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.
Publication Date: 2025-12-03 PubMed ID: 41375541PubMed Central: PMC12691151DOI: 10.3390/ani15233483Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
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
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, Texas A&M University, College Station, TX 77840, USA.
- Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
- College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC 27607, USA.
- Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA.
- Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.
- 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.
References
This article includes 29 references
- Ivester K.M., Couëtil L.L., Zimmerman N.J. Investigating the Link between Particulate Exposure and Airway Inflammation in the Horse. J. Vet. Intern. Med. 2014;28:1653–1665. doi: 10.1111/jvim.12458.
- Couroucé-Malblanc A., Pronost S., Fortier G., Corde R., Rossignol F. Physiological Measurements and Upper and Lower Respiratory Tract Evaluation in French Standardbred Trotters During a Standardised Exercise Test on the Treadmill. Equine Vet. J. 2002;34((Suppl. S34)):402–407. doi: 10.1111/j.2042-3306.2002.tb05456.x.
- Sheats M.K., Davis K.U., Poole J.A. Comparative Review of Asthma in Farmers and Horses. Curr. Allergy Asthma Rep. 2019;19:50. doi: 10.1007/s11882-019-0882-2.
- Pirie R.S. Recurrent Airway Obstruction: A Review. Equine Vet. J. 2014;46:276–288. doi: 10.1111/evj.12204.
- Leclere M., Lavoie-Lamoureux A., Lavoie J.P. Heaves, an Asthma-like Disease of Horses. Respirology. 2011;16:1027–1046. doi: 10.1111/j.1440-1843.2011.02033.x.
- Simões J., Sales Luís J., Tilley P. Contribution of Lung Function Tests to the Staging of Severe Equine Asthma Syndrome in the Field. Res. Vet. Sci. 2019;123:112–117. doi: 10.1016/j.rvsc.2018.12.014.
- Gigue Áre S., Viel L., Lee E., Mackay R.J., Hernandez J., Franchini M. Cytokine Induction in Pulmonary Airways of Horses with Heaves and Effect of Therapy with Inhaled fluticasone Propionate. Vet. Immunol. Immunopathol. 2002;85:147–158. doi: 10.1016/S0165-2427(01)00420-2.
- Ainsworth D.M., Grünig G., Matychak M.B., Young J., Wagner B., Erb H.N., Antczak D.F. Recurrent Airway Obstruction (RAO) in Horses Is Characterized by IFN-γ and IL-8 Production in Bronchoalveolar Lavage Cells. Vet. Immunol. Immunopathol. 2003;96:83–91. doi: 10.1016/S0165-2427(03)00142-9.
- Bullone M., Joubert P., Gagné A., Lavoie J.P., Hélie P. Bronchoalveolar Lavage Fluid Neutrophilia Is Associated with the Severity of Pulmonary Lesions During Equine Asthma Exacerbations. Equine Vet. J. 2018;50:609–615. doi: 10.1111/evj.12806.
- Uberti B., Morán G. Role of Neutrophils in Equine Asthma. Anim. Health Res. Rev. 2018;19:65–73. doi: 10.1017/S146625231800004X.
- Ng H., Havervall S., Rosell A., Aguilera K., Parv K., Von Meijenfeldt F.A., Lisman T., MacKman N., Thålin C., Phillipson M. Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19. Arterioscler. Thromb. Vasc. Biol. 2021;41:988–994. doi: 10.1161/ATVBAHA.120.315267.
- Ponti G., Maccaferri M., Micali S., Manfredini M., Milandri R., Bianchi G., Pellacani G., Kaleci S., Chester J., Conti A., et al. Seminal Cell Free DNA Concentration Levels Discriminate Between Prostate Cancer and Benign Prostatic Hyperplasia. Anticancer Res. 2018;38:5121–5125. doi: 10.21873/anticanres.12833.
- Letendre J.A., Goggs R. Determining Prognosis in Canine Sepsis by Bedside Measurement of Cell-Free DNA and Nucleosomes. J. Vet. Emerg. Crit. Care. 2018;28:503–511. doi: 10.1111/vec.12773.
- Couëtil L.L., Cardwell J.M., Gerber V., Lavoie J.P., Léguillette R., Richard E.A. Inflammatory Airway Disease of Horses-Revised Consensus Statement. J. Vet. Intern. Med. 2016;30:503–515. doi: 10.1111/jvim.13824.
- Charoensappakit A., Sae-khow K., Rattanaliam P., Vutthikraivit N., Pecheenbuvan M., Udomkarnjananun S., Leelahavanichkul A. Cell-Free DNA as Diagnostic and Prognostic Biomarkers for Adult Sepsis: A Systematic Review and Meta-Analysis. Sci. Rep. 2023;13:19624. doi: 10.1038/s41598-023-46663-2.
