve as a platform for discovering effective methods to control equine piroplasmosis.

Cite This Article

APA
Tuvshintulga B, Nugraha AB, Mizutani T, Liu M, Ishizaki T, Sivakumar T, Xuan X, Yokoyama N, Igarashi I. (2021). Development of a stable transgenic Theileria equi parasite expressing an enhanced green fluorescent protein/blasticidin S deaminase. Sci Rep, 11(1), 9107. https://doi.org/10.1038/s41598-021-88594-w

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 11
Issue: 1
Pages: 9107
PII: 9107

Researcher Affiliations

Tuvshintulga, Bumduuren
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
  • Institute of Veterinary Medicine, Mongolian University of Life Sciences, Zaisan, Ulaanbaatar, 17024, Mongolia.
Nugraha, Arifin Budiman
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
  • Department of Animal Infectious Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, IPB University, Jl. Agatis, Kampus IPB Dramaga, Bogor, Jawa Barat, 16680, Indonesia.
Mizutani, Tomoka
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Liu, Mingming
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Ishizaki, Takahiro
  • Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japan.
Sivakumar, Thillaiampalam
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Xuan, Xuenan
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Yokoyama, Naoaki
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. yokoyama@obihiro.ac.jp.
Igarashi, Ikuo
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.

MeSH Terms

  • Aminohydrolases / genetics
  • Gene Expression Regulation
  • Green Fluorescent Proteins / genetics
  • Nucleosides / pharmacology
  • Organisms, Genetically Modified
  • Peptide Elongation Factor 1 / genetics
  • Plasmids
  • Theileria / drug effects
  • Theileria / genetics
  • Transfection

Conflict of Interest Statement

The authors declare no competing interests.

