Influenza A Virus detection in Bulk Tank and Pen Level Milk from Dairies Affected by Highly Pathogenic Avian Influenza H5N1

ABSTRACT Highly pathogenic avian influenza virus H5N1 has been infecting dairy herds in the U.S. since its initial incursion into cows in early 2024. Although national strategies have aimed to detect affected herds, the best way to surveil herds for the H5N1 virus has not been formally studied and we also do not understand herd-level patterns of infection. To understand infection patterns of H5N1 in dairy herds over time, we conducted early surveillance of non-affected farms in California in the Fall of 2024 in an observational study. Daily bulk tank milk (BTM) samples were submitted from each herd and tested for influenza A (IAV) via rRt-PCR. In a subset of herds, IAV testing of multiple excretion types from cattle of different classes and pen-level daily milk was also completed soon after BTM detection. Daily detections of IAV occurred in BTM for a minimum of 33 days, with some herds continuing to have detection beyond a 75-day window. BTM Ct nadirs were seen between 1-3 weeks of detection. In herds that were tested, virus was detected in the milk from all pens of cattle within a very short time frame after BTM detection, or prior to the initiation of pen level sampling. A very low percentage (2.8%) of individual cow samples tested positive for IAV when collected soon after BTM detection, and although the virus was found in all excretion types, a majority of positive samples were from milk. This suggests that BTM may be the best early indicator of herd infection, and that movement of the virus to all lactating pens of cattle after herd incursion is relatively quick. These results also suggest that surveillance strategies with a long interval between BTM testing days may miss herds with short infection windows. Because most herds experienced test days where some submitted BTM samples had virus detected while others did not, and virus was detected in pen level milk samples when the BTM from the herd had become test negative, this work also highlights the necessity of studying the test sensitivity of IAV rRt-PCR detection in aggregate milk samples. Interpretive summary This study characterized daily bulk tank and pen-level milk detections of H5N1 virus on affected dairies. Viral loads in bulk tank samples peaked within three weeks and declined by two months, though some herds showed prolonged detection. Individual cow testing revealed that bulk tank milk testing is more sensitive for H5N1 early outbreak detection, and also identified the likely presence of non-clinically affected cows shedding virus. Pen-level sampling revealed rapid herd-level spread, emphasizing the need for frequent and comprehensive milk sampling to improve detection sensitivity and support effective outbreak management for H5N1 on dairy operations. Competing Interest Statement The authors have declared no competing interest. Funding Statement Data used in this publication was made possible, in part, by an Agreement from the United States Department of Agriculture APHIS VS (APHIS VS, Cooperative Agreement 25-9419-0731) and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016. This publication may not necessarily express the views of APHIS VS. Support was provided by the California Department of Food and Agriculture (CDFA, Sacramento, CA). Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the CDFA. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes The funding statement has been changed Data Availability All data produced in the present work are contained in the manuscript or available online at the following DOI: 10.17605/OSF.IO/TFJ87 Abbreviations - CS - clinical signs of H5N1 disease in cows, - H5N1 - highly pathogenic avian influenza virus of the H5N1 subtype, - IAV - influenza A virus, - BTM - bulk tank milk, - PLM - pen level milk