From Species to Signals: Exploring the Potential of Airborne eDNA and eRNA Analysis for Pathogen-Agnostic Monitoring at One Health Crossover Points

Land-use change is altering patterns of disease spillover between wild and farmed animals, demanding more agile surveillance at One Health interfaces. Indirect surveillance, which detects host presence rather than pathogens directly, could help inform about disease spillover potential. We collected airborne environmental nucleic acids (eNA), in and around a livestock farm to assess its suitability as a surveillance tool to analyze animal, microbial and viral communities. Host animal presence was compared to three complementary methods; camera trapping, bioacoustics monitoring, and collection of insects for invertebrate derived DNA (iDNA). Air sampling methods were benchmarked in 2024 and finally collected (May 2025) at a farm and two nearby nature estates. The eNA was sequenced using metabarcoding and shotgun metagenomics which identified 53 vertebrate, 324 fungi, 2,739 virus, and 2,753 bacterial taxa across three sites, with the greatest diversity at the farm for all groups except fungi. eNA missed two vertebrate species detected by camera traps but captured six additional species. Bioacoustics outperformed eDNA for bird detection (27 vs 10 families), and iDNA yielded eight vertebrate families. Airborne eNA analysis provides a comprehensive picture of animal, microbial, and viral communities at each crossing-over site, establishing its potential for agnostic host and pathogen monitoring.