Sustained High Levels of Antibodies to Avian Influenza Virus H5N1 Clade 2.3.4.4b in Naturally Infected Cattle

Sustained High Levels of Antibodies to Avian Influenza Virus H5N1 Clade 2.3.4.4b in Naturally Infected Cattle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sustained High Levels of Antibodies to Avian Influenza Virus H5N1 Clade 2.3.4.4b in Naturally Infected Cattle Ismaila Shittu, Jessica Rodriguez, Judith U. Oguzie, Claudia M. Trujillo-Vargas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7274486/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Cattle were not previously thought susceptible to highly pathogenic avian influenza (HPAI) H5N1 virus infection until March 2024, when outbreaks were reported among dairy cattle. Data are sparse regarding the immune response of cattle to infections with such viruses. To better understand the systemic humoral immune responses of HPAI H5N1-infected dairy cattle, we conducted a prospective study of 15 cows on an H5N1-affected farm. The cows’ sera were studied using microneutralization (MN), hemagglutination inhibition, and enzyme-linked immunosorbent assays. At four months post-outbreak, MN assays revealed 73% (11/15) of the cattle sera had elevated H5 antibody levels that ranged from 1:40 to 1:640. Eight months post-outbreak, 73% retained elevated MN antibodies, wherein 18% (2/11) had an increased response, 63.6% (7/11) maintained antibody levels, and 18% (2/11) showed a decline. By 13 months post-outbreak, only 50% (5/10) retained elevated titers by the MN assay. The results from all three assays were in agreement; however, the MN assay seemed to be more sensitive. Our findings indicate a correlation among the three assays, with high levels of H5 antibodies persisting for more than a year after natural infections. This information is vital for guiding future use of H5 vaccines in dairy cattle farms. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology avian influenza dairy cattle immune response farm Texas Figures Figure 1 Introduction Until recently, cattle were thought to be somewhat resistant to infection with influenza A virus 1 . Evidence now shows infection with highly pathogenic avian influenza virus (HPAIV) H5N1 is often manifested by an increase in specific antibody titers, a significant drop in milk production, reduction in feed intake, and shedding of H5N1 virus in nasal secretions and milk, and lesions in mammary gland 2 – 6 . In March 2024, outbreaks of HPAIV H5N1 clade 2.3.4.4b genotype B3.13 were reported in dairy cattle on a North Texas farm, following a spillover from poultry that was preceded by reassortment of HPAIV H5N1 2.3.4.4b North American strain with low pathogenic avian influenza virus North American strain in wild birds 7 . Subsequently, several dairy herds were affected and the virus spilled into humans, particularly farm workers 8 , 9 . As of July 2025, more than a thousand confirmed dairy farms have suffered HPAIV infections in 17 US states 10 . Immune responses to HPAIV H5N1 in poultry have been well-studied 11 – 14 . However, limited information is available regarding the systemic, immunoglobulin G (IgG)-mediated immune responses of cattle to infections with the virus, apart from evidence of seroconversion that were uncovered in studies of experimentally infected cattle 2 , 5 , 6 . In this study, we sought to assess the humoral IgG-mediated immune response and duration of antibodies produced in cattle naturally infected with an HPAIV H5N1 clade 2.3.4.4b strain using three different serological assays: microneutralization (MN), hemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISA). Understanding host immune responses is vital for designing an effective vaccination strategy. We hypothesized that cattle infected with the HPAIV H5N1 strain may not elicit a strong and lasting antibody response. Results To better understand the duration of the immune response in cattle exposed to HPAI H5N1 virus, we monitored their specific antibodies over a period of 13 months using three different assays: MN, HI, and ELISA (Table 1 ). The three assays successfully detected H5 antibodies in the infected cows, and the results were largely comparable across all methods (Table 2 ). Microneutralization assay Four months post-outbreak, 73% (11/15) of the cattle had detectable neutralizing antibodies (NAb) to H5N1 virus, with levels ranging from 1:80 to 1:640 (Table 1 ). Serum samples collected eight months post-outbreak showed that 73% (11/15) of the cattle had varying degrees of NAb. Among these cattle, 18% (2/11, samples ID USL086_TX and USL156_TX) exhibited an increase in NAb, 63.6% (7/11) maintained stable levels, while the remaining 18% (2/11, samples ID USL153_TX and USL157_TX) showed a decline in neutralization (Table 1 ). Four of the samples that were negative by MN were also negative by HI (Table 1 ). Table 1 A comparison or serologic response as measured by three different assays among cattle that were naturally exposed to highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b beginning in the spring 2024 and followed for 13 months. Dairy Cow ID Number Four months post H5N1 outbreak on the farm Eight months post H5N1 outbreak on the farm Thirteen months post H5N1 outbreak on the farm MN HI ELISA MN HI ELISA MN HI ELISA USL086_TX 160 40 positive 320 40 positive 40 20 positive USL090_TX 320 160 positive 320 80 positive 40 < 20 positive USL149_TX < 20 < 20 doubtful < 20 < 20 doubtful NA NA NA USL150_TX < 20 < 20 doubtful < 20 < 20 doubtful < 20 < 20 doubtful USL151_TX 160 160 positive 160 40 positive NA NA NA USL152_TX 320 80 positive 320 40 positive NA NA NA USL153_TX 640 160 positive 160 40 positive 80 40 positive USL154_TX < 20 < 20 doubtful < 20 < 20 negative < 20 < 20 negative USL155_TX 160 80 positive 160 NT positive 20 NT positive USL156_TX 320 160 positive 640 160 positive NA NA NA USL157_TX 640 160 positive 320 160 positive 40 < 20 positive USL158_TX 320 80 positive 320 80 positive 20 < 20 positive USL159_TX 80 40 positive 80 40 positive NA NA NA USL160_TX 640 160 positive 640 160 positive 40 20 positive USL161_TX < 20 < 20 doubtful < 20 < 20 negative < 20 < 20 negative MN = microneutralization; HI = hemagglutination inhibition; ELISA = enzyme-linked immunosorbent assay; NA = not available; NT = not tested; doubtful = as defined by the package insert (or borderline) Hemagglutination-inhibition test The HI test was largely in agreement with the MN and ELISA tests (Table 2 ). The HI assay suggested that 73% (11/15) of the cows has elevated HI results at both four- and eight-months after the outbreak (Table 1 ). However, by the thirteenth month, the remaining four samples did not have detectable antibody levels (less than 1:20) and were declared negative. ELISA By four- and eight-months post-outbreak, 73% (11/15) of the infected cows had elevated antibodies against H5. The remaining four samples collected at four months were classified as doubtful (40% < SN < 50%) according to the assay interpretation proposed by the manufacturer (Table 1 ). Also, at eight and thirteen months, two and one samples were identified as doubtful, respectively (Table 1 ). Assay Agreement The results indicate perfect agreement (κ = 1.0) between the MN, HI, and ELISA assays