References

This article includes 39 references
  1. Kappmeyer LS, Thiagarajan M, Herndon DR, Ramsay JD, Caler E, Djikeng A, Gillespie JJ, Lau AO, Roalson EH, Silva JC, Silva MG, Suarez CE, Ueti MW, Nene VM, Mealey RH, Knowles DP, Brayton KA. Comparative genomic analysis and phylogenetic position of Theileria equi.. BMC Genomics 2012 Nov 9;13:603.
    doi: 10.1186/1471-2164-13-603pmc: PMC3505731pubmed: 23137308google scholar: lookup
  2. Wise LN, Kappmeyer LS, Mealey RH, Knowles DP. Review of equine piroplasmosis.. J Vet Intern Med 2013 Nov-Dec;27(6):1334-46.
    doi: 10.1111/jvim.12168pubmed: 24033559google scholar: lookup
  3. Knowles D Jr. Equine babesiosis (piroplasmosis): a problem in the international movement of horses.. Br Vet J 1996 Mar;152(2):123-6.
    doi: 10.1016/s0007-1935(96)80066-2pubmed: 8680834google scholar: lookup
  4. Scoles GA, Ueti MW. Vector ecology of equine piroplasmosis.. Annu Rev Entomol 2015 Jan 7;60:561-80.
    doi: 10.1146/annurev-ento-010814-021110pubmed: 25564746google scholar: lookup
  5. Ribeiro IB, Câmara AC, Bittencourt MV, Marçola TG, Paludo GR, Soto-Blanco B. Detection of Theileria equi in spleen and blood of asymptomatic piroplasm carrier horses.. Acta Parasitol 2013 Jun;58(2):218-22.
    doi: 10.2478/s11686-013-0127-9pubmed: 23666659google scholar: lookup
  6. Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis.. Parasitol Res 2007 Apr;100(5):1165-8.
    doi: 10.1007/s00436-006-0430-6pubmed: 17216488google scholar: lookup
  7. Adams LG. Clinicopathological aspects of imidocarb dipropionate toxicity in horses.. Res Vet Sci 1981 Jul;31(1):54-61.
    doi: 10.1016/S0034-5288(18)32521-9pubmed: 7313320google scholar: lookup
  8. Hines SA, Ramsay JD, Kappmeyer LS, Lau AO, Ojo KK, Van Voorhis WC, Knowles DP, Mealey RH. Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor.. Parasit Vectors 2015 Jan 20;8:33.
    doi: 10.1186/s13071-014-0611-6pmc: PMC4311422pubmed: 25600252google scholar: lookup
  9. Kunz SE, Kemp DH. Insecticides and acaricides: resistance and environmental impact.. Rev Sci Tech 1994 Dec;13(4):1249-86.
    doi: 10.20506/rst.13.4.816pubmed: 7711312google scholar: lookup
  10. Mehlhorn H, Schein E. Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998.. Parasitol Res 1998 Jun;84(6):467-75.
    doi: 10.1007/s004360050431pubmed: 9660136google scholar: lookup
  11. Ramsay JD, Ueti MW, Johnson WC, Scoles GA, Knowles DP, Mealey RH. Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.. PLoS One 2013;8(10):e76996.
    doi: 10.1371/journal.pone.0076996pmc: PMC3792048pubmed: 24116194google scholar: lookup
  12. Shaw MK. Cell invasion by Theileria sporozoites.. Trends Parasitol 2003 Jan;19(1):2-6.
    doi: 10.1016/s1471-4922(02)00015-6pubmed: 12488213google scholar: lookup
  13. Zapf F, Schein E. The development of Babesia (Theileria) equi (Laveran, 1901) in the gut and the haemolymph of the vector ticks, Hyalomma species.. Parasitol Res 1994;80(4):297-302.
    doi: 10.1007/BF02351869pubmed: 8073015google scholar: lookup
  14. Jalovecka M, Hajdusek O, Sojka D, Kopacek P, Malandrin L. The Complexity of Piroplasms Life Cycles.. Front Cell Infect Microbiol 2018;8:248.
    doi: 10.3389/fcimb.2018.00248pmc: PMC6065256pubmed: 30083518google scholar: lookup
  15. De Goeyse I, Jansen F, Madder M, Hayashida K, Berkvens D, Dobbelaere D, Geysen D. Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.. Vet Parasitol 2015 Mar 15;208(3-4):238-41.
    doi: 10.1016/j.vetpar.2015.01.013pubmed: 25660425google scholar: lookup
  16. Adamson R, Lyons K, Sharrard M, Kinnaird J, Swan D, Graham S, Shiels B, Hall R. Transient transfection of Theileria annulata.. Mol Biochem Parasitol 2001 Apr 25;114(1):53-61.
    doi: 10.1016/s0166-6851(01)00238-9pubmed: 11356513google scholar: lookup
