Methods for detecting highly pathogenic avian influenza H5N1 virus in dairy processing environments

The spread of highly pathogenic avian influenza (HPAI) H5N1 into mammals, including U.S. dairy cattle, underscores the need for effective surveillance in milk and environmental samples. This study evaluated molecular methods to detect influenza A virus (IAV) in various milk types and dairy farm surfaces. An aluminum chloride (AlCl₃) precipitation method was optimized for virus concentration in milk, combined with magnetic bead-based nucleic acid extraction and Plant RNA Isolation Aid. Mean recoveries of inactivated HPAI H5N1 were 22.46 ± 11.86% and 17.22 ± 6.15% in pasteurized and raw milk, respectively. Detection limits (LoD_95%) for H3N2 were 2.9 × 10⁵ genome copies (gc)/L (raw) and 8.13 × 10⁴ gc/L (pasteurized); for H5N1, 1.20 × 10⁴ gc/L (raw) and 3.10 × 10⁵ gc/L (pasteurized). Capsid integrity RT-qPCR using propidium monoazide (PMAxx™), platinum (IV) chloride (PtCl₄), and Crosslinker (CL), showed PtCl₄ best eliminated signals from heat-inactivated virus, but only after extreme heat (99 °C, 5 min). For surfaces, ISO 15216:1-based swabbing with PBS outperformed sponge-based methods, especially on stainless steel and silicone. Virus recovery varied by surface and matrix. These results support standardized sampling and concentration protocols for HPAI H5N1 monitoring in dairy environments.