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

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In early 2024, HPAI H5N1 spilled over into dairy cattle in Texas and has since been detected in multiple states, raising concerns about cross-species transmission and zoonotic risk. We assessed the feasibility of serological assays for antibody responses to HPAI H5N1 in dairy cattle by comparing two competitive commercial ELISA assays with a virus neutralization (VN) assay. Samples, including serum and milk matrices, were collected during the acute phase of confirmed outbreaks and 60 days post-outbreak. Results were analyzed for sensitivity, specificity, concordance, and agreement using Kappa statistics and receiver operating characteristic (ROC) analysis. Comparisons to VN revealed substantial agreement for the IDvet ELISA in both serum and milk, while the IDEXX ELISA showed high specificity but lower sensitivity, particularly for low-titer samples. Rennet treatment of milk, required for the VN test, did not affect IDvet performance but reduced agreement for IDEXX. Seroprevalence estimates based on VN and IDvet were similar, indicating consistent detection of exposed animals. In contrast, IDEXX underestimated the number of positives, which may limit surveillance accuracy. These findings emphasize the importance of assay validation in non-conventional species and highlight VN and high-performing ELISAs, as valuable tools for post-outbreak surveillance in cattle. Given the zoonotic potential of H5N1, reliable serological testing is essential for monitoring livestock exposure and assessing risks to human health. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Highly pathogenic avian influenza (HPAI) H5N1 virus, clade 2.3.4.4b (genotypes B3.13 and D1.1), has been detected in U.S. dairy cattle in 2024 ( 1 , 2 ). Since its initial detection, the virus has continued to circulate in this species, posing a major threat to both animal and public health. As of March 16, 2026, the USDA Animal and Plant Health Inspection Service has confirmed 1,088 cases across 19 states using molecular diagnostic assays ( 3 ). However, the true prevalence of HPAI H5N1 virus infection in the US dairy cattle population remains largely unknown. Accurate serological assays are essential for surveillance programs aiming at estimating infection prevalence and identifying at-risk populations. Diagnostic laboratories currently employ a range of techniques to detect H5N1 virus infections, including real-time polymerase chain reaction (qPCR) followed by next-generation sequencing analysis, isolation in chicken embryos or cell culture, and serological assays such as enzyme-linked immunosorbent assay (ELISA) ( 4 )( 5 ). Among these, commercially available competitive ELISA-based assays – suitable for multi-species applications since they don’t use species-specific detection antibodies – are being used to identify previous viral exposure because they are considered high throughput and are rapid ( 6 )( 7 ). However, a major limitation to the widespread adoption of currently available ELISA kits for HPAI H5N1 diagnosis in cattle is the lack of comprehensive data comparing their performance to other standard tests such as virus neutralization (VN) and/or hemagglutination inhibition (HI). Existing studies have been limited in scope and sample size ( 8 ). To address this gap, the present study aimed to evaluate the diagnostic sensitivity and specificity of two commercial ELISA kits relative to virus neutralization assay for detecting HPAI H5N1 antibodies in dairy cattle. METHODS Samples In this study, we tested 929 serum samples from dairy cattle comprising 200 samples collected in 2023 prior the HPAI H5N1 spillover to dairy cows, 92 samples collected from animals in affected farms during acute phase of clinical outbreaks in early 2024, and 637 samples collected at ~ 60 days after an HPAI H5N1 outbreak at a large dairy farm ( 9 ). Additionally, we tested 1,100 milk samples from several affected dairy farms. The 637 samples collected post-HPAI outbreak were collected as part of the study described in ( 9 ), under protocol approved by the Cornell University Institutional Animal Care and Use Committee (IACUC; Protocol No. 2013-0064). Reminiscent samples from these study and other diagnostic samples submitted to the AHDC for HPAI or other routine diagnostic testing were used in the present study. Cells and virus Bovine uterine epithelial cells (Cal-1, developed in-house at the Virology Laboratory at AHDC), which were cultured in minimal essential medium (MEM, Corning Inc., Corning, NY) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. (ThermoFisher Scientific, Waltham, MA; 10 U.mL–1 and 100 µg.mL–1, respectively). These cells were used as indicators of virus replication in the H5N1 VN assay. The VN assay was developed and validated using a recombinant rHPAI H5N1 TX2/24-miniGFP2 was generated as previously described ( 10 , 11 ). The virus used to develop the recombinant rHPAI H5N1 TX2/24-miniGFP2 ( 10 , 11 ), was isolated from the milk of infected dairy cattle in Texas, USA ( 12 ), HPAI H5N1 TX2/24 (A/Cattle/Texas/063224-24-1/2024 and belongs to genotype B3.13, GISAID accession number: EPI_ISL_19155861). Virus neutralization Virus neutralization (VN) test was performed using the rHPAI H5N1 TX2/24-miniGFP2 virus to detect and quantify H5N1 virus-specific neutralizing antibodies (Fig. 5 ). Briefly, 25 µL of each sample were added to a 96-well plate and subjected to two-fold serial dilutions (1:8 to 1:2,048) in MEM. Next, 50 µL of a virus suspension containing 100–200 TCID50 of rHPAI TX2/24-miniGFP2 was added to each well of the 96-well plate and incubated for 1 h at 37°C. Cal-1 cells (100 µL) were added to each well, and the plates were incubated at 37°C for 48 h. The results were visualized using a fluorescence microscope (Hybrid microscope ECHO Revolve 3K) to determine endpoint neutralizing antibody (NAb) titers. The