at 4- and 8-months post-infection (Fig. 1 ). However, by month 13, the agreement notably decreased notably, with ELISA versus HI and HI versus MN showing low to slight agreement (κ = 0.12 and κ = 0.18, respectively). The ELISA versus MN agreement remained moderate at 13 months (κ = 0.60). Considering all the time points combined, the highest overall concordance was observed, once again, between the ELISA and MN assays (κ = 0.88, 95% CI 0.72-1.0), followed by HI and MN (κ = 0.78, 95% CI 0.57–0.98), and ELISA and HI (κ = 0.72, 95% CI 0.50–0.94) (Table 2 ). These results suggest that the ELISA and MN assays maintain better long-term agreement compared to the HI assay in measuring IgG-mediated antibody response following infection over time. Table 2 Pairwise Kappa analysis for dichotomised results of three different serological assays for cattle that were naturally exposed to the highly pathogenic avian influenza H5N1 over a one year period. Test Comparison 4 Months (κ {95% CI} 8 Months (κ {95% CI}) 13 Months (κ {95% CI}) Overall (κ {95% CI}) MN vs HI 1.0 (1.0–1.0) 1.0 (1.0–1.0) 0.18 (-0.16, 0.18) 0.78 (0.57, 0.98) MN vs ELISA 1.0 (1.0–1.0) 1.0 (1.0–1.0) 0.6 (0.14, 0.6) 0.88 (0.72, 1.0) HI vs ELISA 1.0 (1.0–1.0) 1.0 (1.0–1.0) 0.12 (-0.12, 0.12) 0.72 (0.5, 0.94) MN and HI titers, originally continuous, were dichotomized using predefined assay-specific cutoff values to allow for categorical comparison. ELISA data were provided as dichotomous outcomes (positive/negative). Kappa values reflect agreement beyond chance between each pair of assays at each timepoint. MN = microneutralization; HI = hemagglutination inhibition; ELISA = enzyme-linked immunosorbent assay Titer Comparison The log₂-transformed mean antibody titers measured by MN assays and HI and at 4-, 8-, and 13-months post-infection were visually compared (Fig. 1 ). Across all timepoints, MN titers remained consistently higher than HI titers. At 4 months, the mean MN titer was significantly elevated compared to HI, suggesting stronger early detection by the MN assay. This trend persisted at 8 months and was still apparent at 13 months, although the overall titers declined substantially for both assays. Spearman correlation (ρ) between the two assays over the entire time period showed a strong, statistically significant, positive correlation across all time points (ρ = 0.931, p = 2.5 × 10⁻¹⁷), further indicating consistent agreement between both assays. While the correlation was strong at 4-months (ρ = 0.883), it was highest at the 8-month timepoint (ρ = 0.923), and moderately lower at 13-months (ρ = 0.721). The progressive decline in titers over time suggests waning humoral immunity, with MN assay demonstrating greater persistence in detecting antibodies relative to HI assay. Discussion Over the years, the HPAIV H5N1 has evolved into several clades and genotypes, with clade 2.3.4.4b being recently the most prevalent globally. In the United States, genotypes B3.13 and D1.1, both belonging to clade 2.3.4.4b, have been detected in cattle 15 . The outbreak of the H5N1 clade 2.3.4.4b, genotype B3.13 in Texas in 2024 underscored the ability of the H5 A/Goose/Guangdong/1/1996 virus to cross species barriers 3 , 16 . Current efforts are focused on addressing the threats posed by this virus by developing and evaluating vaccines to control the disease in animals. It is essential to understand the antibody responses of livestock to the H5N1 virus, particularly long-lived livestock like cows, such that vaccine strategies can be designed to reduce H5N1 transmission. In our study, we found varying levels of H5N1 antibodies in 73% of the cattle monitored on a farm affected by the H5N1 epizootic. Additionally, we observed a decline in antibody levels to H5 over time among the cattle that seroconverted. However, some of the cows maintained detectable antibody titers for more than a year. To our knowledge, these data represent the first reported effort to monitor antibody decay in dairy cattle that were naturally exposed to H5N1 infection. A previous study with H5 isolates indicated that infected cattle seroconverted, with antibodies detected for up to 90 days 2 . The H5N1 B3.13 strain was found to circulate in cows, and evidence has shown that humans infected with the virus, especially farm workers infected with the virus, also produce NAb 9 . Our findings align with previous studies on antibody persistence following influenza A virus infection in humans 17 , 18 . We found a significant agreement in the Kappa analysis with the antibodies to H5N1, as detected by MN, HI, and ELISA assays, for up to eight months following the outbreak (Table 2 ). Additionally, there was a correlation between the mean antibody titers for HI and MN, with only 1to 2 log 2 differences observed (Fig. 1 ). This finding aligns with previous studies, which indicate that an HI titer of 1:40 is equivalent to an MN titer of 1:160 19,20 . However, by the thirteenth month, a significant reduction in detectable antibodies was observed with the ELISA and HI assays. The HI assay is easy to set up, inexpensive and regarded as the gold standard, but it has been shown to be less sensitive 21 , 22 . The MN assay has been shown to be more sensitive and specific at determining virus NAb 21 , 22 . On the other hand, HI tests are effective at detecting antibodies that prevent viral hemagglutination. It is important to note that while some antibodies can inhibit hemagglutination, they do not always neutralize the virus. Conversely, NAb may not always have the ability to inhibit hemagglutination induced by the virus 23 . We found approximately 27% (4/15) of the cattle showed no detectable antibodies during the monitoring period. This may be due to the innate immune mediators of the cows, which probably limit the replication of the virus at the point of entry, leading to an abortive infection. The innate immune mediators are the first line of defense encountered by influenza virus, which leads to the secretions of signaling proteins, such as interferons, chemokines, cytokines and eicosanoids 24 . Another possibility could be that an infection occurred, but the antibody response was below the detection level of the assays used in the monitoring process. Notably, a few ELISA tests yielded doubtful outcomes, particularly in cases where both MN and HI assays reported some samples as negative. It is important to note that the ELISA results provided a qualitative measurement, unlike the quantitative measures provided by the MN and HI assays. The initial dilution for the ELISA tests was set at 1:10. Given this dilution factor, any results falling below the detection threshold of 1:40 would be interpreted as negative in both the MN and HI assays. Hence, these borderline “doubtful” results are considered negative. Our study is limited by the small number of animals that were enrolled, which restricts the generalizability of our findings. Furthermore, we operated under the assumption that each cow was infected only once with the H5N1 virus. This assumption may not reflect the reality, as the immune response could vary significantly if the cattle experienced multiple exposures to the virus over time. Also, there was a decrease in the number of animals available during