EARLY RELEASE – Persistence of Influenza H5N1 and H1N1 Viruses in Unpasteurized Milk on Milking Unit Surfaces – Volume 30, Issue 8 – August 2024 – Emerging Infectious Diseases Journal

In late March 2024, the highly pathogenic avian influenza A (H5N1) virus was detected in dairy cows in the United States and subsequently spread to herds across multiple states, resulting in at least three confirmed human infections (1Testing of milk from infected dairy cows has shown that unpasteurized milk contains high concentrations of infectious influenza viruses (2LC Caserta et al., unpublished. Date, https://doi.org/10.1101/2024.05.22.595317Exposure of dairy workers to contaminated raw milk during the milking process may increase human H5 virus infections, which may allow H5 viruses to adapt through viral evolution in humans and acquire the ability for human-to-human transmission.

The milking process is largely automated and uses vacuum units, commonly called clusters or claws, which are attached to the cow's teats to collect the milk (Figure 1) (3). However, some steps in the milking process require human intervention, such as forest stripping, in which workers manually extract the first 3-5 bottles of milk from each teat. Forest stripping stimulates the teats for optimal milk production, improves milk quality by removing bacteria, and provides an opportunity to check for abnormal milk. The forest stripping process can result in milk splashing on the milking room floor and surrounding equipment, as well as milk aerosols.

After forestripping, each teat is hand washed and dried before the claw is attached. During milking, a flexible rubber expanding liner housed within the stainless steel shell of the claw opens to allow milk flow and closes to apply pressure to the teat to stop milk flow (Figure 1When the milk flow rate decreases to a certain level, the claws will automatically release (3), at which point any milk remaining in the inflated liner may splash onto dairy workers, equipment, or the surroundings. Of note, milking often occurs at human eye level; humans have a lower physical working space than cows, increasing the likelihood of infectious milk coming into contact with the mucous membranes of human workers. Eye and respiratory protection is not currently mandatory for dairy workers, but recommendations have been published (Four).

Although the persistence of influenza viruses on surfaces in raw milk is unknown, information on virus persistence is important for understanding the risk of virus exposure to dairy workers during the milking process. Therefore, we analyzed the persistence of infectious influenza viruses in raw milk on surfaces such as rubber inflation liners and stainless steel commonly found in milking units (Figure 1).

Infectious strains included influenza A(H5N1) strain A/dairy cattle/TX/8749001/2024 or the surrogate influenza A(H1N1)pdm09 pandemic influenza virus strain A/California/07/2009. Viruses were diluted 1:10 in raw, unpasteurized milk and in phosphate-buffered saline (PBS) as a control. As described in a previous study (Five–7), we pipetted droplets of virus diluted in milk or PBS onto stainless steel or rubber inflation liner coupons inside an environmental chamber. Virus samples were then collected immediately (time 0) or after 1, 3, or 5 h and assayed for 50% tissue culture infectious dose assays (7A persistence study was conducted at 70% relative humidity to mimic the environmental conditions within outdoor milking parlours in the Texas Panhandle region where the virus was detected in dairy herds in March and April 2024.

H5N1 bovine virus remained infectious after 1 h in unpasteurized milk on stainless steel and rubber inflation linings, whereas infectious virus in PBS dropped below the limit of detection after 1 h (Figure 2Panel A). The results indicate that unpasteurized milk containing the H5N1 virus remains infectious on materials in the milking unit. To evaluate whether a less pathogenic influenza virus could be used as an alternative to study virus persistence on milking unit materials, we compared the decay of H5N1 and H1N1 viruses in raw milk over 1 h on rubber inflation liners and stainless steel surfaces (Figure 2Panel B). The two viruses showed similar decay rates on both surfaces, suggesting that H1N1 could be used as a surrogate for the H5N1 bovine virus in studying viral persistence in raw milk. Further experiments investigating H1N1 infectivity over longer periods revealed that the virus persisted in unpasteurized milk on rubber inflation liners for at least 3 hours and on stainless steel for at least 1 hour (Figure 2Panel C). These results indicate that influenza virus is stable in unpasteurized milk and that influenza A virus on milking equipment may remain infectious for more than 3 hours.

Taken together, our data provide compelling evidence that dairy workers are at risk for H5N1 virus infection from contaminated surfaces during the milking process. To reduce shedding of H5N1 virus from dairy cows to humans, farms should enforce the use of personal protective equipment, such as face shields, masks, and eye protection, for workers during milking. Additionally, contaminated rubber inflatable liners may be a source of cow-to-cow transmission observed in dairy farms. Disinfecting liners after each cow milking may help reduce the spread of influenza virus between animals on farms and contain the current outbreak.

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