Highly pathogenic avian influenza virus of the H5N1 subtype has been infecting U.S. dairy cattle and spreading among dairy operations since March 2024. H5N1 surveillance systems for dairies are needed, but information on whether environmental sampling can inform these systems is lacking. To guide a surveillance framework, we determined the environmental sites with Influenza A virus (IAV) detection on H5N1-affected dairies (n = 25) in four states (California, Colorado, Michigan, and Ohio) and explored sample characteristics that may have influenced detection. A total of 581 samples from dairy environmental sites were characterized for IAV RNA via rRT-PCR, and classified into six categories. A total of 94 samples (16.2%) had IAV detected, and the Ct values measured from these samples were typically higher than those measured in bulk tank milk from a subset of sampled herds. A majority of IAV detections were made from the following site categories: milking equipment/personal protective equipment, parlor surfaces, and wastewater/lagoons/manure. These results suggest that environmental sites most likely to be contaminated with IAV on dairies are those with exposure to milk. Meanwhile, mixed effect logistic modeling showed that days into an outbreak that samples were collected was associated with IAV detection. These results provide a framework within which to continue the assessment of environmental sampling as a surveillance tool for dairy H5N1. This framework can be strengthened by studies that perform further IAV viral characterization and collect samples from sites prior to, during, and after H5N1 outbreak periods.

Highly pathogenic avian influenza virus H5N1 has been infecting dairy herds in the US since its initial incursion into cows in early 2024. Although national strategies have aimed to detect affected herds during this outbreak, information regarding longitudinal herd-level viral detection has not been formally studied. To help understand viral shedding of H5N1 in milk from H5N1-affected dairy herds over time, an outbreak investigation was conducted on dairy farms in California in the Fall and Winter of 2024. The primary objective of this observational investigation was to identify initial H5N1 infection in these CA herds and then evaluate viral distribution via longitudinal bulk tank milk (BTM) testing. A secondary objective was to evaluate viral movement via longitudinal pen-level milk (PLM) sampling in a subset of affected herds. Daily BTM samples were submitted from 19 originally non-infected herds that were clients of a single veterinary clinic and volunteered to participate in this study. Samples were tested for influenza A (IAV) via rRT-PCR. In a subset of 5 herds, daily PLM IAV testing was completed soon after initial BTM IAV detection. Overall, a total of 4,749 BTM and 2,922 PLM samples were submitted for testing, and all herds eventually became infected with IAV. After initial detection, daily detections of IAV occurred in BTM for a minimum of 33 d, with some herds continuing to have detection at the end of the sampling period (>75 d). BTM Ct nadirs were seen 1-3 weeks after initial detection. In PLM-tested herds, virus was detected in samples from all pens of cattle soon after BTM detection, and in some cases was seen in all pens on the first day of PLM sampling. Overall, these results support BTM testing as an effective early detection tool for dairy H5N1 outbreaks but suggest that surveillance strategies that do not test aggregate milk from all lactating cows on an operation, or have a long interval between BTM testing days, may fail to identify all affected herds. Future investigations should study farm-level factors associated with long-term viral detection in some herds and assess detection sensitivities of the IAV rRT-PCR assay used on aggregate milk samples.