  17. Suarez CE, McElwain TF. Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites.. Int J Parasitol 2009 Feb;39(3):289-97.
    doi: 10.1016/j.ijpara.2008.08.006pubmed: 18831975google scholar: lookup
  18. Silva MG, Knowles DP, Mazuz ML, Cooke BM, Suarez CE. Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid.. Sci Rep 2018 Apr 17;8(1):6096.
    doi: 10.1038/s41598-018-23010-4pmc: PMC5904164pubmed: 29666434google scholar: lookup
  19. Hakimi H, Yamagishi J, Kegawa Y, Kaneko O, Kawazu S, Asada M. Establishment of transient and stable transfection systems for Babesia ovata.. Parasit Vectors 2016 Mar 23;9:171.
    doi: 10.1186/s13071-016-1439-zpmc: PMC4806448pubmed: 27008652google scholar: lookup
  20. Liu M, Adjou Moumouni PF, Asada M, Hakimi H, Masatani T, Vudriko P, Lee SH, Kawazu SI, Yamagishi J, Xuan X. Establishment of a stable transfection system for genetic manipulation of Babesia gibsoni.. Parasit Vectors 2018 Apr 23;11(1):260.
    doi: 10.1186/s13071-018-2853-1pmc: PMC5914073pubmed: 29685172google scholar: lookup
  21. Talman AM, Blagborough AM, Sinden RE. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.. PLoS One 2010 Feb 10;5(2):e9156.
    doi: 10.1371/journal.pone.0009156pmc: PMC2819258pubmed: 20161781google scholar: lookup
  22. Nishikawa Y, Zhang H, Ibrahim HM, Ui F, Ogiso A, Xuan X. Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein.. Parasitol Int 2008 Jun;57(2):219-22.
    doi: 10.1016/j.parint.2007.10.004pubmed: 18006371google scholar: lookup
  23. Li W, Zhang N, Liang X, Li J, Gong P, Yu X, Ma G, Ryan UM, Zhang X. Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker.. Mol Biochem Parasitol 2009 Dec;168(2):143-8.
    doi: 10.1016/j.molbiopara.2009.07.003pubmed: 19631239google scholar: lookup
  24. Alzan HF, Silva MG, Davis WC, Herndon DR, Schneider DA, Suarez CE. Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line.. Parasit Vectors 2017 May 2;10(1):214.
    doi: 10.1186/s13071-017-2143-3pmc: PMC5414359pubmed: 28464956google scholar: lookup
  25. Hussein HE, Bastos RG, Schneider DA, Johnson WC, Adham FK, Davis WC, Laughery JM, Herndon DR, Alzan HF, Ueti MW, Suarez CE. The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction.. PLoS Negl Trop Dis 2017 Oct;11(10):e0005965.
    doi: 10.1371/journal.pntd.0005965pmc: PMC5646870pubmed: 28985216google scholar: lookup
  26. Suarez CE, Bishop RP, Alzan HF, Poole WA, Cooke BM. Advances in the application of genetic manipulation methods to apicomplexan parasites.. Int J Parasitol 2017 Oct;47(12):701-710.
    doi: 10.1016/j.ijpara.2017.08.002pubmed: 28893636google scholar: lookup
  27. Narlikar L, Ovcharenko I. Identifying regulatory elements in eukaryotic genomes.. Brief Funct Genomic Proteomic 2009 Jul;8(4):215-30.
    doi: 10.1093/bfgp/elp014pmc: PMC2764519pubmed: 19498043google scholar: lookup
  28. Suarez CE, Norimine J, Lacy P, McElwain TF. Characterization and gene expression of Babesia bovis elongation factor-1alpha.. Int J Parasitol 2006 Jul;36(8):965-73.
    doi: 10.1016/j.ijpara.2006.02.022pubmed: 16677650google scholar: lookup
  29. Silva MG, Knowles DP, Suarez CE. Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in Babesia bigemina and Babesia bovis.. Parasit Vectors 2016 Nov 11;9(1):576.
    doi: 10.1186/s13071-016-1859-9pmc: PMC5106780pubmed: 27835993google scholar: lookup
  30. Liu M, Asada M, Cao S, Adjou Moumouni PF, Vudriko P, Efstratiou A, Hakimi H, Masatani T, Sunaga F, Kawazu SI, Yamagishi J, Xuan X. Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter.. Mol Biochem Parasitol 2017 Sep;216:56-59.
    doi: 10.1016/j.molbiopara.2017.07.003pubmed: 28729071google scholar: lookup
  31. Andersen GR, Pedersen L, Valente L, Chatterjee I, Kinzy TG, Kjeldgaard M, Nyborg J. Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha.. Mol Cell 2000 Nov;6(5):1261-6.