the follow-up period, especially at the final stage of the monitoring. In conclusion, our study revealed that antibodies to H5N1 were present in 50% of the infected cattle over a year, as measured by the MN assay. This finding is significant as it suggests that a considerable portion of the cattle developed a relatively strong IgG-mediated immune response to the virus. To ensure long-lasting immunity in cattle, it may be beneficial to administer vaccinations at least every eight months. Materials Ethical Statement Our laboratory work involving avian influenza viruses was reviewed and approved by UTMB Institutional Biosafety Committee. UTMB’s Institutional Animal Care and Use Committee (IACUC) evaluated this work as exempt from formal IACUC ethical review. All procedures, including animal welfare and ethics, and laboratory biosafety procedures were conducted in accordance with the US, ARRIVE, and UTMB relevant guidelines and regulations. Study site and sampling With the permission of the farm management, we sampled and monitored a single dairy farm in Northern Texas for more than a year. The farm spans approximately 100 acres and is home to more than 8,000 cattle of varying ages comprising both dairy and beef cattle. It features a computerized system that monitors different health parameters, such as rumination and milk production within the Holstein dairy herd. In March 2024, we confirmed an outbreak of HPAIV H5N1 on dairy cattle on the farm, as previously reported 8 . At the time of the HPAIV incursion, serum samples were not available but these cows’ manifested signs of acute illness consistent with infection by the virus, such as reduced milk production, fever, respiratory signs, including nasal discharge. From July 2024 to April 2025, we monitored 15 cows on the dairy farm, as this was the number the farmer permitted us to monitor. All the sampled cattle were female, with an average age of 4.5 years. Blood samples were collected from these cows using tail venipuncture without anesthesia with a vacutainer tube and a 21-gauge needle. Serum was then extracted from the blood for serological analysis. Samples were taken at four, eight, and thirteen months following the outbreak. By thirteen months post-outbreak, only ten of the monitored cattle were still on the farm for sampling, as the other cows had been sold. Serum samples processing and serological assays Prior to conducting MN and HI assays, serum samples were treated overnight (18-20 hours) with receptor destroying enzyme II (RDE; Denka Seiken, Tokyo, Japan) at 37°C to eliminate nonspecific inhibitors, following the manufacturer’s recommendations. To inactivate the RDE, the serum samples were then incubated in a water bath at 56°C for 30 minutes. Finally, the serum was diluted in PBS without Mg+2 and Ca+2 (Sigma-Aldrich, Saint Louis, MO) to achieve a final concentration of 1:10. Microneutralization assay To determine the NAb titer, we conducted a MN assay using a recombinant H5N1 virus (rg-A/bald eagle/Florida/ W22-134-OP/2022 of clade 2.3.4.4b), which was kindly provided by Dr. Richard Webby from St. Jude Children's Hospital in Memphis, TN. The assay was performed in an enhanced biosafety level 2 facility, as previously described 25,26 with slight modifications. Briefly, serial two-fold dilutions of the RDE-treated serum samples were performed starting at a dilution of 1:20, in duplicate, using serum-free, high glucose Dulbecco's Modified Eagle Medium with glutamine (Gibco, cat. no. 11965-092) supplemented with 100 units/mL penicillin-streptomycin, 1mM sodium pyruvate, and 2 µg/mL tosyl phenylalanyl chloromethyl ketone-trypsin. The diluted sera were mixed in equal volumes with 100 TCID 50 of the virus and incubated at room temperature (RT) for one hour with continuous rocking. After incubation, 100 µL of the mixture was transferred to a monolayer of Madin-Darby canine kidney (MDCK, ATCC #CRL-CCL34) cells in 96-well plates (GenClone, San Diego, CA, USA) and incubated at 37 o C. After 18-20 hours, the MDCK plates were fixed with cold 80% acetone (Fisher Chemical™, cat. no. A18-1). Thereafter, the acetone-fixed plates were washed three times with a wash buffer (PBS containing 0.3 % (v/v) Tween 20). An ELISA was then performed using a pan-influenza A nucleoprotein (anti-NP) mouse monoclonal antibody (Sino Biological, Wayne, PA, USA. cat. no. 40208-MM03) as the primary antibody (1:2000) and horse-radish peroxidase (HRP) conjugated goat anti-mouse IgG (Thermo Fisher Scientific, cat. No. 31430) as the secondary antibody (1:5000). The anti-NP was added first, and after an incubation period of one hour, the wells were washed three times with the wash solution. The secondary antibody was then added and incubated for another one hour at RT. The plate was washed three more times with the wash buffer. Finally, a stop solution (0.5M sulfuric acid) was added to halt the activity of the HRP enzyme. The absorbance of the resultant solution was measured at 450 nm using the Agilent BioTek Epoch microplate spectrophotometer (Agilent, CA, USA). We defined NAb titer as the reciprocal of the highest serum dilution at which 50% of the serum samples in the wells were inhibited. Hemagglutination-inhibition assay We conducted a HI assay using four hemagglutinating units of the recombinant H5N1 virus (rg-A/bald eagle/Florida/W22-134-OP/2022, clade 2.3.4.4b). The assay was performed in V-bottom 96-well plates using the pre-treated cattle serum samples along with 1% chicken red blood cells (Lampire Biological Laboratories, Pipersville, PA), following a previously described method 27 . We defined the HI titer as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. For both the MN and HI assays, samples with an antibody titer of 1:40 or greater were considered positive. Previous studies have indicated that this threshold can effectively reduce the risk of contracting influenza infection 19,28 . Enzyme-linked immunosorbent assay (ELISA) To determine the influenza immunoglobulin G (IgG) antibody titer in the cattle serum, we used the ID Screen® H5 antibody competition multi-species ELISA kit (Innovative Diagnostic, Grabels, France) following the manufacturer's instructions. Briefly, the serum samples, along with both negative and positive controls, were diluted ten-fold using a dilution buffer in a 96-well microplate that was coated with recombinant H5 protein. The plates were then incubated at 37°C for 1 hour and 30 minutes. After incubation, we washed the plates three times with IX wash solution provided with the kit. Thereafter, a 1X conjugate solution was added, and the plates were incubated for 30 minutes at RT (21 ± 5°C). After this step, the plates were washed three times again with the wash solution. Following the washes, a substrate solution was added and incubated for 15 minutes at RT. Finally, a stop solution was added, and absorbance readings were measured at 450 nm using the Agilent BioTek Epoch microplate spectrophotometer (Agilent, CA, USA). Each sample and controls were tested in duplicate. For the ELISA positive samples, we used a sample-to-negative percentage (S/N %) cutoff of ≤40%, as recommended by the manufacturer. Data analysis We compared MN and HI assay results over the three time periods (4, 