NAb titers are expressed as the reciprocal of the highest serum dilution that completely inhibited HPAI H5N1 virus replication, based on the expression of miniGFP2 by the rHPAI TX2/24-miniGFP2 virus. Specificity assessment for the H5N1 VN assay To assess the specificity and potential cross-reactivity of the H5N1 virus used in our VN assay, we also screened a panel of serum samples known to be positive for antibodies against other influenza A viruses from various species (Table 4 ). These samples were tested against the rHPAI H5N1 TX2/24-miniGFP2 virus as described above. Table 4. Screening on H5N1 virus cross reactivity as assessed by VN assay. Animal species Sample ID Influenza A subtype HPAI H5N1 SN titer Additional information Canine 1 H3N2 (CIV) Neg 1:8 CIV H3N2 Pool 1/6/2020, RTU 2/20/2023 Canine 2 H3N8 (CIV) Neg 1:8 CIV H3N8 Pool 1-6-20 RTU 7/8/2024 Equine 3 H3N8 (EIV) Neg 1:8 EIV Donor Horse 3M059 Swine 4 H1N1 (SIV) Neg 1:8 H1N1 NVSL 0301 IDV 0901 1401 Avian 5 H5N2 (AIV) H7N1 (AIV) Pos 1:256 A/TY/MN/3689-1551/81 (H5N2) and Eq1-Bel (H7N1) - AI AGID Antiserum Avian Avian AI AGID Antiserum 6 H5N2 (AIV) H7N1 (AIV) Pos 1:128 A/TY/MN/3689-1551/81 (H5N2) and Eq1-Bel (H7N1) - AI AGID Antiserum Avian Avian AI AGID Antiserum 7 H5N2 (AIV) H7N1 (AIV) Pos 1:64 A/TY/MN/3689-1551/81 (H5N2) and Eq1-Bel (H7N1) - AI AGID Antiserum Avian Canine 8 H3N2, H3N8 Neg 1:8 216151-18-2, canine serum (H3N8, HI=32; H3N2, HI=16) Canine 9 H3N2, H3N8 Neg 1:8 216151-18-3, canine serum (H3N8, HI=32; H3N2, HI=32) Canine 10 H3N2, H3N8 Neg 1:8 216151-18-4, canine serum (H3N8, HI=64; H3N2, HI=16) Canine 11 H3N2, H3N8 Neg 1:8 216151-18-10, canine serum (H3N8, HI=32; H3N2, HI=16) Canine 12 H3N2, H3N8 Neg 1:8 216151-18-15, canine serum (H3N8, HI=32; H3N2, HI=16) Canine 13 H3N2, H3N8 Neg 1:8 216151-18-20, canine serum (H3N8, HI=8; H3N2, Neg 8) Canine 14 H3N2, H3N8 Neg 1:8 216151-18-24, canine serum (H3N8, HI=64; H3N2, HI=16) Canine 15 H3N2, H3N8 Neg 1:8 216151-18-30, canine serum (H3N8, HI=16; H3N2, Neg 8) Swine 16 H3N2 Neg 1:8 SIV NVSL antiserum Mice 17 H3N2 Neg 1:8 Ace Fluid Mice 18 H1N1 Neg 1:8 Ace Fluid Turkey 19 H3N2 Neg 1:8 Turkey antiserum Turkey 20 H1N1 Neg 1:8 Turkey antiserum Feline 21 H7N2 = P128 Neg 1:8 026558-17-1, feline serum Feline 22 H7N2 = P256 Neg 1:8 026558-17-3, feline serum Feline 23 H7N2 = P16 Neg 1:8 026558-17-15, feline serum Feline 24 H7N2 = P64 Neg 1:8 026558-17-16, feline serum Feline 25 H7N2 = P32 Neg 1:8 026558-17-19, feline serum Feline 26 H7N2 Neg 1:8 Feline Smokey As 1/3/17 ELISA kits Two different competitive ELISA kits were used and evaluated for antibody detection. The assays used included the IDEXX Multi-species Influenza A- and the Innovative Diagnostics (ID) Vet Influenza A Multi-species kits, which detect antibodies against the influenza A nucleoprotein (NP). All serum samples described above were tested side-by-side using these two commercial competitive ELISA kits and the VN assay described above. For testing via ELISA, the serum samples were diluted at 1:5 (IDEXX ELISA) or 1:10 (ID. Vet ELISA) and added to the test well of each kit, respectively. All incubations and washing steps during the ELISA testing were performed following the respective kit’s manufacturer’s instructions. Final ELISA results were interpreted according to the manufacturer's recommended instructions (Table 3 ). Table 3 Comparison of ELISA characteristics Feature IDEXX ELISA ID.Vet ELISA ELISA Type Competitive (Blocking ELISA) Competitive (Blocking ELISA) Target Antigen Influenza A Nucleoprotein (NP) Influenza A Nucleoprotein (NP) Target Species Poultry, swine, horses Poultry, swine, cattle, horses, carnivores (e.g., dogs, cats), others Sample Types Serum, plasma, egg yolk, porcine oral fluid, bovine milk Serum, plasma, egg yolk, porcine oral fluid, bovine milk, bulk tank milk Result Interpretation < 40: Positive 40–<50: Suspicious ≥ 50: Negative < 45: Positive 45–<50: Suspicious ≥ 50: Negative Color Signal Interpretation More color = more antibody (positive) Less color = more antibody (positive) Statistical analysis To assess the agreement between the assays (VN vs ELISAs, ID.Vet ELISA vs IDEXX ELISA), we applied the Cohen's kappa concordance coefficient ( 13 ) using the Visualizing Categorical Data (vcd) package in RStudio software (2024.9.1.394). The analysis was performed on categorical data (positive or negative). Kappa value-based assay agreement was interpreted as follows: kappa between 0.00–0.20 (poor agreement), 0.21–0.40 (fair), 0.41–0.60 (moderate), 0.61–0.80 (good), and 0.81–1.00 (very good) ( 14 ). Additionally, we employed a Receiver Operating Characteristic (ROC) curve analysis using GraphPad Prism 11 software to estimate the Area Under the Curve (AUC) (ranging between 0 and 1), where a score of 1.0 indicated a perfect model and a score of 0.5 being considered no better than random chance. RESULTS Diagnostic specificity of virus neutralization and ELISA kits Testing of the 200 serum samples collected prior to the HPAI H5N1 spillover into dairy cattle showed that all samples tested negative by the virus neutralization assay (200/200; 100%) (Fig. 1 ). Using the IDEXX ELISA, 200/200 (100%) samples also tested negative, corresponding to a diagnostic specificity of 100% and complete agreement with VN results. Using the IDVet ELISA, 199/200 (99.5%) samples tested negative, resulting in a diagnostic specificity of 99.5% and one discrepant result when compared with VN. Comparison between the IDEXX and IDVet ELISAs showed concordant negative results for 199/200 samples (Fig. 1 ). Additionally, testing of a panel of samples known to be antibody positive for influenza A viruses of several subtypes revealed no cross reactivity of antibodies against several H1, H3 and H7 viruses with the H5N1 B3.13 virus used in our VN assay. Importantly, only samples positive for H5N2-specific antibodies presented neutralizing antibodies to the H5N1 B3.13 virus used in our VN assay (Table 4 ). Diagnostic