Multiplex hydrolysis-probe assay for the simultaneous detection of Theileria equi and Babesia caballi infections in equids.
Abstract: Quantitative real-time PCR assays previously developed for the detection of Theileria equi and Babesia caballi, were combined in a single multiplex TaqMan qPCR platform for the simultaneous detection of both heamoprotozoan parasites in equids. The multiplex equine piroplasmosis (M-EP) qPCR assay was shown to be efficient and specific. The detection limit was determined to be 1.4 × 10 % parasitized erythrocytes (PE) for T. equi and 2.8 × 10 % PE for B. caballi. The effect of differential DNA concentrations on the outcome of the M-EP qPCR for each target species was also investigated. The data demonstrated that the assay could reliably detect both targets, over a range of at least 1000-fold difference in target concentrations, without loss of sensitivity. The assay was subsequently evaluated on 243 field samples collected from areas where limited tick control strategies were implemented. The IFAT detected circulating T. equi and B. caballi antibodies in 100% and 92% of the samples, respectively. The M-EP qPCR assay detected T. equi parasite DNA in 98% of the samples, while B. caballi could only be detected in 6% of the samples tested, confirming that B. caballi infections generally occur at extremely low parasitaemias that rarely exceed 1%. The developed M-EP qPCR assay therefore serves as a reliable tool for the rapid diagnosis and epidemiological survey of equine piroplasmosis.
Copyright © 2018 Elsevier B.V. All rights reserved.
Publication Date: 2018-03-28 PubMed ID: 29773138DOI: 10.1016/j.vetpar.2018.03.022Google Scholar: Lookup
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Summary
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The research article presents a Multiplex hydrolysis-probe assay that can simultaneously detect Theileria equi and Babesia caballi infections in equines, identifying them as efficient and specific. The researchers also delve into the effect of varying DNA concentrations on the method, indicating the assay’s reliability over a wide range of target concentrations.
Objective of the Research
- The main focus of this research was to combine the previously separated real-time PCR assays for Theileria equi and Babesia caballi detection in a single multiplex TaqMan qPCR platform.
- This was done to allow for the simultaneous detection of both protozoan parasites in equids, making the process more efficient.
Findings of the Research
- The study confirmed the multiplex equine piroplasmosis (M-EP) qPCR assay’s efficiency and specificity. It revealed detection limits of 1.4 × 10% for T. equi and 2.8 × 10% for B. caballi.
- The research also revealed how differences in DNA concentrations affected the M-EP qPCR results. The assay was reliable in detecting both targets, even with up to a 1000-fold difference in target concentrations, without a loss in sensitivity.
Evaluation and Practical Application
- The assay developed in this study was applied on 243 field samples from areas with limited tick control strategies. Using Indirect Fluorescent Antibody Test (IFAT), the researchers detected circulating T. equi and B. caballi antibodies in 100% and 92% of the samples, respectively.
- The M-EP qPCR assay spotted T. equi parasite DNA in 98% of the tested samples, whereas B. caballi was detected only in 6% of them. The lower detection rate for B. caballi implies the parasite usually breeds at really low parasitaemias, rarely more than 1%.
- Therefore, the developed M-EP qPCR assay offers a dependable tool for quick diagnosis and epidemiological survey of equine piroplasmosis.
Cite This Article
APA
Bhoora RV, Pienaar R, Cornelius F, Josemans A, Matthee O, Marumo R, Troskie C, Mans BJ.
(2018).
Multiplex hydrolysis-probe assay for the simultaneous detection of Theileria equi and Babesia caballi infections in equids.
Vet Parasitol, 255, 61-68.
https://doi.org/10.1016/j.vetpar.2018.03.022 Publication
Researcher Affiliations
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa. Electronic address: Bhoorar@arc.agric.za.
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Bacteriology - Diagnostic services, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Vaccine production, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa.
- Epidemiology, Parasites and Vectors, Agricultural Research Council, Onderstepoort Veterinary Research, Onderstepoort, 0110, South Africa; Department of Life and Consumer Sciences, University of South Africa, South Africa.
MeSH Terms
- Animals
- Babesia / isolation & purification
- Babesiosis / diagnosis
- Babesiosis / parasitology
- Horse Diseases / diagnosis
- Horse Diseases / parasitology
- Horses
- Multiplex Polymerase Chain Reaction / methods
- Multiplex Polymerase Chain Reaction / veterinary
- Theileria / isolation & purification
- Theileriasis / diagnosis
- Theileriasis / parasitology
Citations
This article has been cited 4 times.- Lv K, Zhang Y, Yang Y, Liu Z, Deng L. Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.. Front Vet Sci 2022;9:873190.
- Nardini R, Bartolomé Del Pino LE, Cersini A, Manna G, Viola MR, Antognetti V, Autorino GL, Scicluna MT. Comparison of PCR-based methods for the detection of Babesia caballi and Theileria equi in field samples collected in Central Italy.. Parasitol Res 2021 Jun;120(6):2157-2164.
- Tirosh-Levy S, Gottlieb Y, Fry LM, Knowles DP, Steinman A. Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny.. Pathogens 2020 Nov 8;9(11).
- Smith RM, Bhoora RV, Kotzé A, Grobler JP, Lee Dalton D. Translocation a potential corridor for equine piroplasms in Cape mountain zebra (Equus zebra zebra).. Int J Parasitol Parasites Wildl 2019 Aug;9:130-133.
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