- Ware S.A., Kliment C.R., Giordano L., Redding K.M., Rumsey W.L., Bates S., Zhang Y., Sciurba F.C., Nouraie S.M., Kaufman B.A. Cell-Free DNA Levels Associate with COPD Exacerbations and Mortality. Respir. Res. 2024;25:42. doi: 10.1186/s12931-023-02658-1.
- Medina J.E., Dracopoli N.C., Bach P.B., Lau A., Scharpf R.B., Meijer G.A., Andersen C.L., Velculescu V.E. Cell-Free DNA Approaches for Cancer Early Detection and Interception. J. Immunother. Cancer. 2023;11:e006013. doi: 10.1136/jitc-2022-006013.
- Fingerhut L., Ohnesorge B., von Borstel M., Schumski A., Strutzberg-Minder K., Mörgelin M., Deeg C.A., Haagsman H.P., Beineke A., von Köckritz-Blickwede M., et al. Neutrophil Extracellular Traps in the Pathogenesis of Equine Recurrent Uveitis (ERU) Cells. 2019;8:1528. doi: 10.3390/cells8121528.
- Bayless R.L., Cooper B.L., Sheats M.K. Extracted Plasma Cell-Free DNA Concentrations Are Elevated in Colic Patients with Systemic Inflammation. Vet. Sci. 2024;11:427. doi: 10.3390/vetsci11090427.
- Bayless R.L., Cooper B.L., Sheats M.K. Investigation of Plasma Cell-Free DNA as a Potential Biomarker in Horses. J. Vet. Diagn. Investig. 2022;34:402–406. doi: 10.1177/10406387221078047.
- Frippiat T., Art T., Tosi I. Airway Hyperresponsiveness, but Not Bronchoalveolar Inflammatory Cytokines Profiles, Is Modified at the Subclinical Onset of Severe Equine Asthma. Animals. 2023;13:2485. doi: 10.3390/ani13152485.
- Rossi H., Virtala A.M., Raekallio M., Rahkonen E., Rajamäki M.M., Mykkänen A. Comparison of Tracheal Wash and Bronchoalveolar Lavage Cytology in 154 Horses with and without Respiratory Signs in a Referral Hospital over 2009–2015. Front. Vet. Sci. 2018;5:61. doi: 10.3389/fvets.2018.00061.
- Tullie S., Asiri A., Acharjee A., Moiemen N.S., Lord J.M., Harrison P., Hazeldine J. Day One Cell-Free DNA Levels as an Objective Prognostic Marker of Mortality in Major Burns Patients. Cells. 2025;14:821. doi: 10.3390/cells14110821.
- Hampson P., Dinsdale R.J., Wearn C.M., Bamford A.L., Bishop J.R., Hazeldine J., Moiemen N.S., Harrison P., Lord J.M. Neutrophil Dysfunction, Immature Granulocytes, and Cell-free DNA are Early Biomarkers of Sepsis in Burn-injured Patients: A Prospective Observational Cohort Study. Ann. Surg. 2017;265:1241–1249. doi: 10.1097/SLA.0000000000001807.
- Janssen P., Tosi I., Hego A., Maréchal P., Marichal T., Radermecker C. Neutrophil Extracellular Traps Are Found in Bronchoalveolar Lavage Fluids of Horses with Severe Asthma and Correlate with Asthma Severity. Front. Immunol. 2022;13:921077. doi: 10.3389/fimmu.2022.921077.
- Neuberger E.W.I., Brahmer A., Ehlert T., Kluge K., Philippi K.F.A., Boedecker S.C., Weinmann-Menke J., Simon P. Validating Quantitative PCR Assays for CfDNA Detection Without DNA Extraction in Exercising SLE Patients. Sci. Rep. 2021;11:13581. doi: 10.1038/s41598-021-92826-4.
- Chen X., Chen C., Tu Z., Guo Z., Lu T., Li J., Wen Y., Chen D., Lei W., Wen W., et al. Intranasal PAMAM-G3 Scavenges Cell-Free DNA Attenuating the Allergic Airway Inflammation. Cell Death Discov. 2024;10:213. doi: 10.1038/s41420-024-01980-x.
- Hu Z., Chen H., Long Y., Li P., Gu Y. The Main Sources of Circulating Cell-Free DNA: Apoptosis, Necrosis and Active Secretion. Crit. Rev. Oncol./Hematol. 2021;157:103166. doi: 10.1016/j.critrevonc.2020.103166.
- Papayannopoulos V. Neutrophil Extracellular Traps in Immunity and Disease. Nat. Rev. Immunol. 2018;18:134–147. doi: 10.1038/nri.2017.105.
Citations
This article has been cited 0 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
