Detection of airborne wild waterbird-derived DNA demonstrates potential transmission of avian influenza virus via air-inlets into poultry houses Alex Bossers, Myrna M.T. de Rooij, Isabella van Schothorst, Francisca C. Velkers and Lidwien A.M. Smit Background Outbreaks of highly pathogenic avian influenza (HPAI) in poultry farms and wild birds worldwide are still increasing despite intensified control measures. HPAI is causing unprecedented mortality in bird populations, and is increasingly affecting mammalian species. Better understanding of HPAI introduction pathways into farms are needed for targeted disease prevention and control. The relevance of airborne transmission has been suggested but research involving air sampling is limited and unequivocal evidence on transmission routes is lacking. Aim To investigate whether HPAI-virus from wild birds can potentially enter poultry houses through air-inlets by characterizing host materials by eukaryote DNA sequencing. Methods Particulate matter (PM) samples were collected in, and around three HPAI affected poultry farms which were cleared and decontaminated prior to sampling. Indoor measurements (N=61) were taken directly in the air-flow entering through air-inlets, while outdoor air samples (N=60) were collected around the poultry house. Positive controls were obtained from a bird rehabilitation shelter. Metabarcoding was performed on environmental DNA by deep-sequencing 18S-rRNA gene amplicons. Results Waterbird DNA was detected in air inside all three, and outside of two poultry farms. Sequences annotated at species level included swans and tufted ducks. Waterbird DNA was present in all indoor and outdoor air samples from the bird shelter. Conclusion HPAI-virus can potentially be introduced in poultry houses through air-inlets via airborne contaminated wild bird-derived matter. The eDNA metabarcoding provided a comprehensive tool to assess biosecurity for HPAI-virus and other pathogens potentially transmitted through air via detection of their corresponding hosts.