sensitivity of virus neutralization and ELISA kits Among the 92 samples collected during the acute phase of clinical H5N1 virus outbreaks in dairy farms, 46 (50.0%) tested positive by the virus neutralization test (VNT) (Fig. 2 ). Using the IDvet ELISA, 53 (57.60%) samples tested positive, exceeding the number of VNT-positive samples. In contrast, the IDEXX ELISA detected 25 (27.17%) positives, fewer than with VNT (Fig. 2 ). Of the 637 samples collected on day 60 post-clinical H5N1 outbreak, the VNT identified 570 (89.48%) positives, corroborating the high positivity observed with the IDvet ELISA, which detected 553 (86.8%) positives. The IDEXX ELISA detected markedly fewer positives, with only 204 (32%) samples testing positive (Fig. 3 ). Agreement between virus neutralization and ELISA kits The agreement between VN and IDvet ELISA was the highest at 93.6%, while agreement between VN and IDEXX ELISA was much lower at 42.2%. Agreement between the two ELISAs themselves was 43.3% (Table 1 ). Table 1 ROC (Receiver Operating Characteristic) of VN and ELISA kits used in the present study. Assay Area Under the Curve (AUC) Standard error 95% Confidence interval p -value VN 1.0000 0.0000 1.0000–1.0000 < 0.0001 Serum ID Vet 0.9933 0.001782 0.9898–0.9968 < 0.0001 Serum IDEXX 0.7419 0.01743 0.7077–0.7761 < 0.0001 Milk ID Vet 0.9517 0.01058 0.9309 to 0.9724 < 0.0001 Milk IDEXX 0.5660 0.02800 0.5111 to 0.6208 < 0.01942 Kappa coefficients (κ) further evaluated the agreement beyond chance: VN and IDvet ELISA showed substantial agreement (κ = 0.695, p-value = 0), VN and IDEXX ELISA showed slight agreement (κ = 0.1, p-value = 1.49e-08), and agreement between the two ELISAs was also slight (κ = 0.124, p-value = 3.15e-11) (Table 2 ). Table 2 Cohen's Kappa coefficient comparison between serological assays performed in serum and milk samples. Comparison Agreement (%) Kappa Interpretation p-value Serum VN vs. IDEXX 42.2% 0.100 Slight agreement 1.49 × 10⁻⁸ VN vs. ID.Vet 93.6% 0.695 Substantial agreement 0 IDEXX vs. ID.Vet 43.3% 0.124 Slight agreement 3.15 × 10⁻¹¹ Milk VN vs. IDEXX 82.7 0.23 Fair 0 VN vs. ID.Vet 93.6 0.811 Almost perfect agreement 0 IDEXX vs. ID.Vet 82.5 0.243 Fair 0 Rennet IDEXX vs. IDEXX 34.8 -0.332 Poor 0.0755 Rennet IDVet vs. IDVet 92.6 0.471 Moderate 0.00395 Rennet IDEXX vs. VN 30.8 0.029 Slight agreement 0.619 Rennet IDVet vs. VN 89.3 0.364 Fair 0.0126 Detection of H5N1 antibodies in milk For detection of antibodies in milk we first evaluated the impact of rennet treatment, as this was required prior to VN testing due to toxicity of untreated milk samples to the indicator cells. We evaluated whether this treatment influenced the performance of the two ELISAs by testing subsets of 23 and 27 milk samples with each assay, both with and without rennet treatment. For the VN comparison, among rennet-treated milk samples, the IDvet ELISA showed high concordance with VN (89.3% agreement; κ = 0.364; p = 0.0126), indicating fair agreement. In contrast, the IDEXX ELISA exhibited much lower concordance with VN (30.8% agreement; κ = 0.029; p = 0.619), indicating slight agreement (Table 2 ). When assessing the impact of rennet treatment alone, the IDvet ELISA performance was largely unaffected, showing high agreement between treated and untreated samples (92.6% agreement; κ = 0.471; p = 0.00395). The IDEXX ELISA, however, showed poor stability following rennet treatment, with low agreement between treated and untreated milk samples (34.8% agreement; κ = − 0.332; p = 0.0755) (Table 2 ). Overall milk sample screening results further reflected these performance trends. Using VN, 868 (78.9%) samples tested negative and 233 (21.18%) tested positive. The IDvet ELISA identified 837 (76.09%) negatives, 236 (21.45%) positives, and 28 (2.54%) suspect samples. In comparison, the IDEXX ELISA classified 1,049 (95.36%) negatives, 39 (3.54%) positives, and 12 (1.09%) suspects (Fig. 4 ). DISCUSSION Detecting specific antibodies against the highly pathogenic avian influenza (HPAI) H5N1 virus in the serum of dairy cows represents a critical epidemiological tool, particularly considering the incursion and continuous circulation of clade 2.3.4.4b in this mammalian host. The findings of this study revealed substantial differences in diagnostic sensitivity and specificity between an in-house VNT and two commercial ELISA kits evaluated. The IDvet assay demonstrated markedly superior performance and stronger concordance with the virus neutralization test, a recognized gold standard for detecting neutralizing antibodies. These results underscore the need for targeted validation of diagnostic assays in non-conventional species such as cattle. Ensuring high diagnostic accuracy is essential for effective surveillance, timely outbreak detection, and the implementation of appropriate control measures. Results from our comparative analysis indicate that the IDvet ELISA exhibits higher sensitivity than the IDEXX ELISA, during both the acute and convalescent phases of infection. A likely explanation for the low sensitivity observed with the IDEXX assay could be that the assay was developed and validated for use in avian species (e.g., chickens, ducks, geese, turkeys), and assay parameters such as target antigen and antibody concentrations were chosen based on the immune responses of a different non-mammalian species, potentially resulting in lower sensitivity to detect bovine antibodies ( 15 ). Nevertheless, it is important to note that both ELISAs achieved high specificity during the acute phase, approaching or equaling 100%, a critical feature for minimizing false-positive results. However, the sensitivity of the VNT and ID.vet ELISA’s is a strong performance feature in favor of the use of these assays. This study also used Kappa analysis ( 14 ) to assess the performance of the two ELISA assays in terms of specificity, sensitivity, percentage agreement, and concordance. The virus neutralization (VN) test showed good