    doi: 10.1016/s1097-2765(00)00122-2pubmed: 11106763google scholar: lookup
  32. Vinkenoog R, Sperança MA, van Breemen O, Ramesar J, Williamson DH, Ross-MacDonald PB, Thomas AW, Janse CJ, del Portillo HA, Waters AP. Malaria parasites contain two identical copies of an elongation factor 1 alpha gene.. Mol Biochem Parasitol 1998 Jul 1;94(1):1-12.
    doi: 10.1016/s0166-6851(98)00035-8pubmed: 9719506google scholar: lookup
  33. Gardner MJ, Bishop R, Shah T, de Villiers EP, Carlton JM, Hall N, Ren Q, Paulsen IT, Pain A, Berriman M, Wilson RJ, Sato S, Ralph SA, Mann DJ, Xiong Z, Shallom SJ, Weidman J, Jiang L, Lynn J, Weaver B, Shoaibi A, Domingo AR, Wasawo D, Crabtree J, Wortman JR, Haas B, Angiuoli SV, Creasy TH, Lu C, Suh B, Silva JC, Utterback TR, Feldblyum TV, Pertea M, Allen J, Nierman WC, Taracha EL, Salzberg SL, White OR, Fitzhugh HA, Morzaria S, Venter JC, Fraser CM, Nene V. Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes.. Science 2005 Jul 1;309(5731):134-7.
    doi: 10.1126/science.1110439pubmed: 15994558google scholar: lookup
  34. Hayashida K, Hara Y, Abe T, Yamasaki C, Toyoda A, Kosuge T, Suzuki Y, Sato Y, Kawashima S, Katayama T, Wakaguri H, Inoue N, Homma K, Tada-Umezaki M, Yagi Y, Fujii Y, Habara T, Kanehisa M, Watanabe H, Ito K, Gojobori T, Sugawara H, Imanishi T, Weir W, Gardner M, Pain A, Shiels B, Hattori M, Nene V, Sugimoto C. Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of Theileria-induced leukocyte transformation.. mBio 2012;3(5):e00204-12.
    doi: 10.1128/mBio.00204-12pmc: PMC3445966pubmed: 22951932google scholar: lookup
  35. Silva MG, Ueti MW, Norimine J, Florin-Christensen M, Bastos RG, Goff WL, Brown WC, Oliva A, Suarez CE. Babesia bovis expresses Bbo-6cys-E, a member of a novel gene family that is homologous to the 6-cys family of Plasmodium.. Parasitol Int 2011 Jan;60(1):13-8.
    doi: 10.1016/j.parint.2010.09.004pubmed: 20884375google scholar: lookup
  36. Liu Y, Tewari R, Ning J, Blagborough AM, Garbom S, Pei J, Grishin NV, Steele RE, Sinden RE, Snell WJ, Billker O. The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.. Genes Dev 2008 Apr 15;22(8):1051-68.
    doi: 10.1101/gad.1656508pmc: PMC2335326pubmed: 18367645google scholar: lookup
  37. Barriga OO. A review on vaccination against protozoa and arthropods of veterinary importance.. Vet Parasitol 1994 Oct;55(1-2):29-55.
    doi: 10.1016/0304-4017(94)90054-Xpubmed: 7886919google scholar: lookup
  38. Avarzed A, Igarashi I, De Waal DT, Kawai S, Oomori Y, Inoue N, Maki Y, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N. Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis.. J Clin Microbiol 1998 Jul;36(7):1835-9.
    doi: 10.1128/JCM.36.7.1835-1839.1998pmc: PMC104937pubmed: 9650921google scholar: lookup
  39. Tuvshintulga B, AbouLaila M, Davaasuren B, Ishiyama A, Sivakumar T, Yokoyama N, Iwatsuki M, Otoguro K, Ōmura S, Igarashi I. Clofazimine Inhibits the Growth of Babesia and Theileria Parasites In Vitro and In Vivo.. Antimicrob Agents Chemother 2016 May;60(5):2739-46.
    doi: 10.1128/AAC.01614-15pmc: PMC4862543pubmed: 26883713google scholar: lookup

Citations

This article has been cited 2 times.
  1. Fang T, Ben Mamoun C. Babesia duncani, a Model Organism for Investigating Intraerythrocytic Parasitism and Novel Antiparasitic Therapeutic Strategies. J Infect Dis 2024 Jul 25;230(1):263-270.
    doi: 10.1093/infdis/jiae191pubmed: 39052743google scholar: lookup
  2. Aida H, Foreman JH, Ochi A, Takizawa Y, Yamanaka T. A case of equine piroplasmosis in the Tokyo 2020 Olympic Games. J Equine Sci 2023 Sep;34(3):93-99.
    doi: 10.1294/jes.34.93pubmed: 37781566google scholar: lookup
Subscribe to our newsletter
Join 99,103 horse owners like you who receive our email updates on horse health and nutrition.