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

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.