alignment with the hemagglutination inhibition (HI) assay (data not shown). The VN is a reliable and highly sensitive method, capable of serving as a strong comparator. Building on the Kappa analysis, VNT was subsequently used as the reference standard for evaluating the ELISAs. The IDvet ELISA demonstrated substantial agreement with VNT (κ = 0.695), indicating good diagnostic performance. In contrast, the IDEXX ELISA showed only slight agreement with VN (κ = 0.1), reflecting poor alignment and raising concerns about the use of this kit in bovine samples due to is low sensitivity - particularly in samples with low antibody titers (< 128). Agreement between the two ELISA assays was also low (κ = 0.124), despite both targeting anti-NP antibodies. These results highlight the markedly different diagnostic capabilities of the assays, with VNT showing greater reliability for diagnostics followed by the IDvet ELISA. Receiver operating characteristic (ROC) analysis (Table 1 ) of the IDEXX ELISA indicated that the assay is highly specific - approximately 100% - across the evaluated range, with virtually no false positives. The positive likelihood ratio (LR) also increased alongside specificity, reinforcing that a positive result from this assay is a strong indicator of infection/previous exposure (Supplementary Table 1). However, the assay’s sensitivity was notably low. Using the manufacturer’s recommended cut-off (< 0.51, positive), sensitivity increased modestly to 34% without compromising specificity (Supplementary Table 1), yet this level still risks missing a substantial number of true positive cases. Furthermore, adjusting the cut-off to achieve a sensitivity of approximately 80% resulted in a dramatic reduction in specificity to 41.50% (< 0.92, positive), significantly increasing the potential for false positives (Supplementary Table 1). These findings highlight the trade-off between sensitivity and specificity in the IDEXX ELISA, particularly when applied to bovine samples. In milk samples, VNT detected antibody-positive cases at rates of approximately 21–22%, serving as the benchmark for performance comparison. The IDvet ELISA showed close agreement with VNT, producing similar positivity rates and a comparable qualitative distribution of results. The IDEXX ELISA detected only 3.5% positives, which was lower than VNT, indicating reduced alignment in this matrix. This difference in milk samples was consistent with patterns observed in serum testing. Overall, these findings highlight VNT’s central role in accurately identifying H5N1 antibody–positive samples, while illustrating how ELISA performance can vary depending on assay design and potentially, species-specific validation. When examining the impact of rennet treatment, VNT results provided a robust baseline for comparison. The IDvet ELISA maintained high agreement with VNT regardless of treatment, suggesting stable performance in milk matrices. The IDEXX ELISA showed a decline in agreement following rennet treatment; however, this may reflect a combination of assay characteristics and matrix effects rather than solely the clarification step. Maintaining strong concordance with VNT under varying sample conditions is an important attribute for assays intended for field surveillance. From a broader surveillance standpoint, assays that align closely with VNT can provide more accurate prevalence estimates and aid in detecting antibodies in animals with subclinical infections, especially after an outbreak. While differences in sensitivity and specificity were observed between the ELISAs, both demonstrated high specificity during acute infection, minimizing false positives. Accurate detection is particularly important given the zoonotic nature of the H5N1 virus, which poses potential risks to rural workers and those in the milk production chain. These findings emphasize the value of validated serological tools that perform well against standard methods like VNT to ensure reliable assessment of herd exposure and human infection risk. Declarations Acknowledgements We thank the producer and veterinarian Dr. William Leone for allowing sample collection in his farm. The authors also thank Dr. Dennis Summers and his staff and veterinarians, and Lauren E. Meyer for their help with sample collection. The study was funded in part by the Cornell University Animal Health Diagnostic Center Virology Laboratory activity funds. References Caserta LC, Frye EA, Butt SL, Laverack M, Nooruzzaman M, Covaleda LM, Thompson AC, Prarat Koscielny M, Cronk B, Johnson A, Kleinhenz K, Edwards EE, Gomez G, Hitchener G, Martins M, Kapczynski DR, Suarez DL, Alexander Morris ER, Hensley T, Beeby JS, Lejeune M, Swinford AK, Elvinger F, Dimitrov KM, Diel DG (2024) Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. 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Nat Commun 16:6520 Peña-Mosca F, Frye EA, MacLachlan M, Rebelo AR, de Oliveira PSB, Nooruzzaman M, Koscielny MP, Zurakowski M, Lieberman ZR, Leone WM, Elvinger F, Nydam DV, Diel DG (2025) The impact of influenza A H5N1 virus infection in dairy cows. https://doi.org/10.21203/rs.3.rs-6101018/v1 Nooruzzaman M, de Oliveira PSB, Feng C, Diel DG (2026) Recombinant influenza A H5N1 viruses expressing fluorescent and. https://doi.org/10.64898/2026.01.17.700109 . luminescent reporter proteins Caserta LC, Frye EA, Butt SL, Laverack M, Nooruzzaman M, Covaleda LM, Thompson AC, Koscielny MP, Cronk B, Johnson A, Kleinhenz K, Edwards EE, Gomez G, Hitchener G, Martins M, Kapczynski DR, Suarez DL, Alexander Morris ER, Hensley T, Beeby JS, Lejeune M, Swinford AK, Elvinger F, Dimitrov KM, Diel DG (2024) Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. Nature 634:669–676 Cohen J (1960) A Coefficient of Agreement for Nominal Scales. Educ Psychol Meas 20:37–46 McHugh ML (2012) Interrater reliability: the kappa statistic. Biochem Med (Zagreb) 22:276–282 Ludi AB, Morris A, Gubbins S, Asfor A, Afzal M, Browning CF, Grazioli S, Foglia EA, Wilsden G, Burman A, Brocchi E, Paton DJ, King DP (2022) Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus. Viruses 14:1495 Additional Declarations The authors declare potential competing interests as follows: The virus neutralization test described in the study is currently offered at the Animal Health Diagnostic Center (AHDC) at Cornell on a fee-for-service basis. ARR, EF and DGD are employees at the AHDC. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-9163114","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608505687,"identity":"762d5b6c-2db6-4259-8682-2f4eb72dbb0a","order_by":0,"name":"Pablo S.B. de Oliveira","email":"","orcid":"","institution":"Cornell College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"S.B.","lastName":"de Oliveira","suffix":""},{"id":608505688,"identity":"34643074-8fae-4f14-8a90-2f560933e20d","order_by":1,"name":"Ana Rita Rebelo","email":"","orcid":"","institution":"Cornell College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Rita","lastName":"Rebelo","suffix":""},{"id":608505689,"identity":"e459b77c-99d1-430c-85b9-ef67cb5bb873","order_by":2,"name":"Elisha Frye","email":"","orcid":"","institution":"Cornell College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Elisha","middleName":"","lastName":"Frye","suffix":""},{"id":608505690,"identity":"89da9865-0434-443e-8df1-4ab4a59d62d9","order_by":3,"name":"Mohammed Nooruzzaman","email":"","orcid":"","institution":"Cornell College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"","lastName":"Nooruzzaman","suffix":""},{"id":608505691,"identity":"949ceb94-2165-4f7a-a93c-f62e17fd7da4","order_by":4,"name":"Diego G. Diel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCAwwGBxgKbCCcBwVgEWK0GKRBOAkGRGphYDA4TIIWvvOLNx74YHBenl/67MEHQC1yfDcS8GuRvPGs4OAMg9uGM/vykg2AWowlCWkxuHHG4DCPwe0EgzM8ZhJALYkbiNLyx+AcSIv5D6CWesJazvcA/W5wAGwLyPsJBoT9wlZwsMcg2XBmD48x0GEShjPPPMCvhe/84c0fflTYyfPz8Bh++FBhI893nIAtDBKoCiQIKAcB/gNEKBoFo2AUjIKRDQBCn0q6/0lNQAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3237-8940","institution":"Cornell College of Veterinary Medicine","correspondingAuthor":true,"prefix":"","firstName":"Diego","middleName":"G.","lastName":"Diel","suffix":""}],"badges":[],"createdAt":"2026-03-18 21:52:31","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9163114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9163114/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105035376,"identity":"c0319168-7937-4e64-9403-cec6ab611f99","added_by":"auto","created_at":"2026-03-20 07:25:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80646,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity assessment of VNT and ELISA kits using bovine serum samples collected in 2023, before the HPAI H5N1 outbreak and spillover into dairy cattle (n = 200 samples).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/448cd5955bfe67b47259fa7d.jpg"},{"id":105031560,"identity":"bb1b2a55-fd4d-431d-9759-7727f2a269ca","added_by":"auto","created_at":"2026-03-20 06:43:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97410,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic performance comparison between VNT and the ELISA kits using serum samples collected from dairy cows during the acute phase of HPAI H5N1 outbreak. A. ELISA test results. B. VN test results (left) and neutralizing antibody titer distribution of the positive samples. (n = 92 samples).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/e6abbd40133661ee9ee123ba.jpg"},{"id":105031561,"identity":"247d5467-a458-4041-b797-4b5d4253e251","added_by":"auto","created_at":"2026-03-20 06:43:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108076,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic performance comparison between VNT and the ELISA kits using serum samples collected from dairy cows at approximately 60 days post-clinical HPAI H5N1 virus outbreak. A. ELISA test results. B. VN test results (left) and neutralizing antibody titer distribution of the positive samples. (n = 637 samples).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/3dcb69895ae8a4599ddcda2b.jpg"},{"id":105035852,"identity":"27991e90-777c-4daa-92cd-7c2933460b31","added_by":"auto","created_at":"2026-03-20 07:26:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111353,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic performance comparison between VNT and the ELISA kits using milk samples collected from dairy cows at approximately 60 days post-clinical HPAI H5N1 virus outbreak. A. ELISA test results. B. VN test results (left) and neutralizing antibody titer distribution of the positive samples. (n = 1,100 samples).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/d46346ee6ce6f390fb6f9a51.jpg"},{"id":105035898,"identity":"2d663b61-3da2-4be2-acb9-63a3587299ed","added_by":"auto","created_at":"2026-03-20 07:26:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":139605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC curve. \u003c/strong\u003eThe left panels show a dot diagram where each dot represents an individual sample; the X-axis displays the positive (P) or negative (N) classification of samples based on the VN used as the reference standard in this study, and the Y-axis shows S/P ration for IDEXX (A) and IDvet ELISA kits (B). The right panels show an ROC curve analysis based on the same data. Panels C and D display the same analyses applied to milk samples, illustrating the performance of IDEXX (C) and IDVet (D).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/d686d567c7056e0fb4672c26.jpg"},{"id":105031564,"identity":"a96b482a-53ce-4b5f-a531-e8bf161be3c8","added_by":"auto","created_at":"2026-03-20 06:43:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":166309,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the virus neutralization test used in this study. Following incubation of serial 2-fold dilutions of serum, a constant amount (100-200 TCID50) of rHPAI H5N1 TX2/24-miniGFP2 virus is added and incucated with serum for 1 h. Following incubation, cal-1 cells are added and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24-48 h. Final read is performed under a fluorescence microscope. Absence of miniGFP2 signal represents neutralization, while presence of miniGFP2 signal represents lack of neutralization. Neutralizing antibody titers are determined as the reciprocal of the highest serum dilution to completely neutralize the virus.