8, and 13 months) and overall, with a Spearman's rank correlation test. Additionally, we compared MN and ELISA and HI and ELISA assays over time by dichotomized (positive or negative) the MN, HI, and ELISA data. In dichotomization, we defined a MN titer of ≥ 1:40 19 as positive, a HI titer ≥ 1:40 19 as positive, and a ELISA cutoff of ≤40% as positive. We employed a pairwise Cohen’s Kappa test in comparing the dichotomized three assays over time (Table 2). For quantitative analysis, titers below the lower limit of quantification (<20) were assigned a value of 10. All numerical titer values were log₂-transformed prior to analysis. Statistical analyses and data visualization were performed using RStudio (version 4.4.2; R Core Team, 2024). Declarations Acknowledgements We appreciate the management and staff of the farm for their support. Authors contributions Conceptualization: I.S., G.C.G.; Methodology: I.S.; Investigation: I.S., J.R., J.U.O., C.M.T-V., L.V.M., T.N.T., N.E.S., J.A.L., G.C.G.; Data analysis: I.S., J.R.; Visualization: I.S., J.R.; Original draft.: I.S., G.C.G.; Revision: I.S., J.R., J.U.O., C.M.T-V., L.V.M., T.N.T., N.E.S., J.A.L., G.C.G.; Project administration: G.C.G; Funding acquisition: G.C.G.; Supervision: G.C.G. All authors read and approved the final version of the manuscript. Data availability Data needed to evaluate the conclusions in the paper are present in the paper. Additional data or specimens may be requested from the corresponding author. The sharing of additional data or specimens will require the signing of a materials transfer agreement. Funding This project was supported in part by the Agriculture and Food Research Initiative Competitive Grant from the American Rescue Plan Act (award number 2023-70432-39558) through USDA APHIS, and by Professor Gregory C. Gray's startup funding from the University of Texas Medical Branch. The findings and conclusions in this report are those of the authors and should not be construed to represent any official USDA or US Government determination or policy. Competing interests The authors declare no competing interests. References Sreenivasan, C. C., Thomas, M., Kaushik, R. S., Wang, D. & Li, F. Influenza A in Bovine Species: A Narrative Literature Review. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 22 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Editor invited by journal 03 Sep, 2025 Submission checks completed at journal 29 Aug, 2025 First submitted to journal 29 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7274486","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":512717427,"identity":"9c0b7b12-076d-484c-b9f9-e65e05d8eb56","order_by":0,"name":"Ismaila Shittu","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Ismaila","middleName":"","lastName":"Shittu","suffix":""},{"id":512717428,"identity":"31bc2170-3ca4-498b-b855-339b31a3b328","order_by":1,"name":"Jessica Rodriguez","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Rodriguez","suffix":""},{"id":512717429,"identity":"33361590-5651-4b9e-bcb8-bdf846e66e71","order_by":2,"name":"Judith U. Oguzie","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"U.","lastName":"Oguzie","suffix":""},{"id":512717430,"identity":"85ad9713-74fb-434a-889d-384be618a9a3","order_by":3,"name":"Claudia M. Trujillo-Vargas","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"M.","lastName":"Trujillo-Vargas","suffix":""},{"id":512717431,"identity":"5c22daf1-142e-4b86-a256-6c8209c522e6","order_by":4,"name":"Lyudmyla V. Marushchak","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Lyudmyla","middleName":"V.","lastName":"Marushchak","suffix":""},{"id":512717432,"identity":"36d2febe-98ff-4a54-b97e-3b80d8c8165e","order_by":5,"name":"Thang Nguyen-Tien","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Thang","middleName":"","lastName":"Nguyen-Tien","suffix":""},{"id":512717433,"identity":"080caae1-3791-49c0-ba01-0d9ebe6799a6","order_by":6,"name":"Nicholas E. Schneider","email":"","orcid":"","institution":"Schneider Veterinary Services","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"E.","lastName":"Schneider","suffix":""},{"id":512717434,"identity":"9fd9201d-5f02-4be2-a12f-575a29b1f607","order_by":7,"name":"John A. Lednicky","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"A.","lastName":"Lednicky","suffix":""},{"id":512717435,"identity":"c0764cab-bc32-4722-8a95-1a4433be97fc","order_by":8,"name":"Gregory C. Gray","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYPACGyDmAWI24rWkATWQqOUwCVrk3Y9f+/Cz7XzifrGzBxg+lB0mrMXwTE7xzN6224k90nkJjDPOEaNlBk8yAy9YS44BM28bkVoY/7adg2j5S4wWeQn2w0DDD0C0MBKjxYAnh5lZ5lyycc/tvISDPefSibCl/fhjxjdldrLts3MPPvhRZk2ELQd4DBgYodFxgLB6kC0N7A8YGP4QpXYUjIJRMApGKgAAcrE654shpZoAAAAASUVORK5CYII=","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":true,"prefix":"","firstName":"Gregory","middleName":"C.","lastName":"Gray","suffix":""}],"badges":[],"createdAt":"2025-08-01 22:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7274486/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7274486/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90982482,"identity":"edabb8bd-ee1a-49b7-b8c8-27f2f38eb1ec","added_by":"auto","created_at":"2025-09-10 09:29:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206614,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal comparison of mean log\u003csub\u003e2\u003c/sub\u003e-transformed MN and HI antibody titers among dairy cattle at 4-, 8-, and 13-months post-HPAI H5N1 infection.\u003c/p\u003e\n\u003cp\u003eTiter values below the lower limit of quantification (\u0026lt;20) were assigned a value of 10 prior to transformation. Lines represent mean titers across timepoints; error bars represent standard error of the mean.\u003c/p\u003e\n\u003cp\u003eMN = microneutralization; HI = hemagglutination inhibition\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7274486/v1/58029560f69534c884551477.jpeg"},{"id":90985626,"identity":"879c07f4-21e2-4898-a64a-6ff9574c2034","added_by":"auto","created_at":"2025-09-10 09:53:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1030913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7274486/v1/6809d098-5a13-4418-9bd5-62d35dcc2477.