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Parasites & vectors2023; 16(1); 435; doi: 10.1186/s13071-023-06045-z

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

Abstract: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. Methods: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. Results: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. Conclusions: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.
© 2023. The Author(s).
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Publication Date: 2023-11-25 PubMed ID: 38007442PubMed Central: PMC10675911DOI: 10.1186/s13071-023-06045-zGoogle Scholar: Lookup
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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.

Overview

  • This study developed and validated polymerase chain reaction (PCR) tests that can specifically identify different genetic types (genotypes) of the parasite Theileria equi, which causes a disease in horses known as equine piroplasmosis.
  • The new PCR tests are more sensitive and able to detect multiple genotypes in infected animals, improving disease detection and potentially aiding better disease management.

Introduction and Background

  • Theileria equi is a protozoan parasite responsible for equine piroplasmosis, a significant infectious disease affecting horses and other equids worldwide, leading to economic losses.
  • The parasite is classified into five genotypes (A, B, C, D, and E) based on variations in its 18S ribosomal RNA (18S rRNA) gene sequence.
  • Knowing the genotype involved in an infection is important because different genotypes may vary in their pathogenicity, geographic distribution, and response to treatment, which impacts disease control strategies.
  • Prior to this study, standard PCR methods could detect the presence of T. equi but could not differentiate between these genotypes easily.

Research Objectives

  • To design specific PCR assays that can selectively amplify the 18S rRNA sequences unique to each of the five genotypes (A–E) of T. equi.
  • To evaluate the specificity and sensitivity of these genotype-specific PCR assays using known reference materials and field samples.
  • To compare the new PCR assays’ ability to detect genotypes with previous PCR sequencing approaches.

Methods

  • Primer Design: Researchers designed pairs of forward and reverse primers customized to selectively bind to unique sequences within the 18S rRNA gene region for each genotype.
  • Specificity Testing: The PCR assays were tested on plasmids (circular DNA constructs) containing 18S rRNA inserts representing each genotype to confirm that each assay only amplifies its target genotype.
  • Sample Testing: The assays were then applied to 270 blood DNA samples from T. equi-infected equids—92 from donkeys in Sri Lanka and 178 from horses in Paraguay.
  • Phylogenetic Validation: PCR products (amplicons) were sequenced, and the sequences were analyzed phylogenetically to confirm that they clustered with the correct genotype clades, thereby validating assay accuracy.

Results

  • Assay Specificity: Each genotype-specific PCR assay successfully detected only its intended T. equi genotype without amplifying DNA from other genotypes, from Babesia caballi (another equine parasite), or uninfected horse blood.
  • Improved Sensitivity: Compared to prior PCR sequencing approaches, the new assays detected more genotypes within the samples—four genotypes in Sri Lankan samples (A, C, D, E) and all five genotypes in Paraguayan samples.
  • Diverse Genotype Detection: Almost all samples (100% Sri Lankan; 93.3% Paraguayan) tested positive for at least one genotype, demonstrating excellent sensitivity.
  • Co-infection Detection: The assays revealed multiple simultaneous infections with different T. equi genotypes in many samples—90.2% of Sri Lankan and 22.5% of Paraguayan samples showed co-infections with two or more genotypes.
  • Phylogenetic Confirmation: Sequences from PCR products grouped into their respective genotypic clades, confirming the PCR assays’ reliability and specificity in genotype identification.

Conclusions and Implications

  • This study successfully developed a set of PCR assays capable of differentiating T. equi genotypes rapidly and reliably based on 18S rRNA gene sequences.
  • These genotype-specific assays provide superior sensitivity, detecting a broader range of genotypes and mixed infections that previous methods missed.
  • Improved detection of T. equi genotypes supports better epidemiological studies, infection tracking, and potentially informs tailored treatment and control strategies in equine populations.
  • These assays represent valuable diagnostic tools for veterinarians and researchers working to control equine piroplasmosis globally.

Cite This Article

APA
Ahedor B, Otgonsuren D, Zhyldyz A, Guswanto A, Ngigi NMM, Valinotti MFR, Kothalawala H, Kalaichelvan N, Silva SSP, Kothalawala H, Acosta TJ, Sivakumar T, Yokoyama N. (2023). Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes. Parasit Vectors, 16(1), 435. https://doi.org/10.1186/s13071-023-06045-z

Publication

Parasites & vectors
ISSN: 1756-3305
NlmUniqueID: 101462774
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 435
PII: 435

Researcher Affiliations

Ahedor, Believe
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
  • Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana.
Otgonsuren, Davaajav
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
Zhyldyz, Atambekova
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
Guswanto, Azirwan
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
Ngigi, Noel Muthoni Mumbi
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
Valinotti, Maria Fátima Rodríguez
  • Centro de Diagnostico Veterinario, San Lorenzo, Paraguay.
Kothalawala, Hemal
  • Veterinary Research Institute, Peradeniya, Sri Lanka.
Kalaichelvan, Nizanantha
  • Department of Farm Animal Production and Health, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka.
Silva, Seekkuge Susil Priyantha
  • Department of Animal Production and Health, Peradeniya, Sri Lanka.
Kothalawala, Hemali
  • Department of Animal Production and Health, Peradeniya, Sri Lanka.
Acosta, Tomás Javier
  • Universidad Nacional de Canendiyu, Salto del Guaira, Paraguay.
  • Field Center of Animal Science and Agriculture, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
Sivakumar, Thillaiampalam
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan.
Yokoyama, Naoaki
  • National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido, 080-8555, Japan. yokoyama@obihiro.ac.jp.
  • WOAH Reference Laboratory for Equine Piroplasmosis, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan. yokoyama@obihiro.ac.jp.

MeSH Terms

  • Cattle
  • Horses
  • Animals
  • Theileria / genetics
  • Theileriasis / diagnosis
  • Babesiosis / diagnosis
  • RNA, Ribosomal, 18S / genetics
  • Phylogeny
  • DNA, Protozoan / genetics
  • Horse Diseases / diagnosis
  • Polymerase Chain Reaction
  • Equidae
  • Genotype
  • Cattle Diseases

Grant Funding

  • Open Partnership Joint Project of the JSPS Bilateral Joint Research Projects (PJSBP120229902) / Japan Society for the Promotion of Science
  • 22H02511 / Japan Society for the Promotion of Science
  • 22KK0095 / Japan Society for the Promotion of Science

Conflict of Interest Statement

The authors declare that they have no competing interests.

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Citations

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