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/690d50c62a2badfe1214d002.jpg"},{"id":105036936,"identity":"d6c01771-175e-489f-9906-b583883b292a","added_by":"auto","created_at":"2026-03-20 07:36:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1485089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9163114/v1/7563b214-5949-4503-a95d-5a0d774e6237.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: The virus neutralization test described in the study is currently offered at the Animal Health Diagnostic Center (AHDC) at Cornell on a fee-for-service basis. ARR, EF and DGD are employees at the AHDC.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eComparison of serological assays to monitor exposure to highly pathogenic avian influenza H5N1 virus\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHighly pathogenic avian influenza (HPAI) H5N1 virus, clade 2.3.4.4b (genotypes B3.13 and D1.1), has been detected in U.S. dairy cattle in 2024 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Since its initial detection, the virus has continued to circulate in this species, posing a major threat to both animal and public health. As of March 16, 2026, the USDA Animal and Plant Health Inspection Service has confirmed 1,088 cases across 19 states using molecular diagnostic assays (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, the true prevalence of HPAI H5N1 virus infection in the US dairy cattle population remains largely unknown.\u003c/p\u003e \u003cp\u003eAccurate serological assays are essential for surveillance programs aiming at estimating infection prevalence and identifying at-risk populations. Diagnostic laboratories currently employ a range of techniques to detect H5N1 virus infections, including real-time polymerase chain reaction (qPCR) followed by next-generation sequencing analysis, isolation in chicken embryos or cell culture, and serological assays such as enzyme-linked immunosorbent assay (ELISA) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Among these, commercially available competitive ELISA-based assays \u0026ndash; suitable for multi-species applications since they don\u0026rsquo;t use species-specific detection antibodies \u0026ndash; are being used to identify previous viral exposure because they are considered high throughput and are rapid (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, a major limitation to the widespread adoption of currently available ELISA kits for HPAI H5N1 diagnosis in cattle is the lack of comprehensive data comparing their performance to other standard tests such as virus neutralization (VN) and/or hemagglutination inhibition (HI). Existing studies have been limited in scope and sample size (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address this gap, the present study aimed to evaluate the diagnostic sensitivity and specificity of two commercial ELISA kits relative to virus neutralization assay for detecting HPAI H5N1 antibodies in dairy cattle.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eIn this study, we tested 929 serum samples from dairy cattle comprising 200 samples collected in 2023 prior the HPAI H5N1 spillover to dairy cows, 92 samples collected from animals in affected farms during acute phase of clinical outbreaks in early 2024, and 637 samples collected at ~\u0026thinsp;60 days after an HPAI H5N1 outbreak at a large dairy farm (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Additionally, we tested 1,100 milk samples from several affected dairy farms. The 637 samples collected post-HPAI outbreak were collected as part of the study described in (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), under protocol approved by the Cornell University Institutional Animal Care and Use Committee (IACUC; Protocol No. 2013-0064). Reminiscent samples from these study and other diagnostic samples submitted to the AHDC for HPAI or other routine diagnostic testing were used in the present study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCells and virus\u003c/h3\u003e\n\u003cp\u003eBovine uterine epithelial cells (Cal-1, developed in-house at the Virology Laboratory at AHDC), which were cultured in minimal essential medium (MEM, Corning Inc., Corning, NY) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. (ThermoFisher Scientific, Waltham, MA; 10 U.mL\u0026ndash;1 and 100 \u0026micro;g.mL\u0026ndash;1, respectively). These cells were used as indicators of virus replication in the H5N1 VN assay. The VN assay was developed and validated using a recombinant rHPAI H5N1 TX2/24-miniGFP2 was generated as previously described (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The virus used to develop the recombinant rHPAI H5N1 TX2/24-miniGFP2 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), was isolated from the milk of infected dairy cattle in Texas, USA (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), HPAI H5N1 TX2/24 (A/Cattle/Texas/063224-24-1/2024 and belongs to genotype B3.13, GISAID accession number: EPI_ISL_19155861).