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Viruses2024; 16(7); 1091; doi: 10.3390/v16071091

Evaluation of Non-Invasive Sampling Techniques for the Molecular Surveillance of Equid Herpesviruses in Yearling Horses.

Abstract: Equid alphaherpesvirus 1 (EHV-1) is a highly contagious respiratory tract pathogen of horses, and infection may be followed by myeloencephalopathy or abortion. Surveillance and early detection have focused on PCR assays using less tolerated nasal swabs. Here, we assess non-invasive non-contact sampling techniques as surveillance tools in naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1. Horses were individually housed for 10 h periods on 2 consecutive days. Sampling included nasal swabs, nostril wipes, environmental swabs, droplet-catching devices, and air sampling. The latter was completed via two strategies: a combined air sample collected while going from horse to horse and a collective air sample collected at a stationary central point for 6 h. Samples were screened through quantitative PCR and digital PCR. Nine horses on day 1 and 11 horses on day 2 were positive for EHV-1; overall, 90.9% of the nostril wipes, 81.8% of the environmental surfaces, and 90.9% of the droplet-catching devices were found to be positive. Quantitative analysis showed that the mean DNA copies detection per cm of nostril wipe sampled concentration (4.3 × 10 per day) was significantly ( < 0.05) comparable to that of nasal swabs (3.6 × 10 per day) followed by environmental swabs (4.3 × 10 per day) and droplet catchers (3.5 × 10 per day), respectively. Overall, 100% of the air samples collected were positive on both qPCR and dPCR. In individual air samples, a mean concentration of 1.0 × 10 copies of DNA were detected in per m air sampled per day, while in the collective air samples, the mean concentration was 1.1 × 10. Environmental samples look promising in replacing direct contact sampling. Environmental and air sampling could become efficient surveillance tools at equestrian events; however, it needs threshold calculations for minimum detection levels.
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Publication Date: 2024-07-07 PubMed ID: 39066254PubMed Central: PMC11281437DOI: 10.3390/v16071091Google Scholar: Lookup
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  • Journal Article
  • Evaluation Study
  • Research Support
  • 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.

Objective Overview

  • This study evaluates non-invasive, non-contact methods for detecting equid herpesviruses in yearling horses, aiming to find effective alternatives to traditional nasal swabs for molecular surveillance.
  • It particularly focuses on the detection of equid alphaherpesvirus 1 (EHV-1), a contagious respiratory pathogen, by using surrogate sampling techniques in naturally virus-shedding horses.

Background and Importance

  • Equid alphaherpesvirus 1 (EHV-1) is a significant respiratory pathogen in horses causing infections that can lead to serious complications such as myeloencephalopathy (neurological disease) or abortion.
  • Early detection and continuous surveillance of EHV-1 are critical to control its spread, especially in equestrian environments.
  • Traditional surveillance relies on PCR tests from nasal swabs, which require direct contact and can be distressing or less tolerated by horses.
  • Non-invasive and non-contact sampling methods are desirable to improve animal welfare and ease of monitoring.

Study Design and Methods

  • The study used naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1 detection, which allows safer and controlled evaluation of sampling techniques.
  • Horses were individually housed for 10-hour periods on two consecutive days to standardize exposure and sampling conditions.
  • The researchers collected a variety of sample types from each horse, including:
    • Nasal swabs (traditional, direct contact samples)
    • Nostril wipes (non-invasive, non-contact samples)
    • Environmental swabs from surfaces around the horses
    • Droplet-catching devices aimed at collecting respiratory droplets
    • Air samples collected by two strategies:
      • Combined air sampling collected while moving from horse to horse
      • Collective air sampling at a fixed central location over 6 hours
  • Samples were analyzed using quantitative PCR (qPCR) and digital PCR (dPCR) to detect and quantify viral DNA.

