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Parasites & vectors2018; 11(1); 125; doi: 10.1186/s13071-018-2751-6

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

Abstract: Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing. We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR. The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.
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Publication Date: 2018-03-02 PubMed ID: 29499748PubMed Central: PMC5834856DOI: 10.1186/s13071-018-2751-6Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Validation Study

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.

This research paper discusses the development and validation of a new method to diagnose equine piroplasmosis, a significant infection in horses caused by the tick-borne protozoa Theileria equi and Babesia caballi. The researchers developed a rapid, sensitive test, a real-time quantitative PCR (qPCR) assay, which effectively identified both causes of the disease in horse samples and was found to be more cost-effective than existing methods. The test was reliable in confirming results of serological tests for T. equi but not for B. caballi, warranting further research.

Development and Evaluation of The Assay

  • The researchers developed a duplex qPCR assay that targets two specific genes, ema-1 gene of T. equi and the 18S rRNA gene of B. caballi, which are unique to each protozoa.
  • The aim was to simultaneously detect both causative agents of equine piroplasmosis, enhancing testing efficiency and accuracy.
  • After the development, the assay underwent a series of evaluations to determine its sensitivity and reliability.

Results of The Validation Process

  • The researchers ran the duplex qPCR test on samples from 362 horses from Canada and the United States, found negative for equine piroplasmosis using the enzyme-linked immunosorbent assay (cELISA). The duplex qPCR did not yield any false-positive results, indicating its high specificity.
  • Further validation occurred using horse samples from an endemic area in Brazil. The results of the qPCR were compared to two alternative tests; an 18S rRNA qPCR for T. equi developed in Japan, and another 18S rRNA qPCR for B. caballi.
  • The reliability of the cELISA was also assessed by comparing its results to those of the duplex qPCR.
  • Results from the Brazilian horse samples yielded higher infection rates with both protozoa, with all three tests (duplex qPCR, single-target qPCR, and cELISA) producing similar results for T. equi. However, for B. caballi, there was a significant discrepancy between the duplex qPCR/single-target qPCR and the cELISA.

Implications of The Findings

  • The duplex qPCR performed comparably to existing single-target qPCR assays for both T. equi and B. caballi in terms of detection efficiency, but was found to be more cost-effective in terms of response time and reagent costs, making it a good choice for disease control and investigations.
  • Unfortunately, the qPCR test did not correlate well with the cELISA results when testing for B. caballi, suggesting it should not be used as a standalone confirmatory test for this protozoa.
  • Though the new test is highly effective for T. equi detection, the research affirms further investigation is needed to understand the transmission risk of B. caballi in horses that test negative through PCR but possess detectable antibodies.

Cite This Article

APA
Lobanov VA, Peckle M, Massard CL, Brad Scandrett W, Gajadhar AA. (2018). Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis. Parasit Vectors, 11(1), 125. https://doi.org/10.1186/s13071-018-2751-6

Publication

Parasites & vectors
ISSN: 1756-3305
NlmUniqueID: 101462774
Country: England
Language: English
Volume: 11
Issue: 1
Pages: 125
PII: 125

Researcher Affiliations

Lobanov, Vladislav A
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada. Vladislav.Lobanov@inspection.gc.ca.
Peckle, Maristela
  • Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil.
Massard, Carlos L
  • Department of Animal Parasitology, Veterinary Institute, Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil.
Brad Scandrett, W
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.
Gajadhar, Alvin A
  • Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.

MeSH Terms

  • Animals
  • Babesia / genetics
  • Babesia / isolation & purification
  • Babesiosis / diagnosis
  • Babesiosis / epidemiology
  • Babesiosis / parasitology
  • Brazil / epidemiology
  • Canada / epidemiology
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Horse Diseases / diagnosis
  • Horse Diseases / parasitology
  • Horses
  • Japan / epidemiology
  • Protozoan Infections, Animal / diagnosis
  • Protozoan Infections, Animal / epidemiology
  • RNA, Ribosomal, 18S / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / veterinary
  • Reproducibility of Results
  • Risk Factors
  • Seroepidemiologic Studies
  • Theileria / genetics
  • Theileria / isolation & purification
  • Ticks

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The collection of blood samples from horses in the state of Rio de Janeiro, Brazil was approved by the Ethics Committee on Animal Research of the Federal Rural University of Rio de Janeiro (COMEP / UFRRJ), under process number 23083.001257/2012-53 (protocol number 201/2012). Collections of blood from horses located in Canada and the United States were approved by the University of Saskatchewan Committee on Animal Care and Supply and by the NVSL/Center for Veterinary Biologics Institutional Animal Care and Use Committee, respectively. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Citations

This article has been cited 6 times.
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