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Animals : an open access journal from MDPI2025; 15(23); 3483; doi: 10.3390/ani15233483

A Portable Fluorometer Detects Significantly Elevated Cell-Free DNA in Tracheal Wash and Bronchoalveolar Lavage Fluid in Horses with Severe Asthma.

Abstract: Severe equine asthma (sEA) is characterized by increased lower airway neutrophils that contribute to dysregulated inflammation through the release of cytokines, reactive oxygen species and neutrophil extracellular traps (NETs). NETs are composed of cell-free DNA (cfDNA) intercalated with enzymatic proteins and are known to be increased in the lower airway of asthmatic horses. The objectives of this study were two-fold: 1. Determine if cfDNA can be accurately measured in equine bronchoalveolar lavage fluid (BALF) and tracheal wash (TW) with a Qubit 4 fluorometer. 2. Determine whether Qubit-measured cfDNA in BALF or TW is significantly different in horses with sEA, mild/moderate neutrophilic equine asthma, mastocytic equine asthma and healthy horses. A total of sixty-three horses received a physical examination and clinical score followed by a BAL +/- TW. Cell-free DNA was measured using three methods in unfiltered BAL and TW as well as BAL and TW supernatant. Cell-free DNA concentrations were highly correlated between the Qubit 4 fluorometer and NanoDrop spectrophotometer as well as between the Qubit 4 fluorometer and SYTOX green plate-based assay. Cell-free DNA concentrations were highly correlated between unfiltered TW and TW supernatant as well as between unfiltered BALF and BAL supernatant. Cell-free DNA concentrations in BAL and TW supernatant were significantly higher in horses with sEA compared to healthy horses or horses with mild/moderate equine asthma. Cell-free DNA is a biomarker of sEA that can be easily measured in the field with the small portable Qubit 4 fluorometer in BAL and TW fluid. These findings support further investigation of NETs as a biomarker and potential therapeutic target for severe equine asthma.
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Publication Date: 2025-12-03 PubMed ID: 41375541PubMed Central: PMC12691151DOI: 10.3390/ani15233483Google Scholar: Lookup
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  • Journal Article

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 investigated whether a portable device called the Qubit 4 fluorometer can accurately measure cell-free DNA (cfDNA) in airway fluid samples from horses.
  • It found that horses with severe equine asthma have significantly higher levels of cfDNA in their airway fluids compared to healthy horses or those with milder asthma.

Background

  • Severe Equine Asthma (sEA): A chronic respiratory disease in horses characterized by increased neutrophils— a type of immune cell—in the lower airways.
  • Neutrophil Extracellular Traps (NETs): Structures composed mainly of cell-free DNA combined with enzymatic proteins released by neutrophils during inflammation.
  • Importance of NETs: They contribute to the dysregulated inflammation seen in sEA and their levels are elevated in affected horses, making them promising markers for disease severity.

Study Objectives

  • To assess whether cfDNA can be accurately quantified in bronchoalveolar lavage fluid (BALF) and tracheal wash (TW) samples from horses using the Qubit 4 fluorometer.
  • To determine if cfDNA levels measured by Qubit 4 differ significantly among horses with severe equine asthma, mild/moderate neutrophilic or mastocytic equine asthma, and healthy controls.

Methods

  • Subjects: Sixty-three horses undergoing physical examination and clinical scoring.
  • Sample Collection: Bronchoalveolar lavage (BAL) and/or tracheal wash (TW) fluids collected for analysis.
  • cfDNA Measurement:
    • Three different methods were used to measure cfDNA: Qubit 4 fluorometer, NanoDrop spectrophotometer, and SYTOX green plate-based assay.
    • Measurements taken from both unfiltered samples and supernatants (fluid after centrifugation) of BAL and TW.

Key Findings

  • cfDNA concentrations measured by Qubit 4 showed strong correlation with measurements from NanoDrop spectrophotometer and SYTOX green assay, affirming the accuracy of the Qubit 4.
  • cfDNA levels were highly correlated between unfiltered and supernatant samples, indicating cfDNA is accessible in both sample types.
  • Horses with severe equine asthma had significantly elevated cfDNA levels in both BAL and TW supernatant compared to healthy horses and those with mild/moderate asthma.

Implications

  • The Qubit 4 fluorometer, a small and portable device, can reliably measure cfDNA in airway samples, making it practical for field use in diagnosing and monitoring severe equine asthma.
  • Elevated cfDNA serves as a valuable biomarker of severe equine asthma, reflecting NET formation and ongoing neutrophil-driven inflammation.
  • These results encourage further research on NETs not only as biomarkers but also as potential targets for novel therapies to treat severe equine asthma.

Conclusion

  • Rapid, accurate field measurement of cfDNA by the Qubit 4 fluorometer provides a useful tool for distinguishing severe equine asthma from milder forms and healthy states.
  • This approach enhances understanding of disease mechanisms and supports improved diagnosis and management strategies in veterinary medicine.

Cite This Article

APA
Cooper BL, Hobbs KJ, Bayless R, Stinson-Miller A, Gruber E, Hepworth-Warren K, Lavoie JP, Sheats MK. (2025). A Portable Fluorometer Detects Significantly Elevated Cell-Free DNA in Tracheal Wash and Bronchoalveolar Lavage Fluid in Horses with Severe Asthma. Animals (Basel), 15(23), 3483. https://doi.org/10.3390/ani15233483

Publication

Animals : an open access journal from MDPI
ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 15
Issue: 23
PII: 3483

Researcher Affiliations

Cooper, Bethanie L
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
Hobbs, Kallie J
  • Department of Clinical Sciences, College of Veterinary Medicine, Texas A&M University, College Station, TX 77840, USA.
Bayless, Rosemary
  • Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
Stinson-Miller, Austen
  • College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC 27607, USA.
Gruber, Erika
  • Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
Hepworth-Warren, Kate
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
Lavoie, Jean-Pierre
  • Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.
Sheats, M Katie
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.

Grant Funding

  • 532001 / American Association of Equine Practitioners
  • 2022-100029 / Hong Kong Jockey Club

Conflict of Interest Statement

The authors declare no conflicts of interest.

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