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Methods for detecting highly pathogenic avian influenza H5N1 virus in dairy processing environments.
Pablo Puchades-Colera1, Inés Girón-Guzmán1, Alba Pérez-Cataluña1
1VISAFELab, Department of Preservation and Food Safety Technologies, Institute of Agrochemistry and Food Technology, IATA-CSIC, Paterna, Valencia, Spain.
Detecting avian influenza in milk and on dairy farms is crucial. This study optimized molecular methods for virus detection in milk and on surfaces, improving surveillance strategies for highly pathogenic avian influenza H5N1.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Food Safety
Background:
- Highly pathogenic avian influenza (HPAI) H5N1 poses a significant threat to dairy cattle and public health.
- Effective surveillance in milk and environmental samples is essential for early detection and control.
- Existing molecular detection methods require optimization for diverse dairy matrices.
Purpose of the Study:
- To evaluate and optimize molecular detection methods for influenza A virus (IAV) in various milk types and on dairy farm surfaces.
- To assess virus recovery rates and detection limits for HPAI H5N1 and other influenza strains.
- To identify the most effective methods for sample concentration and nucleic acid extraction.
Main Methods:
- Optimization of an aluminum chloride (AlCl3) precipitation method for virus concentration in milk.
- Utilized magnetic bead-based nucleic acid extraction combined with Plant RNA Isolation Aid.
- Evaluated capsid integrity real-time quantitative PCR (RT-qPCR) using propidium monoazide (PMAxx™) and various chemical crosslinkers (PtCl4, CL).
- Assessed swabbing methods (ISO 15216:1) versus sponge-based methods for surface sampling.
Main Results:
- Mean recoveries of inactivated HPAI H5N1 were 22.46% in pasteurized and 17.22% in raw milk.
- Established detection limits (LoD95%) for H3N2 and H5N1 in different milk types.
- Platinum (IV) chloride (PtCl4) demonstrated the best elimination of signals from heat-inactivated virus after extreme heat treatment.
- ISO 15216:1-based swabbing with PBS was superior for surface sampling, particularly on stainless steel and silicone.
Conclusions:
- Developed and validated optimized molecular methods for HPAI H5N1 detection in milk and on dairy farm surfaces.
- The optimized methods provide a foundation for standardized surveillance protocols.
- Improved detection capabilities are critical for managing HPAI outbreaks in the dairy industry.
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