Low Levels of Neutralizing Antibodies to Influenza A (H5N1) and D Viruses Among Cattle and Cattle Workers on US Farms, 2024–2025

Bovine respiratory disease complex (BRDC) is a major disease problem in cattle production systems. Numerous pathogens have been implicated as causing BRDC, including the recently discovered influenza D virus (IDV) [1]. IDV has been found to be highly enzootic in cattle across multiple continents [1, 2], and there is some evidence of spillover to livestock workers [3, 4]. In March 2024, unprecedented outbreaks of influenza A virus (IAV) H5N1 clade 2.3.4.4b were reported in the United States [5] with occasional spillover to dairy and poultry workers [6, 7]. Given the occupational threats of both IDV and IAV H5N1, we sought to assess the dynamics of antibodies to IAV H5N1 and IDV in farm workers and cattle.

As part of our One Health surveillance initiative in the United States and Mexico [6, 8], between April 2024 and May 2025 we enrolled a total of seven dairy and beef cattle farms in Indiana (n = 1), Kentucky (n = 3), and Texas (n = 2) (Table 1). Our visits to these farms took place on four separate occasions, permitting us to prospectively follow the farms. We obtained informed consent from every farm worker who participated in the study. Based on availability, we collected 5 to 10 mL of blood from both the cattle and the farm workers through venipuncture using a plain vacutainer tube (BD, Franklin Lakes, NJ, USA). After collection, the blood samples were centrifuged at 600 g for 15 min, and the serum was harvested and stored in microtubes at −20°C. Throughout the four visits, we collected 270 serum samples, which comprised 142 samples from cattle and 128 samples from farm workers (Table 1). Ethical approval for the study was granted by the University of Texas Medical Branch's Institutional Review Board (23-0085).

To determine the presence of neutralizing antibodies (NAbs) against IAV-H5 and IDV in both the cattle and farm workers sera, we conducted microneutralization (MN) assays. Briefly, serum samples were treated overnight (18–20 h) with Receptor Destroying Enzyme II (RDE; Denka Seiken, Tokyo, Japan) according to the manufacturer's instructions. This treatment was necessary to eliminate nonspecific inhibitors that could interfere with the assay. Starting with a dilution of 1:20 to 1:2560 RDE-treated sera, we used a recombinant H5N1 virus (rg-A/bald eagle/Florida/W22-134-OP/2022 of clade 2.3.4.4b) and a bovine-origin IDV strain (D/Bovine/Kansas/1-35/2010) on a monolayer of Madin-Darby canine kidney (MDCK; ATCC cat. no. CRL-CCL34) cells in 96-well plates following standard procedures with minor modifications [4, 9].

As measured by the MN assay, none of the enrolled beef cattle farm workers had NAbs antibodies to IAV-H5 during any of the four visits to the farms in Indiana, Kentucky, and Texas, except for the first visit to Texas (Table 1). During this visit, two dairy farm workers were found to have NAbs to IAV-H5 [6]. Furthermore, NAbs to IAV-H5 were not detected in any beef cattle tested during the four field visits in Indiana, Kentucky, and Texas. However, 19 of the 22 (73.3%) Texas dairy cattle on one farm showed elevated NAbs to IAV-H5 (Table 1). Additionally, at least 7 of 15 (46.7%) out of these dairy cattle maintained elevated NAbs levels during subsequent visits for over a year (Shittu et al., manuscript under journal review).

We found NAbs to IDV in one of 128 farm worker serum samples (0.8%). Notably, antibodies to IDV were consistently detected in the serum of this worker at every visit. This indicates that NAbs in individuals infected with IDV can persist for more than a year. To the best of our knowledge, this is the first report documenting the persistence of antibodies to IDV in farm workers. This finding highlights the importance of monitoring immune responses in this population, who are often exposed to various pathogens in their work environment.

Understanding how long antibodies remain detectable can provide valuable insights into the immune system's effectiveness to clear invading pathogens and guide future health strategies for workers in agricultural settings. In the cattle, during the winter of 2024 (third visit), we detected NAbs against IDV exclusively in the beef cattle (Table 1). In Indiana, 2 of 6 beef cows (33.3%) tested positive for IDV NAbs. In Kentucky, 1 of 19 beef cows (5.3%) had IDV NAbs, while in Texas, 10 of 30 beef cows (22.2%) were found to have IDV NAbs (Table 1). Unlike IAV and other respiratory viruses with high incidences during winter [10], little is known about the seasonality of IDV. More extensive research should be conducted in establishing the seasonality of IDV in cattle to aid in control strategies. In conclusion, our study documents a low prevalence of neutralizing antibodies to IAV H5N1 and IDV in both cattle and cattle workers.

Ismaila Shittu: investigation, methodology, validation, writing – original draft, writing – review and editing, data curation, formal analysis, conceptualization, visualization. Daniel B. Cummings: investigation, methodology, writing – review and editing. John T. Groves: investigation, methodology, writing – review and editing. Alex G. Hagan: investigation, methodology, writing – review and editing. Gregory C. Gray: writing – original draft, writing – review and editing, conceptualization, funding acquisition, project administration, resources, supervision, methodology, investigation.

The authors declare no conflicts of interest.