Evaluation of serologic assays for detection and quantification of influenza antibodies in milk samples from H5N1 highly pathogenic avian influenza virus infected dairy cows

Authors: IBRAHIM, SHERIF - Orise Fellow; Lee, Youngsub; POSEY, ERIN - Animal And Plant Health Inspection Service (APHIS); TORCHETTI, MIA - Animal And Plant Health Inspection Service (APHIS); Olivier, Timothy; Spackman, Erica; Suarez, David; Lee, Chang

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2026
Publication Date: 7/28/2026
Citation: Ibrahim, S., Lee, Y., Posey, E., Torchetti, M.K., Olivier, T.L., Spackman, E., Suarez, D.L., Lee, C.W. 2026. Evaluation of serologic assays for detection and quantification of influenza antibodies in milk samples from H5N1 highly pathogenic avian influenza virus infected dairy cows. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2026.1902594.
DOI: https://doi.org/10.3389/fvets.2026.1902594

Interpretive Summary: H5N1 highly pathogenic avian influenza (HPAI) virus has been circulating in US dairy cattle with significant implications for milk production and cow health. Thus, effective surveillance and containment efforts are essential to prevent and control outbreaks, prevent potential zoonotic infections in humans, and maintain sustainable dairy operations. Reliable diagnostic and surveillance tools are crucial for effective HPAI control. In this study, we evaluated new test (NI-ELLA) for detection and quantification of antibody in individual milk samples from H5N1 virus infected cows as well as aggregate bulk tank milk samples and compared its performance with other existing tests. Our study showed that NI-ELLA is an effective tool for supporting H5N1 HPAI infection monitoring in dairy cattle and the evaluation of their antibody response in milk samples in relation to infection stages and protective immunity.

Technical Abstract: Since the confirmation of H5N1 HPAI virus in US dairy cattle on March 25, 2024, the bovine HPAI outbreak continues and more than 1,000 farms with confirmed herds in 19 states reported as of February 24, 2026. To ensure the ongoing monitoring of HPAI spread additional serologic assays were evaluated to complement the existing commercial ELISA and molecular tools which underpin the epidemiological approaches being employed. We have previously established N1 antibody- specific neuraminidase inhibition (NI)-enzyme linked lectin assay (ELLA) for DIVA (differentiation of infected from vaccinated animals) surveillance for the detection of H5N1 infection in vaccinated poultry. In this study, we applied NI-ELLA for the detection and quantification of N1 antibody in individual (n = 150) and bulk tank (n = 104) bovine milk samples. We demonstrated that both untreated samples and infranatant collected by centrifugation followed by heat inactivation can be used for different serologic assays depending on the sample quality. There was a strong correlation in antibody detection between NI-ELLA and commercial ELISAs. Endpoint titer determined by NI-ELLA also correlated well with virus neutralization titer, which is used to evaluate protective antibody levels in milk samples. Detection and quantification of N1 antibodies in bulk tank milk samples further showed a higher detection rates and stronger antibody titers among virus isolation-negative samples (regardless of presence or absence of viral RNA detected by PCR) compared to virus isolation-positive samples. The higher level of neutralizing antibody likely contributed to the lack of detection of infectious virus depending on the stages of viral infection when milk was collected. Future studies are warranted to describe the antibody duration and response dynamics in milk samples over extended periods of time across individual cows, and NI-ELLA will serve as a valuable quantification tool for evaluating antibody response in those studies.