Detection of antibodies against influenza A viruses in cattle

Abstract

Unexpected outbreaks caused by the H5N1 highly pathogenic avian influenza virus (HPAIV) in dairy cows in the United States (US) have raised significant veterinary and public health concerns. When and how the H5N1 HPAIV was introduced into dairy cows and the broader epidemiology of influenza A virus (IAV) infections in cattle in the US remain unclear. Herein, we performed a retrospective study to screen more than 1,700 cattle serum samples collected from different bovine breeds in the US from January 2023 to May 2024 using an enzyme-linked immunosorbent assay (ELISA) targeting the nucleoprotein (NP) to detect IAV infections, and the positive samples were further tested by hemagglutination inhibition (HI) assay. Results showed that 586 of 1,724 samples (33.99%) from 15 US states were seropositive by the NP ELISA assay, including 78 samples collected in 2024 and 508 samples collected in 2023. Moreover, the HI assay revealed that 45 of these ELISA-positive samples were positive to human seasonal H1N1 and H3N2 and swine H3N2 and H1N2 viruses, and some were positive to two or three tested IAVs. Surprisingly, none of these ELISA-positive samples were HI positive for the circulating bovine H5N1 strain. Our results demonstrate that IAVs other than H5N1 can infect cattle, infections are not limited to dairy cows, and that bovine infections with swine and human IAVs have occurred prior to the H5N1 outbreaks. All results highlight the value in monitoring IAV epidemiology in cattle, as the viruses might adapt to cattle and/or reassort with the currently circulating H5N1 HPAIV, increasing risk to humans.IMPORTANCEInfluenza A virus (IAV) is an important zoonotic pathogen that can infect different species. Although cattle were not historically considered vulnerable to IAV infections, an unexpected outbreak caused by H5N1 highly pathogenic avian influenza virus in dairy cows in the United States (US) in early 2024 has raised significant concerns. When and how the virus was introduced into dairy cows and the wider impact of IAV infections in cattle in the US remain unclear. Our retrospective serological screen provided evidence of human and swine H1 and H3 IAV infections in different cattle breeds in addition to dairy cows, although no H5N1 infection was detected. Our results underline the necessity to monitor IAV epidemiology in cattle, as reassortment of IAVs from different species may occur in cattle, generating novel viruses that pose threats to public and animal health.

Keywords: ELISA antibody; HI antibody; IAV infection; cattle; surveillance.

Fig 1

Numbers of bovine serum samples collected in each month from Jan 2023 to May 2024 and seropositive numbers in each month tested by IAV NP ELISA assay.

Fig 2

Geographic distribution of bovine serum samples tested and positive sample numbers in different states. Colored areas indicate the states where bovine serum samples were collected. Numbers shown in each state represent the numbers of positive samples/total samples in each state. The map was created using OpenStreet maps via Tableau Public.

Fig 3

Prevalence of IAV seropositivity in different breeds of cattle. Red colored lines represent NP ELISA-positive rates for each breed. HI titers were measured for NP ELISA-positive samples. For each sample, the reactivity of sera was assessed against six IAVs, including rgH5N1, seasonal huH1N1 and huH3N2, swH3N2 (Clusters I and IV), and swH1N2v. No samples were HI-positive against the rgH5N1. Open circled blue dots denote seropositive samples against seasonal huH1N1. Open blue squares indicate seropositive samples against seasonal huH3N2. Solid yellow dots denote seropositive samples against Cluster I swH3N2. Open green circles represent seropositive samples against Cluster IV swH3N2. Solid red circles denote seropositive samples against swH1N2v.

Fig 4

Venn diagram of HI-positive bovine serum samples against human and swine IAVs. Forty-five out of 586 ELISA-positive bovine serum samples were HI-positive to tested human and swine IAVs, including seasonal huH1N1 and huH3N2 viruses, cluster I and IV swH3N2 as well as swH1N2v viruses. None had an HI titer against the tested rgH5N1. The sample IDs were presented in the brackets if samples were dual or triple positive against tested IAV strains, and the geomean of the HI titer against each tested virus was presented.

Fig 5

Western blot detection of HI-positive bovine serum samples. Purified NP, HI-positive bovine serum samples (1:50) and an HRP-conjugated mouse anti-bovine IgG (H + L) as the second antibody (1:500) were used for the Western blot assay. A positive serum sample H3N2pc from a calf infected with the TX98 H3N2 at 21 days post-infection and a bovine serum sample negative by HI and ELISA assays served as the positive and negative controls, respectively. The HI titer of each tested positive serum sample was shown.