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sustained High Levels of Antibodies to Avian Influenza Virus H5N1 Clade 2.3.4.4b in Naturally Infected Cattle","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUntil recently, cattle were thought to be somewhat resistant to infection with influenza A virus \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Evidence now shows infection with highly pathogenic avian influenza virus (HPAIV) H5N1 is often manifested by an increase in specific antibody titers, a significant drop in milk production, reduction in feed intake, and shedding of H5N1 virus in nasal secretions and milk, and lesions in mammary gland \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In March 2024, outbreaks of HPAIV H5N1 clade 2.3.4.4b genotype B3.13 were reported in dairy cattle on a North Texas farm, following a spillover from poultry that was preceded by reassortment of HPAIV H5N1 2.3.4.4b North American strain with low pathogenic avian influenza virus North American strain in wild birds \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Subsequently, several dairy herds were affected and the virus spilled into humans, particularly farm workers \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. As of July 2025, more than a thousand confirmed dairy farms have suffered HPAIV infections in 17 US states \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Immune responses to HPAIV H5N1 in poultry have been well-studied \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, limited information is available regarding the systemic, immunoglobulin G (IgG)-mediated immune responses of cattle to infections with the virus, apart from evidence of seroconversion that were uncovered in studies of experimentally infected cattle \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we sought to assess the humoral IgG-mediated immune response and duration of antibodies produced in cattle naturally infected with an HPAIV H5N1 clade 2.3.4.4b strain using three different serological assays: microneutralization (MN), hemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISA). Understanding host immune responses is vital for designing an effective vaccination strategy. We hypothesized that cattle infected with the HPAIV H5N1 strain may not elicit a strong and lasting antibody response.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo better understand the duration of the immune response in cattle exposed to HPAI H5N1 virus, we monitored their specific antibodies over a period of 13 months using three different assays: MN, HI, and ELISA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The three assays successfully detected H5 antibodies in the infected cows, and the results were largely comparable across all methods (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMicroneutralization assay\u003c/h2\u003e\u003cp\u003eFour months post-outbreak, 73% (11/15) of the cattle had detectable neutralizing antibodies (NAb) to H5N1 virus, with levels ranging from 1:80 to 1:640 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Serum samples collected eight months post-outbreak showed that 73% (11/15) of the cattle had varying degrees of NAb. Among these cattle, 18% (2/11, samples ID USL086_TX and USL156_TX) exhibited an increase in NAb, 63.6% (7/11) maintained stable levels, while the remaining 18% (2/11, samples ID USL153_TX and USL157_TX) showed a decline in neutralization (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Four of the samples that were negative by MN were also negative by HI (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eA comparison or serologic response as measured by three different assays among cattle that were naturally exposed to highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b beginning in the spring 2024 and followed for 13 months.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDairy Cow ID Number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eFour months post H5N1 outbreak on the farm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eEight months post H5N1 outbreak on the farm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e\u003cp\u003eThirteen months post H5N1 outbreak on the farm\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eELISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eELISA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eHI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eELISA\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL086_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003edoubtful\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003edoubtful\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL151_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd 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colname=\"c6\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL153_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL154_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003edoubtful\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL155_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL156_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL157_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL158_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL159_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL160_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUSL161_TX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003edoubtful\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003eMN\u0026thinsp;=\u0026thinsp;microneutralization; HI\u0026thinsp;=\u0026thinsp;hemagglutination inhibition; ELISA\u0026thinsp;=\u0026thinsp;enzyme-linked immunosorbent assay; NA\u0026thinsp;=\u0026thinsp;not available; NT\u0026thinsp;=\u0026thinsp;not tested; doubtful\u0026thinsp;=\u0026thinsp;as defined by the package insert (or borderline)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHemagglutination-inhibition test\u003c/h3\u003e\n\u003cp\u003eThe HI test was largely in agreement with the MN and ELISA tests (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The HI assay suggested that 73% (11/15) of the cows has elevated HI results at both four- and eight-months after the outbreak (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, by the thirteenth month, the remaining four samples did not have detectable antibody levels (less than 1:20) and were declared negative.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eBy four- and eight-months post-outbreak, 73% (11/15) of the infected cows had elevated antibodies against H5. The remaining four samples collected at four months were classified as doubtful (40% \u0026lt; SN\u0026thinsp;\u0026lt;\u0026thinsp;50%) according to the assay interpretation proposed by the manufacturer (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Also, at eight and thirteen months, two and one samples were identified as doubtful, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAssay Agreement\u003c/h3\u003e\n\u003cp\u003eThe results indicate perfect agreement (κ\u0026thinsp;=\u0026thinsp;1.0) between the MN, HI, and ELISA assays at 4- and 8-months post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, by month 13, the agreement notably decreased notably, with ELISA versus HI and HI versus MN showing low to slight agreement (κ\u0026thinsp;=\u0026thinsp;0.12 and κ\u0026thinsp;=\u0026thinsp;0.18, respectively). The ELISA versus MN agreement remained moderate at 13 months (κ\u0026thinsp;=\u0026thinsp;0.60). Considering all the time points combined, the highest overall concordance was observed, once again, between the ELISA and MN assays (κ\u0026thinsp;=\u0026thinsp;0.88, 95% CI 0.72-1.0), followed by HI and MN (κ\u0026thinsp;=\u0026thinsp;0.78, 95% CI 0.57\u0026ndash;0.98), and ELISA and HI (κ\u0026thinsp;=\u0026thinsp;0.72, 95% CI 0.50\u0026ndash;0.94) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggest that the ELISA and MN assays maintain better long-term agreement compared to the HI assay in measuring IgG-mediated antibody response following infection over time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePairwise Kappa analysis for dichotomised results of three different serological assays for cattle that were naturally exposed to the highly pathogenic avian influenza H5N1 over a one year period.