Wild Bird Surveillance for H5N1 and H5N2 Avian Influenza in North American Flyways: Seroprevalence, Phylogeography, and Waterfowl Species Risk Stratification

This study investigates seroprevalence, phylogeographic distribution, and waterfowl species-level risk stratification for H5N1 and H5N2 highly pathogenic avian influenza along North American migratory flyways within the context of veterinary epidemiology and avian influenza surveillance, an area of growing scientific importance given its implications for HPAI early warning system design, poultry biosecurity buffer zone delineation, and wild bird surveillance protocol prioritization. Using cloacal and oropharyngeal swab RT-PCR surveillance combined with anti-nucleoprotein ELISA serology and whole-genome sequencing of positive isolates for phylogeographic analysis, we examine migratory waterfowl serving as long-distance HPAI reservoir hosts, with reassortment at shared wintering and staging grounds enabling intercontinental gene segment exchange and novel HPAI genotype emergence in 18,420 wild birds sampled from 84 wetland sites across 4 flyways (Pacific, Central, Mississippi, Atlantic) during fall 2014, spring 2015, fall 2015, fall 2016, spring 2017 drawn from 84 wetland sentinel sites stratified across 4 North American migratory flyways with U.S. Fish and Wildlife Service sampling cooperation. Results indicate that RT-PCR prevalence of 2.84% (range 0.8-8.4% by flyway) with seroprevalence of 18.4%, significantly higher in Mississippi flyway (seroprevalence 28.4%); dabbling ducks show 3.2x higher prevalence than diving ducks across all flyways (p < 0.001), with 2.84% RT-PCR prevalence; 18.4% seroprevalence; 3.2x higher risk in dabbling vs. diving ducks as the primary quantitative benchmark. Concordance between primary and confirmatory measurement approaches exceeded 93%, validating the analytical framework. These findings contribute empirically to veterinary epidemiology and avian influenza surveillance and carry actionable implications for the design of programs and policies targeting HPAI early warning system design, poultry biosecurity buffer zone delineation, and wild bird surveillance protocol prioritization.