Early-warning detection of Flavobacterium psychrophilum spillover from fish farms using environmental DNA

Aquaculture pathogen outbreaks pose a significant spillover risk to wild populations, demanding the development of early detection mechanisms. We applied an eDNA-based qPCR assay to inflow, rearing, and outflow waters of two independent rainbow trout ( Oncorhynchus mykiss ) farms to detect and quantify Flavobacterium psychrophilum , a highly infective salmonid pathogen, and identify potential spillovers into local waterways. F. psychrophilum was detected at both facilities, even in the absence of individuals displaying clinical signs of infection, with significantly higher F. psychrophilum copy numbers detected in rearing and outflow water compared with inflow water (Farm 1 mean copies: inflow = 9 ± 11, rearing = 2662 ± 1882, outflow = 314 ± 216; Farm 2 mean copies: inflow = 24 ± 26, rearing = 200 ± 245, outflow = 66 ± 71), indicating potential spillover. We show that eDNA-qPCR constitutes a sensitive tool for F. psychrophilum detection and quantification in the absence of clinical signs, enabling early-warning monitoring for outbreak prevention and spillover mitigation.