Environmental samples, a sensitive and practical alternative to individual bird sampling in the surveillance of H9N2 vaccinated turkey flocks

Surveillance for influenza A virus infections in vaccinated poultry flocks remains challenging due to animal welfare, logistical and financial constraints, particularly under current EU regulations governing high pathogenicity avian influenza (HPAI) vaccination. In this field study, we evaluated two environmental sampling (ES) methods – bedding boot swabs and drinker wipes – as alternatives to legally mandated individual bird testing, which involved monthly swabbing of 60 healthy birds (active surveillance, AS) and weekly swabbing of dead or sick birds (passive surveillance, PS). A total of 56 turkey flocks from 23 holdings in Lower Saxony, Germany, were monitored throughout the fattening period following single H9N2 vaccination at hatch. Semiquantitative reverse transcription polymerase chain reaction (RT-qPCR) revealed that, despite vaccination, H9N2 virus incursions occurred at least once in 76.8% (43/56) of flocks during the subsequent fattening period. Influenza A virus detection rate on the basis of individual samples was significantly higher by ES (24.1%) than through AS (10.65%; P <0.0001) or PS (15.6%, P =0.001). Overall, ES demonstrated superior performance in identifying 42/43 infected flocks (99.67%) compared with 30/43 (69.97%) by AS and 38/43 (88.37%) by PS. Heatmap and event-time analyses confirmed that ES reliably identified infection events very early and remained positive longer after initial detection. Non-invasive, animal-friendly ES was easy to implement and well accepted by farmers. Costs for ES surveillance were reduced by 73.5% compared to AS and PS. ES was found to be a sensitive, cost-effective and very practical alternative to conventional surveillance in influenza-vaccinated poultry, with direct relevance for future surveillance strategies in HPAI vaccination programmes.