Development of eNA Sampling for Early Detection of Pathogens in On‐Farm Water Sources

Early detection of livestock pathogens is critical for mitigating risk and implementing timely control or eradication responses. Conventional surveillance methods rely on host sampling, which constrains both spatial and temporal coverage. Environmental nucleic acid (eNA) sampling from livestock water troughs offers a promising alternative for detecting microbial and viral communities associated with livestock health and may facilitate early warning of disease presence. In this study, we evaluated four eNA water sampling methods: syringe, cartridge, funnel and passive filter, for their effectiveness in capturing bacterial and viral eNA communities from livestock troughs. Quantitative PCR was used to assess eNA yield and amplification performance, while 16S rRNA gene metabarcoding and RNA‐based viromic (metatranscriptomic) sequencing were used to characterize bacterial and viral community composition and evaluate the effects of sampling method, pore size, and filter material. Filter pore size had the strongest effect on filtered volume but did not significantly influence downstream analysis of microbial and viral community composition. Sampling method and site had the greatest influence on bacterial diversity, whereas viral diversity was influenced by sampling method and RNA concentration. Syringe filters were the most cost‐effective method, supporting their suitability for large‐scale, industry or landholder led sampling programs. Cartridge filters filtered greater volumes and provided higher richness and may be more suitable for detecting low‐abundance targets. Passive filters, which integrate eNA over longer deployment periods, may improve detection of intermittently shed pathogens, though logistical constraints remain. By comparing cost, volume, nucleic acid yield, and community profile, we provide practical guidance for selecting eNA sampling methods to support early pathogen detection at livestock water sources. We recommend that eNA‐based surveillance can complement existing diagnostic systems to strengthen biosecurity monitoring.