\u003c/p\u003e\n\u003ch3\u003eVirus neutralization\u003c/h3\u003e\n\u003cp\u003eVirus neutralization (VN) test was performed using the rHPAI H5N1 TX2/24-miniGFP2 virus to detect and quantify H5N1 virus-specific neutralizing antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Briefly, 25 \u0026micro;L of each sample were added to a 96-well plate and subjected to two-fold serial dilutions (1:8 to 1:2,048) in MEM. Next, 50 \u0026micro;L of a virus suspension containing 100\u0026ndash;200 TCID50 of rHPAI TX2/24-miniGFP2 was added to each well of the 96-well plate and incubated for 1 h at 37\u0026deg;C. Cal-1 cells (100 \u0026micro;L) were added to each well, and the plates were incubated at 37\u0026deg;C for 48 h. The results were visualized using a fluorescence microscope (Hybrid microscope ECHO Revolve 3K) to determine endpoint neutralizing antibody (NAb) titers. The NAb titers are expressed as the reciprocal of the highest serum dilution that completely inhibited HPAI H5N1 virus replication, based on the expression of miniGFP2 by the rHPAI TX2/24-miniGFP2 virus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSpecificity assessment for the H5N1 VN assay\u003c/h3\u003e\n\u003cp\u003eTo assess the specificity and potential cross-reactivity of the H5N1 virus used in our VN assay, we also screened a panel of serum samples known to be positive for antibodies against other influenza A viruses from various species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These samples were tested against the rHPAI H5N1 TX2/24-miniGFP2 virus as described above.\u003c/p\u003e \n\u003cp\u003eTable 4. Screening on H5N1 virus cross reactivity as assessed by VN assay.\u003c/p\u003e\n\u003ctable style=\"width:467.75pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAnimal species\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eSample ID\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eInfluenza A subtype\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eHPAI H5N1 SN titer\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New 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windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCIV H3N8 Pool 1-6-20 RTU 7/8/2024\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eEquine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eEIV Donor Horse 3M059\u003c/span\u003e\u003c/p\u003e\n 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style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAvian\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH5N2 (AIV) H7N1 (AIV)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:#EE0000;'\u003ePos 1:256\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eA/TY/MN/3689-1551/81 (H5N2) and Eq1-Bel (H7N1) - AI AGID Antiserum Avian\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAvian AI AGID Antiserum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH5N2 (AIV) H7N1 (AIV)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:#EE0000;'\u003ePos 1:128\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eA/TY/MN/3689-1551/81 (H5N2) and Eq1-Bel (H7N1) - AI AGID Antiserum 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windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-2, canine serum (H3N8, HI=32; H3N2, HI=16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-3, canine serum (H3N8, HI=32; H3N2, HI=32)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-4, canine serum (H3N8, HI=64; H3N2, HI=16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-10, canine serum (H3N8, HI=32; H3N2, HI=16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-15, canine serum (H3N8, HI=32; H3N2, HI=16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-20, canine serum (H3N8, HI=8; H3N2, Neg 8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-24, canine serum (H3N8, HI=64; H3N2, HI=16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCanine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e15\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH3N2, H3N8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e216151-18-30, canine serum (H3N8, HI=16; H3N2, Neg 8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eSwine\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 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none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH7N2 = P128\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e026558-17-1, feline serum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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\u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e026558-17-3, feline serum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH7N2 = P16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e026558-17-15, feline serum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH7N2 = P64\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e026558-17-16, feline serum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH7N2 = P32\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e026558-17-19, feline serum\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89.75pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eH7N2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(217, 217, 217);padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eNeg 1:8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 166.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0cm 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;font-size:11.0pt;font-family:\"Aptos\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFeline Smokey As 1/3/17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3\u003eELISA kits\u003c/h3\u003e\n\u003cp\u003eTwo different competitive ELISA kits were used and evaluated for antibody detection. The assays used included the IDEXX Multi-species Influenza A- and the Innovative Diagnostics (ID) Vet Influenza A Multi-species kits, which detect antibodies against the influenza A nucleoprotein (NP). All serum samples described above were tested side-by-side using these two commercial competitive ELISA kits and the VN assay described above. For testing via ELISA, the serum samples were diluted at 1:5 (IDEXX ELISA) or 1:10 (ID. Vet ELISA) and added to the test well of each kit, respectively. All incubations and washing steps during the ELISA testing were performed following the respective kit\u0026rsquo;s manufacturer\u0026rsquo;s instructions. Final ELISA results were