Methodological limitations in eDNA-based detection of Bonamia ostreae: Implications for non-invasive oyster health monitoring

The European flat oyster ( Ostrea edulis ) plays a vital role in marine ecosystems and aquaculture, but populations have been severely impacted by Bonamia ostreae , an intracellular parasite which causes high mortality. Environmental DNA (eDNA) offers a non-invasive alternative to traditional tissue diagnostics, though protocol optimisation is needed for reliable detection. This study validated quantitative PCR (qPCR) assays using synthetic oligonucleotide standards (oligos) and evaluated water, faeces, and passive eDNA sampling detectors (PEDS) — mixed-cellulose ester (MCEF45), glass-fibre filters (GFP), and cellulose sponges (CS) — under varying incubation times, temperatures, water volumes and hatchery tank systems. Bonamia -positive flat oysters were placed individually in static tanks and tested for the likelihood of B. ostreae detection in eDNA samples over 2–14 days of incubation in 11–15 °C seawater, with an additional 14-day sub-treatment at −1 °C. Oysters were also placed in groups in flow-through tanks at 20 °C and tested for the likelihood of parasite detection in eDNA samples. MCEF45 and GFP PEDS achieved 88–90% detection sensitivity at 20 °C, exceeding CS (60%). Optimal detection in PEDS occurred at 12–20 °C; −1 °C markedly reduced sensitivity. Our findings reveal that detection success is governed by host infection intensity, temperature, and incubation duration: heavily infected oysters shed detectable parasites regardless of protocol, while low-intensity infections required favourable temperatures and extended environmental incubation to capture sporadic shedding events. Water samples outperformed faeces significantly despite lower total DNA levels, indicating that pathogen-specific shedding routes — not biomass — determine detection success. Detection rates from 50 to 72% across 2–5 day incubations with water combined with faeces, increasing to 90% at 14 days ( p < 0.01), particularly benefiting low-intensity infections (Cq >25) which showed near-zero detection at shorter periods. Our findings show that eDNA assays remain constrained by biological and environmental factors that prevent the predictable release of B. ostreae into the environment, thus limiting their immediate replacement of tissue diagnostics. While eDNA methods have not undergone the staged diagnostic validation described by the WOAH for animal-derived samples — and positive environmental detections cannot establish whether a susceptible host is infected — the detection capability demonstrated in this study supports the use of eDNA as a cost-effective, non-lethal screening for routine hatchery monitoring and surveillance of wild populations, provided that positive results are followed up with confirmatory testing on animal-derived samples as specified in the relevant WOAH recommendations.