Comparison of serological assays to monitor exposure to highly pathogenic avian influenza H5N1 virus

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 virus in the United States in 2022, it has caused the deaths of millions of wild and domestic birds. In early 2024, HPAI H5N1 spilled over into dairy cattle in Texas and has since been detected in multiple states, raising concerns about cross-species transmission and zoonotic risk. We assessed the feasibility of serological assays for antibody responses to HPAI H5N1 in dairy cattle by comparing two competitive commercial ELISA assays with a virus neutralization (VN) assay. Samples, including serum and milk matrices, were collected during the acute phase of confirmed outbreaks and 60 days post-outbreak. Results were analyzed for sensitivity, specificity, concordance, and agreement using Kappa statistics and receiver operating characteristic (ROC) analysis. Comparisons to VN revealed substantial agreement for the IDvet ELISA in both serum and milk, while the IDEXX ELISA showed high specificity but lower sensitivity, particularly for low-titer samples. Rennet treatment of milk, required for the VN test, did not affect IDvet performance but reduced agreement for IDEXX. Seroprevalence estimates based on VN and IDvet were similar, indicating consistent detection of exposed animals. In contrast, IDEXX underestimated the number of positives, which may limit surveillance accuracy. These findings emphasize the importance of assay validation in non-conventional species and highlight VN and high-performing ELISAs, as valuable tools for post-outbreak surveillance in cattle. Given the zoonotic potential of H5N1, reliable serological testing is essential for monitoring livestock exposure and assessing risks to human health.