Introduction: Highly pathogenic avian influenza virus (HPAIV), a potential pandemic pathogen (PPP), was discovered in a new species, cattle, in March 2024 in the United States. Soon thereafter it was discovered that infectious virus could be present in milk at very high titers (up to 10 log10 50% egg infectious doses per mL). Therefore, "emergency" testing to confirm that current milk processing methods used to reduce bacterial pathogens were sufficient to inactivate HPAIV was needed. Continuous flow pasteurization (CFP) is the industry standard, and the conditions defined in the Pasteurized Milk Ordinance of the U.S. Food and Drug Administration needed to be validated with HPAIV. To accomplish this, biosafety procedures for an experimental design that required a pilot-scale CFP with high volumes of HPAIV-contaminated milk needed to be developed.

Methods: Numerous information sources, including subject matter experts, were utilized to acquire information for risk assessments and to develop procedures for a new, large-scale piece of equipment that required 5L of a PPP at a high titer per test.

Results: Application-specific equipment such as sample and waste vessels with sealed ports, tubing with valved connection, and high efficiency particulate air-filtered vents were designed with corresponding safety procedures, such as seal integrity testing of the sample vessels.

Conclusions: The use of high volumes of infectious material, the scale of the equipment, and time constraints presented unique challenges to balance biosafety and research objectives. Innovative engineering and procedural approaches allowed the research to be completed successfully in a time-sensitive manner while mitigating biosafety risk.