Sampling on 14 California dairy farms positive for H5N1 avian influenza uncovers infectious virus in the air of milking parlors and wastewater, viral RNA in cows’ exhaled breath and milk, and antibodies in the milk of seemingly healthy cows, per a study published today in PLOS Biology.

A team led by Emory University researchers performed air, wastewater, and milk sampling on H5N1-positive dairy farms in two regions of California in 2024 and 2025. The findings, they said, highlight potential alternative sources and viral transmission modes on farms, as well as the presence of viral strains in air and wastewater that may be able to infect people.

“Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b B3.13 virus was first detected in dairy cattle in March of 2024 and has since spread to 16 states,” they wrote. “Transmission routes of highly pathogenic H5N1 between cows or to humans remain unclear due to limited data from affected dairy farms.”

Role of submicron and larger aerosols 

In the first phase of the study on five farms, H5N1 viral RNA was found in milk tanks on farms before cows showed signs of illness. Of 71 air samples analyzed for H5N1 viral RNA, four from the milking parlor and two from cows’ exhaled breath tested positive.

Dairy parlors, which are often enclosed spaces and where aerosolization of milk occurs, pose the greatest threat from inhalation of the virus to dairy farm workers. 

Additional surveillance performed on nine farms showed H5N1 viral RNA in 21 of 35 air samples. Particle-size analysis showed the particles generated in milking-parlor air were both submicron and larger aerosols; four samples contained infectious virus. 

H5N1 viral RNA was detected throughout the wastewater stream, including in manure lagoons used by migratory birds and in fields with grazing cows. Two samples subjected to viral titration contained detectable infectious virus.

“Together, these results highlight the extensive environmental contamination of H5N1 on affected dairy farms and identify additional sources of viral exposure for cows, peridomestic wildlife, and humans,” the authors wrote. “Dairy parlors, which are often enclosed spaces and where aerosolization of milk occurs, pose the greatest threat from inhalation of the virus to dairy farm workers.” 

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