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest Comparison\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 Months\u003c/p\u003e\u003cp\u003e(κ {95% CI}\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 Months\u003c/p\u003e\u003cp\u003e(κ {95% CI})\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13 Months\u003c/p\u003e\u003cp\u003e(κ {95% CI})\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOverall\u003c/p\u003e\u003cp\u003e(κ {95% CI})\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMN vs HI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.18 (-0.16, 0.18)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.78 (0.57, 0.98)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMN vs ELISA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.6 (0.14, 0.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.88 (0.72, 1.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHI vs ELISA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.0 (1.0\u0026ndash;1.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.12 (-0.12, 0.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.72 (0.5, 0.94)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eMN and HI titers, originally continuous, were dichotomized using predefined assay-specific cutoff values to allow for categorical comparison. ELISA data were provided as dichotomous outcomes (positive/negative). Kappa values reflect agreement beyond chance between each pair of assays at each timepoint. MN\u0026thinsp;=\u0026thinsp;microneutralization; HI\u0026thinsp;=\u0026thinsp;hemagglutination inhibition; ELISA\u0026thinsp;=\u0026thinsp;enzyme-linked immunosorbent assay\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eTiter Comparison\u003c/h3\u003e\n\u003cp\u003eThe log₂-transformed mean antibody titers measured by MN assays and HI and at 4-, 8-, and 13-months post-infection were visually compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Across all timepoints, MN titers remained consistently higher than HI titers. At 4 months, the mean MN titer was significantly elevated compared to HI, suggesting stronger early detection by the MN assay. This trend persisted at 8 months and was still apparent at 13 months, although the overall titers declined substantially for both assays. Spearman correlation (ρ) between the two assays over the entire time period showed a strong, statistically significant, positive correlation across all time points (ρ\u0026thinsp;=\u0026thinsp;0.931, p\u0026thinsp;=\u0026thinsp;2.5 \u0026times; 10⁻\u0026sup1;⁷), further indicating consistent agreement between both assays. While the correlation was strong at 4-months (ρ\u0026thinsp;=\u0026thinsp;0.883), it was highest at the 8-month timepoint (ρ\u0026thinsp;=\u0026thinsp;0.923), and moderately lower at 13-months (ρ\u0026thinsp;=\u0026thinsp;0.721). The progressive decline in titers over time suggests waning humoral immunity, with MN assay demonstrating greater persistence in detecting antibodies relative to HI assay.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the years, the HPAIV H5N1 has evolved into several clades and genotypes, with clade 2.3.4.4b being recently the most prevalent globally. In the United States, genotypes B3.13 and D1.1, both belonging to clade 2.3.4.4b, have been detected in cattle \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The outbreak of the H5N1 clade 2.3.4.4b, genotype B3.13 in Texas in 2024 underscored the ability of the H5 A/Goose/Guangdong/1/1996 virus to cross species barriers \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Current efforts are focused on addressing the threats posed by this virus by developing and evaluating vaccines to control the disease in animals. It is essential to understand the antibody responses of livestock to the H5N1 virus, particularly long-lived livestock like cows, such that vaccine strategies can be designed to reduce H5N1 transmission. In our study, we found varying levels of H5N1 antibodies in 73% of the cattle monitored on a farm affected by the H5N1 epizootic. Additionally, we observed a decline in antibody levels to H5 over time among the cattle that seroconverted. However, some of the cows maintained detectable antibody titers for more than a year.\u003c/p\u003e\u003cp\u003eTo our knowledge, these data represent the first reported effort to monitor antibody decay in dairy cattle that were naturally exposed to H5N1 infection. A previous study with H5 isolates indicated that infected cattle seroconverted, with antibodies detected for up to 90 days \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The H5N1 B3.13 strain was found to circulate in cows, and evidence has shown that humans infected with the virus, especially farm workers infected with the virus, also produce NAb \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our findings align with previous studies on antibody persistence following influenza A virus infection in humans \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe found a significant agreement in the Kappa analysis with the antibodies to H5N1, as detected by MN, HI, and ELISA assays, for up to eight months following the outbreak (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, there was a correlation between the mean antibody titers for HI and MN, with only 1to 2 log\u003csub\u003e2\u003c/sub\u003e differences observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This finding aligns with previous studies, which indicate that an HI titer of 1:40 is equivalent to an MN titer of 1:160 \u003csup\u003e19,20\u003c/sup\u003e. However, by the thirteenth month, a significant reduction in detectable antibodies was observed with the ELISA and HI assays. The HI assay is easy to set up, inexpensive and regarded as the gold standard, but it has been shown to be less sensitive \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The MN assay has been shown to be more sensitive and specific at determining virus NAb \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. On the other hand, HI tests are effective at detecting antibodies that prevent viral hemagglutination. It is important to note that while some antibodies can inhibit hemagglutination, they do not always neutralize the virus. Conversely, NAb may not always have the ability to inhibit hemagglutination induced by the virus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe found approximately 27% (4/15) of the cattle showed no detectable antibodies during the monitoring period. This may be due to the innate immune mediators of the cows, which probably limit the replication of the virus at the point of entry, leading to an abortive infection. The innate immune mediators are the first line of defense encountered by influenza virus, which leads to the secretions of signaling proteins, such as interferons, chemokines, cytokines and eicosanoids \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Another possibility could be that an infection occurred, but the antibody response was below the detection level of the assays used in the monitoring process.