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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.
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Publication Date: 2021-04-27 PubMed ID: 33907262PubMed Central: PMC8079379DOI: 10.1038/s41598-021-88594-wGoogle Scholar: Lookup
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  • 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.

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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.
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  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
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