High Pathogenicity Avian Influenza (HPAI) A (H5N1) circulates in wild birds, causing outbreaks in domestic poultry, but recently became enzootic in the United States spilling over into various mammals, including cattle. The zoonotic potential of H5N1 clade 2.3.4.4b emphasizes the need for tools for surveillance in mammalian and avian species. We developed two species-agnostic assays using the MESO SCALE DISCOVERY? (MSD) platform to enhance current surveillance methods: a sandwich immunoassay for influenza A nucleoprotein (NP) antigen, and a bridging serology assay for influenza A/H5 specific antibodies. We assessed the assays by testing milk and serum from cattle herds experiencing H5N1 incursions, serum from H5N1-vaccinated humans and chickens, and negative control samples from cattle, chickens, and humans.

Antibodies and antigen were detected in milk and serum from H5-exposed cattle herds, but not in samples from putatively na?ve herds. The sensitivity for identifying exposed animals improved by combining antigen and antibody testing. The bridging serology assay accurately differentiated serum from H5-vaccinated chickens and humans, relative to H5-na?ve controls. High specificity in human samples was achieved by using H1 hemagglutinin to block potentially cross-reactive activity. The studies demonstrate the applicability of these methods to diverse species and facilitate their use in surveillance studies.