\u003c/p\u003e\u003cp\u003eNotably, a few ELISA tests yielded doubtful outcomes, particularly in cases where both MN and HI assays reported some samples as negative. It is important to note that the ELISA results provided a qualitative measurement, unlike the quantitative measures provided by the MN and HI assays. The initial dilution for the ELISA tests was set at 1:10. Given this dilution factor, any results falling below the detection threshold of 1:40 would be interpreted as negative in both the MN and HI assays. Hence, these borderline \u0026ldquo;doubtful\u0026rdquo; results are considered negative.\u003c/p\u003e\u003cp\u003eOur study is limited by the small number of animals that were enrolled, which restricts the generalizability of our findings. Furthermore, we operated under the assumption that each cow was infected only once with the H5N1 virus. This assumption may not reflect the reality, as the immune response could vary significantly if the cattle experienced multiple exposures to the virus over time. Also, there was a decrease in the number of animals available during the follow-up period, especially at the final stage of the monitoring.\u003c/p\u003e\u003cp\u003eIn conclusion, our study revealed that antibodies to H5N1 were present in 50% of the infected cattle over a year, as measured by the MN assay. This finding is significant as it suggests that a considerable portion of the cattle developed a relatively strong IgG-mediated immune response to the virus. To ensure long-lasting immunity in cattle, it may be beneficial to administer vaccinations at least every eight months.\u003c/p\u003e"},{"header":"Materials","content":"\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur laboratory work involving avian influenza viruses was reviewed and approved by UTMB Institutional Biosafety Committee. UTMB\u0026rsquo;s Institutional Animal Care and Use Committee (IACUC) evaluated this work as exempt from formal IACUC ethical review. All procedures, including animal welfare and ethics, and laboratory biosafety procedures were conducted in accordance with the US, ARRIVE, and UTMB relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy site and sampling\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith the permission of the farm management, we sampled and monitored a single dairy farm in Northern Texas for more than a year. The farm spans approximately 100 acres and is home to more than 8,000 cattle of varying ages comprising both dairy and beef cattle. It features a computerized system that monitors different health parameters, such as rumination and milk production within the Holstein dairy herd. In March 2024, we confirmed an outbreak of HPAIV H5N1 on dairy cattle on the farm, as previously reported \u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the time of the HPAIV incursion, serum samples were not available but these cows\u0026rsquo; manifested signs of acute illness consistent with infection by the virus, such as reduced milk production, fever, respiratory signs, including nasal discharge. From July 2024 to April 2025, we monitored 15 cows on the dairy farm, as this was the number the farmer permitted us to monitor. All the sampled cattle were female, with an average age of 4.5 years. Blood samples were collected from these cows using tail venipuncture without anesthesia with a vacutainer tube and a 21-gauge needle. Serum was then extracted from the blood for serological analysis. Samples were taken at four, eight, and thirteen months following the outbreak. By thirteen months post-outbreak, only ten of the monitored cattle were still on the farm for sampling, as the other cows had been sold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSerum samples processing and serological assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to conducting MN and HI assays, serum samples were treated overnight (18-20 hours) with receptor destroying enzyme II (RDE; Denka Seiken, Tokyo, Japan) at 37\u0026deg;C to eliminate nonspecific inhibitors, following the manufacturer\u0026rsquo;s recommendations. To inactivate the RDE, the serum samples were then incubated in a water bath at 56\u0026deg;C for 30 minutes. Finally, the serum was diluted in PBS without Mg+2 and Ca+2 (Sigma-Aldrich, Saint Louis, MO) to achieve a final concentration of 1:10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicroneutralization assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the NAb titer, we conducted a MN assay using a recombinant H5N1 virus (rg-A/bald eagle/Florida/ W22-134-OP/2022 of clade 2.3.4.4b), which was kindly provided by Dr. Richard Webby from St. Jude Children\u0026apos;s Hospital in Memphis, TN. The assay was performed in an enhanced biosafety level 2 facility, as previously described \u003csup\u003e25,26\u003c/sup\u003e with slight modifications. Briefly, serial two-fold dilutions of the RDE-treated serum samples were performed starting at a dilution of 1:20, in duplicate, using serum-free, high glucose Dulbecco\u0026apos;s Modified Eagle Medium with glutamine (Gibco, cat. no. 11965-092) supplemented with 100 units/mL penicillin-streptomycin, 1mM sodium pyruvate, and 2 \u0026micro;g/mL tosyl phenylalanyl chloromethyl ketone-trypsin. The diluted sera were mixed in equal volumes with 100 TCID\u003csub\u003e50\u003c/sub\u003e of the virus and incubated at room temperature (RT) for one hour with continuous rocking. After incubation, 100 \u0026micro;L of the mixture was transferred to a monolayer of Madin-Darby canine kidney (MDCK, ATCC #CRL-CCL34) cells in 96-well plates (GenClone, San Diego, CA, USA) and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC. After 18-20 hours, the MDCK plates were fixed with cold 80% acetone (Fisher Chemical\u0026trade;, cat. no. A18-1). Thereafter, the acetone-fixed plates were washed three times with a wash buffer (PBS containing 0.3 % (v/v) Tween 20). \u0026nbsp;An ELISA was then performed using a pan-influenza A nucleoprotein (anti-NP) mouse monoclonal antibody (Sino Biological, Wayne, PA, USA. cat. no. 40208-MM03) as the primary antibody (1:2000) and horse-radish peroxidase (HRP) conjugated goat anti-mouse IgG (Thermo Fisher Scientific, cat. No. 31430) as the secondary antibody (1:5000). The anti-NP was added first, and after an incubation period of one hour, the wells were washed three times with the wash solution. The secondary antibody was then added and incubated for another one hour at RT. The plate was washed three more times with the wash buffer. Finally, a stop solution (0.5M sulfuric acid) was added to halt the activity of the HRP enzyme. The absorbance of the resultant solution was measured at 450 nm using the Agilent BioTek Epoch microplate spectrophotometer (Agilent, CA, USA). We defined NAb titer as the reciprocal of the highest serum dilution at which 50% of the serum samples in the wells were inhibited.