During the 2015 outbreak of highly pathogenic avian influenza virus
(HPAI) on poultry farms in the midwestern United States, concern was raised
about the potential for HPAI to contaminate groundwater. Our study objective was to evaluate the occurrence of HPAI in the groundwater supply wells on 13 outbreak-affected poultry farms in Iowa and Wisconsin. We sampled 20 wells, six waste-storage lagoons, and one pond. Three wells and one lagoon were positive for the matrix gene indicative of influenza A virus. Using a semi-nested qPCR assay specific to the H5 HPAI outbreak strain, one well was H5-positive, matching the outbreak virus hemagglutinin gene. Matrix gene-positive samples analyzed for avian influenza virus (AIV) by cell culture and embryonating egg culture were negative. Seven wells were positive by PCR for a poultry-specific parvovirus, thus providing corroborating evidence of virus transport pathways between poultry fecal wastes and groundwater. Our data suggest it is possible for AIV to be transported to groundwater, and during an outbreak, the potential for poultry farm wells to become contaminated with AIV should be considered.