Evaluation of targeted next-generation sequencing for detection of equine pathogens in clinical samples.
Abstract: Equine infectious disease outbreaks may have profound economic impact, resulting in losses of millions of dollars of revenue as a result of horse loss, quarantine, and cancelled events. Early and accurate diagnosis is essential to limit the spread of infectious diseases. However, laboratory detection of infectious agents, especially the simultaneous detection of multiple agents, can be challenging to the clinician and diagnostic laboratory. Next-generation sequencing (NGS), which allows millions of DNA templates to be sequenced simultaneously in a single reaction, is an ideal technology for comprehensive testing. We conducted a proof-of-concept study of targeted NGS to detect 62 common equine bacterial, viral, and parasitic pathogens in clinical samples. We designed 264 primers and constructed a bioinformatics tool for the detection of targeted pathogens. The designed primers were able to specifically detect the intended pathogens. Results of testing 27 clinical samples with our targeted NGS assay compared with results of routine tests (assessed as a group) yielded positive percent agreement of 81% and negative percent agreement of 83%, overall agreement of 81%, and kappa of 0.56 (moderate agreement). This moderate agreement was likely the result of low sensitivity of some primers. However, our NGS assay successfully detected multiple pathogens in the clinical samples, including some pathogens missed by routine techniques.
Publication Date: 2020-12-11 PubMed ID: 33305693PubMed Central: PMC7953111DOI: 10.1177/1040638720978381Google Scholar: Lookup
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- Evaluation Study
- Journal Article
Summary
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This research focused on using next-generation sequencing (NGS) to detect equine bacterial, viral, and parasitic pathogens in clinical samples. The study found moderate success with the NGS technology, leading to its potential for greater accuracy compared to routine tests.
Introduction
- The study began with the premise that equine infectious diseases can cause significant economic loss due to lost revenue, quarantine measures, and event cancellations.
- The researchers noted the importance of early and accurate diagnosis to prevent the spread of such diseases, a challenge especially when it comes to the simultaneous detection of multiple agents.
- The study’s goal was to evaluate the effectiveness of next-generation sequencing (NGS) technology, which can sequence millions of DNA templates simultaneously, for the comprehensive detection of infectious diseases.
Methodology
- The researchers used a proof-of-concept study to test NGS’s ability to detect 62 common equine bacterial, viral, and parasitic pathogens in clinical samples.
- They developed 264 primers, tools used in DNA synthesis, to detect these pathogens.
- Alongside these primers, a bioinformatics tool was constructed to aid in pathogen detection.
Results
- The researchers tested 27 clinical samples using the targeted NGS assay and the results were compared with routine tests.
- The NGS test’s positive percent agreement was 81%; negative percent agreement was 83%; overall agreement was 81% and the kappa was 0.56, indicating moderate agreement.
- While the results of the NGS test and routine tests moderately agreed, it was noted that the level of this agreement could be explained by low sensitivity with some primers.
- Impressively, the NGS test was able to successfully detect multiple pathogens in the clinical samples, including some pathogens that regular tactics overlooked.
Conclusion
- The results underscore the potential of NGS technology for detecting multiple equine pathogens at once. While the primers had a moderate success rate, the research indicated its advantage over routine testing methods in detecting multiple pathogens.
- The results also hint that the sensitivity of the primers might need improvement for better disease detection rates.
Cite This Article
APA
Anis E, Ilha MRS, Engiles JB, Wilkes RP.
(2020).
Evaluation of targeted next-generation sequencing for detection of equine pathogens in clinical samples.
J Vet Diagn Invest, 33(2), 227-234.
https://doi.org/10.1177/1040638720978381 Publication
Researcher Affiliations
- Department of Pathobiology, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA.
- Department of Virology, Faculty of Veterinary Medicine, University of Sadat, El Beheira Governorate, Sadat City, Egypt.
- Tifton Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA.
- Department of Pathobiology, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA.
- Tifton Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA.
- Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN.
MeSH Terms
- Animals
- High-Throughput Nucleotide Sequencing / methods
- High-Throughput Nucleotide Sequencing / veterinary
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horse Diseases / parasitology
- Horse Diseases / virology
- Horses
Conflict of Interest Statement
Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Citations
This article has been cited 4 times.- Luo J, Lu W, Liu R, Zhang S, Cao J, Ma C. From Panels to Pathogen Networks: The Expanding Role of Targeted Sequencing in Veterinary Medicine. Biology (Basel) 2025 Aug 18;14(8).
- Zeng R, Wang G, Zhou F. Analysis of gene polymorphisms in patients with pulmonary infections based on next-generation sequencing technology and their prognostic predictive value. Front Med (Lausanne) 2025;12:1599791.
- Wernery U, Teng JLL, Ma Y, Kinne J, Yeung ML, Anas S, Lau SKP, Woo PCY. Usefulness of Next-Generation Sequencing in Excluding Bovine Leukemia Virus as a Cause of Adult Camel Leukosis in Dromedaries. Pathogens 2023 Jul 29;12(8).
- Goodman L, Lahmers K. Special issue on applied next-generation sequencing in veterinary diagnostic laboratories. J Vet Diagn Invest 2021 Mar;33(2):177-178.
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