Key Results

  • On day 1, 9 horses tested positive for the virus; on day 2, 11 horses were positive.
  • Detection rates for non-invasive samples were high:
    • 90.9% of nostril wipes were positive
    • 81.8% of environmental swabs were positive
    • 90.9% of droplet-catching devices were positive
  • Quantitative viral DNA measurements per cm of sampled material showed:
    • Nostril wipes had a mean DNA copy number of approximately 4.3 × 105 per day, which was statistically comparable to nasal swabs (3.6 × 105)
    • Environmental swabs and droplet catchers showed slightly lower but still significant detection levels (4.3 × 104 and 3.5 × 104, respectively)
  • Air sampling proved highly effective:
    • All air samples (100%) tested positive by both qPCR and dPCR
    • Individual air samples contained a mean of 1.0 × 102 DNA copies per meter of air sampled per day
    • Collective air samples had slightly higher mean concentration at 1.1 × 102

Interpretation and Implications

  • The study demonstrates that non-invasive techniques, especially nostril wipes and air sampling, appear as reliable alternatives to nasal swabs for molecular surveillance of equid herpesviruses.
  • Environmental swabs, nostril wipes, and droplet-catching devices provide sufficiently high detection rates to be considered as less stressful, contact-free options.
  • Air sampling, both mobile and stationary strategies, showed excellent sensitivity, potentially enabling surveillance without direct horse handling.
  • These methods could facilitate virus monitoring in large settings such as equestrian events, improving biosecurity and disease management.
  • However, standardization steps, including establishing detection thresholds, are necessary for practical implementation.

Conclusions

  • Non-invasive sampling approaches are promising surveillance tools to detect equid herpesviruses effectively.
  • Replacing traditional nasal swabs with nostril wipes or using environmental and air samples may enhance surveillance efficiency and horse welfare.
  • Further research is required for establishing minimum detection levels and to confirm applicability in broader field conditions.

Cite This Article

APA
Khan A, Olajide E, Friedrich M, Holt A, Goehring LS. (2024). Evaluation of Non-Invasive Sampling Techniques for the Molecular Surveillance of Equid Herpesviruses in Yearling Horses. Viruses, 16(7), 1091. https://doi.org/10.3390/v16071091

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 16
Issue: 7
PII: 1091

Researcher Affiliations

Khan, Amjad
  • Department of Veterinary Science, Martin-Gatton College of Agriculture, Food and the Environment, University of Kentucky, Lexington, KY 40506, USA.
  • Department of Public Health & Nutrition, University of Haripur, Haripur 22600, Pakistan.
Olajide, Edward
  • Department of Veterinary Science, Martin-Gatton College of Agriculture, Food and the Environment, University of Kentucky, Lexington, KY 40506, USA.
Friedrich, Madeline
  • College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752-8245, USA.
Holt, Anna
  • College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752-8245, USA.
Goehring, Lutz S
  • Department of Veterinary Science, Martin-Gatton College of Agriculture, Food and the Environment, University of Kentucky, Lexington, KY 40506, USA.

MeSH Terms

  • Animals
  • Horses / virology
  • Horse Diseases / virology
  • Horse Diseases / diagnosis
  • Herpesviridae Infections / veterinary
  • Herpesviridae Infections / virology
  • Herpesviridae Infections / diagnosis
  • Herpesvirus 1, Equid / isolation & purification
  • Herpesvirus 1, Equid / genetics
  • Specimen Handling / methods
  • Female
  • Virus Shedding

Grant Funding

  • 3048116163 / INTERNATIONAL EQUESTRIAN FEDERATION

Conflict of Interest Statement

The authors declare no conflicts of interest.

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Citations

This article has been cited 2 times.
  1. Arshad A, Reif AH, Cavalleri JV, Desvars-Larrive A. Zoonotic pathogens in equids in Central Europe: a systematic review. BMC Vet Res 2025 Jul 8;21(1):451.
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  2. Pusterla N, Lawton K, Barnum S, Magdesian KG. Comparison of Nose Wipes, Stall Sponges, and Air Samples with Nasal Secretions for the Molecular Detection of Equine Influenza Virus in Clinically and Subclinically Infected Horses. Viruses 2025 Mar 20;17(3).
    doi: 10.3390/v17030449pubmed: 40143375google scholar: lookup
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