Infectious H5N1 virus detected in dairy farm air and wastewater, alongside evidence of hidden cattle infections, suggests an outbreak may be spreading through more routes than previously recognized.
Study: Surveillance on California dairy farms reveals multiple possible sources of H5N1 influenza virus transmission. Image credit: Parilov/Shutterstock.com
In a recent study published in PLOS Biology, researchers conducted a comprehensive surveillance study of California dairy farms to assess the current status of Highly Pathogenic Avian Influenza (HPAI) H5N1 in the State. The study tested multiple transmission modalities (e.g., air, wastewater, and cattle breath) across 14 H5N1-positive dairy farms and uncovered previously overlooked routes of viral transmission.
California dairy outbreak raises new transmission concerns
When Highly Pathogenic Avian Influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) was first detected in United States (US) dairy cattle in March 2024, it subsequently spread to 16 states. By September 2025, California, the nation’s leading dairy producer, had reported 771 herds testing positive for the virus, making it one of the most affected states.
The initial consensus among experts was that the virus spread primarily through direct contact with unpasteurized milk, a hypothesis supported by observations of high viral load in tested milk samples. However, subsequent reports of dairy workers contracting the virus after indirect contact and the detection of viral RNA on surfaces suggested that the environment itself might be contaminated, challenging the earlier ideas.
Furthermore, it remained unclear whether the H5N1 virus could maintain infectivity when aerosolized during milking procedures or whether reclaimed water used for flushing pens and irrigation served as a viable environmental reservoir.
Researchers track H5N1 across 14 dairy farms
The present study aimed to address these knowledge gaps by conducting an extensive environmental and biological sampling program on 14 H5N1-positive farms in California. The study’s air sample data were obtained through three distinct air sampling technologies: (1) open-face polytetrafluoroethylene (PTFE) filter cassettes for personal exposure modeling at a flow rate of 5 L/min; (2) the handheld MD8 Airport sampler using gelatin filters at 50 L/min; and (3) the stationary AirPrep Cub 210 at 200 L/min.
Simultaneously, environmental water samples were collected from four points along the reclaimed water stream: milk line cleanouts, sump pumps, manure lagoons, and irrigation fields. Air and water samples thus obtained were genetically classified and quantified using real-time quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (ddPCR).
Furthermore, the study assessed intra-animal viral distribution through a 7-day longitudinal study involving 14 cows. Milk was collected from individual udder quarters separately. Finally, whole-genome sequencing (WGS) analyses were used to identify and flag emerging viral variants in air and water.
Infectious H5N1 detected in milking-parlor air
The study’s surveillance efforts successfully detected H5N1 viral RNA in 21 out of 35 air samples analyzed. Alarmingly, air assay results revealed that in milking parlors, concentrations (Cair) reached 104 genome copies per liter (gc/L), and four of the positive air samples contained infectious virus. In exhaled breath samples from rows of cows, the study detected low viral concentrations ranging from 4 to 41 gc/L.
Wastewater analysis findings were also alarming, with all sampled sites (including manure lagoons) containing detectable viral contamination. Samples with viral concentrations exceeding 650 gc/mL were selected for infectivity testing, and two were found to contain infectious virus.
Genomic sequencing analyses identified an air sample from farm “EC” carrying an N189D mutation (H3 numbering 193) in the hemagglutinin (HA) protein, a site previously associated with changes in receptor-binding specificity and improved binding to human-like 2,6-linked sialic acids in prior studies. However, the authors emphasized that whether this specific mutation increases human tropism or zoonotic risk remains unknown.
Furthermore, the study’s separate antibody analysis on Farm FB revealed that 6 out of 10 animals that appeared perfectly healthy had developed H5-specific antibodies in their milk (evidence of prior viral exposure and immune response) without depicting mastitis (inflammation of the mammary gland) or a significant drop in milk production, traits typically used in infection diagnosis.
Finally, the pattern of infection in cow udders was surprisingly heterogeneous rather than confined to a consistent quarter pattern; instead of just one quarter being infected, as is typical in bacterial mastitis, cows often had multiple infected quarters, which remained virus-positive over the seven-day test period. This observation suggests that shared milking equipment may not be the sole mechanism by which the virus is moving from cow to cow.
Multiple transmission routes complicate outbreak containment
This study’s identification of infectious aerosols in milking parlors and persistent viral loads in wastewater lagoons indicates these locations as critical sites for biosafety interventions. Its discovery of the N189D mutation further highlights the possibility that the virus could acquire adaptations relevant to mammalian or human receptor binding, although the functional significance of this mutation remains uncertain.
The authors recommend that future efforts focus on rapid, cow-level diagnostics and on implementing respiratory PPE for workers in enclosed parlor spaces to mitigate inhalation risks.
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