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHemagglutination-inhibition assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a HI assay using four hemagglutinating units of the recombinant H5N1 virus (rg-A/bald eagle/Florida/W22-134-OP/2022, clade 2.3.4.4b). The assay was performed in V-bottom 96-well plates using the pre-treated cattle serum samples along with 1% chicken red blood cells (Lampire Biological Laboratories, Pipersville, PA), following a previously described method \u003csup\u003e27\u003c/sup\u003e. We defined the HI titer as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. For both the MN and HI assays, samples with an antibody titer of 1:40 or greater were considered positive. Previous studies have indicated that this threshold can effectively reduce the risk of contracting influenza infection \u003csup\u003e19,28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the influenza immunoglobulin G (IgG) antibody titer in the cattle serum, we used the ID Screen\u0026reg; H5 antibody competition multi-species ELISA kit (Innovative Diagnostic, Grabels, France) following the manufacturer\u0026apos;s instructions. Briefly, the serum samples, along with both negative and positive controls, were diluted ten-fold using a dilution buffer in a 96-well microplate that was coated with recombinant H5 protein. The plates were then incubated at 37\u0026deg;C for 1 hour and 30 minutes. After incubation, we washed the plates three times with IX wash solution provided with the kit. Thereafter, a 1X conjugate solution was added, and the plates were incubated for 30 minutes at RT (21 \u0026plusmn; 5\u0026deg;C). After this step, the plates were washed three times again with the wash solution. Following the washes, a substrate solution was added and incubated for 15 minutes at RT. Finally, a stop solution was added, and absorbance readings were measured at 450 nm using the Agilent BioTek Epoch microplate spectrophotometer (Agilent, CA, USA). Each sample and controls were tested in duplicate. For the ELISA positive samples, we used a sample-to-negative percentage (S/N %) cutoff of \u0026le;40%, as recommended by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe compared MN and HI assay results over the three time periods (4, 8, and 13 months) and overall, with a Spearman\u0026apos;s rank correlation test. \u0026nbsp;Additionally, we compared MN and ELISA and HI and ELISA assays over time by dichotomized (positive or negative) the MN, HI, and ELISA data. In dichotomization, we defined a MN titer of \u0026ge; 1:40\u003csup\u003e19\u003c/sup\u003e as positive, a HI titer \u0026ge; 1:40\u003csup\u003e19\u003c/sup\u003e as positive, and a ELISA cutoff of \u0026nbsp;\u0026le;40% as positive. We employed a pairwise Cohen\u0026rsquo;s Kappa test in comparing the dichotomized three assays over time (Table 2). For quantitative analysis, titers below the lower limit of quantification (\u0026lt;20) were assigned a value of 10. All numerical titer values were log₂-transformed prior to analysis. Statistical analyses and data visualization were performed using RStudio (version 4.4.2; R Core Team, 2024).\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the management and staff of the farm for their support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: I.S., G.C.G.; Methodology: I.S.; Investigation: I.S., J.R., J.U.O., C.M.T-V., L.V.M., T.N.T., N.E.S., J.A.L., G.C.G.; Data analysis: \u0026nbsp;I.S., J.R.; Visualization: I.S., J.R.; Original draft.: I.S., G.C.G.; Revision: I.S., J.R., J.U.O., C.M.T-V., L.V.M., T.N.T., N.E.S., J.A.L., G.C.G.; Project administration: G.C.G; Funding acquisition: G.C.G.; Supervision: G.C.G. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData needed to evaluate the conclusions in the paper are present in the paper. Additional data or specimens may be requested from the corresponding author. The sharing of additional data or specimens will require the signing of a materials transfer agreement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported in part by the Agriculture and Food Research Initiative Competitive Grant from the American Rescue Plan Act (award number 2023-70432-39558) through USDA APHIS, and by Professor Gregory C. Gray\u0026apos;s startup funding from the University of Texas Medical Branch. 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Vaccines\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 519\u0026ndash;536. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1586/erv.13.35\u003c/span\u003e\u003cspan address=\"10.1586/erv.13.35\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"avian influenza, dairy cattle, immune response, farm, Texas","lastPublishedDoi":"10.21203/rs.3.rs-7274486/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7274486/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCattle were not previously thought susceptible to highly pathogenic avian influenza (HPAI) H5N1 virus infection until March 2024, when outbreaks were reported among dairy cattle. Data are sparse regarding the immune response of cattle to infections with such viruses. To better understand the systemic humoral immune responses of HPAI H5N1-infected dairy cattle, we conducted a prospective study of 15 cows on an H5N1-affected farm. The cows’ sera were studied using microneutralization (MN), hemagglutination inhibition, and enzyme-linked immunosorbent assays. At four months post-outbreak, MN assays revealed 73% (11/15) of the cattle sera had elevated H5 antibody levels that ranged from 1:40 to 1:640. Eight months post-outbreak, 73% retained elevated MN antibodies, wherein 18% (2/11) had an increased response, 63.6% (7/11) maintained antibody levels, and 18% (2/11) showed a decline. By 13 months post-outbreak, only 50% (5/10) retained elevated titers by the MN assay. The results from all three assays were in agreement; however, the MN assay seemed to be more sensitive. Our findings indicate a correlation among the three assays, with high levels of H5 antibodies persisting for more than a year after natural infections. This information is vital for guiding future use of H5 vaccines in dairy cattle farms.\u003c/p\u003e","manuscriptTitle":"Sustained High Levels of Antibodies to Avian Influenza Virus H5N1 Clade 2.3.4.4b in Naturally Infected Cattle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 09:29:32","doi":"10.21203/rs.3.rs-7274486/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T10:55:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T19:01:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T20:29:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85739897500829735020835881151124874752","date":"2025-09-09T15:17:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243455079229330086117620939597569760221","date":"2025-09-04T01:26:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-04T01:22:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T01:21:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-03T11:41:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-29T10:05:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-29T10:02:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d38a7ea5-44c5-4940-a509-10f5e86e5e27","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T10:55:55+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54464761,"name":"Health sciences/Diseases"},{"id":54464762,"name":"Biological sciences/Immunology"},{"id":54464763,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-15T07:23:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 09:29:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7274486","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7274486","identity":"rs-7274486","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}