interpreted according to the manufacturer's recommended instructions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\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 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of ELISA characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIDEXX ELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eID.Vet ELISA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eELISA Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompetitive (Blocking ELISA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompetitive (Blocking ELISA)\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\u003eTarget Antigen\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInfluenza A Nucleoprotein (NP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInfluenza A Nucleoprotein (NP)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTarget Species\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoultry, swine, horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePoultry, swine, cattle, horses, carnivores (e.g., dogs, cats), others\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSample Types\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum, plasma, egg yolk, porcine oral fluid, bovine milk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSerum, plasma, egg yolk, porcine oral fluid, bovine milk, bulk tank milk\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eResult Interpretation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;40: Positive\u003c/p\u003e \u003cp\u003e40\u0026ndash;\u0026lt;50: Suspicious\u003c/p\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50: Negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;45: Positive\u003c/p\u003e \u003cp\u003e45\u0026ndash;\u0026lt;50: Suspicious\u003c/p\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50: Negative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eColor Signal Interpretation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMore color\u0026thinsp;=\u0026thinsp;more antibody (positive)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLess color\u0026thinsp;=\u0026thinsp;more antibody (positive)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTo assess the agreement between the assays (VN vs ELISAs, ID.Vet ELISA vs IDEXX ELISA), we applied the Cohen's kappa concordance coefficient (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) using the Visualizing Categorical Data (vcd) package in RStudio software (2024.9.1.394). The analysis was performed on categorical data (positive or negative). Kappa value-based assay agreement was interpreted as follows: kappa between 0.00\u0026ndash;0.20 (poor agreement), 0.21\u0026ndash;0.40 (fair), 0.41\u0026ndash;0.60 (moderate), 0.61\u0026ndash;0.80 (good), and 0.81\u0026ndash;1.00 (very good) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Additionally, we employed a Receiver Operating Characteristic (ROC) curve analysis using GraphPad Prism 11 software to estimate the Area Under the Curve (AUC) (ranging between 0 and 1), where a score of 1.0 indicated a perfect model and a score of 0.5 being considered no better than random chance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDiagnostic specificity of virus neutralization and ELISA kits\u003c/h2\u003e \u003cp\u003eTesting of the 200 serum samples collected prior to the HPAI H5N1 spillover into dairy cattle showed that all samples tested negative by the virus neutralization assay (200/200; 100%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Using the IDEXX ELISA, 200/200 (100%) samples also tested negative, corresponding to a diagnostic specificity of 100% and complete agreement with VN results. Using the IDVet ELISA, 199/200 (99.5%) samples tested negative, resulting in a diagnostic specificity of 99.5% and one discrepant result when compared with VN. Comparison between the IDEXX and IDVet ELISAs showed concordant negative results for 199/200 samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, testing of a panel of samples known to be antibody positive for influenza A viruses of several subtypes revealed no cross reactivity of antibodies against several H1, H3 and H7 viruses with the H5N1 B3.13 virus used in our VN assay. Importantly, only samples positive for H5N2-specific antibodies presented neutralizing antibodies to the H5N1 B3.13 virus used in our VN assay (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDiagnostic sensitivity of virus neutralization and ELISA kits\u003c/h2\u003e \u003cp\u003eAmong the 92 samples collected during the acute phase of clinical H5N1 virus outbreaks in dairy farms, 46 (50.0%) tested positive by the virus neutralization test (VNT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Using the IDvet ELISA, 53 (57.60%) samples tested positive, exceeding the number of VNT-positive samples. In contrast, the IDEXX ELISA detected 25 (27.17%) positives, fewer than with VNT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the 637 samples collected on day 60 post-clinical H5N1 outbreak, the VNT identified 570 (89.48%) positives, corroborating the high positivity observed with the IDvet ELISA, which detected 553 (86.8%) positives. The IDEXX ELISA detected markedly fewer positives, with only 204 (32%) samples testing positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAgreement between virus neutralization and ELISA kits\u003c/h2\u003e \u003cp\u003eThe agreement between VN and IDvet ELISA was the highest at 93.6%, while agreement between VN and IDEXX ELISA was much lower at 42.2%. Agreement between the two ELISAs themselves was 43.3% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eROC (Receiver Operating Characteristic) of VN and ELISA kits used in the present study.\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\u003eAssay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea Under the Curve (AUC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% Confidence interval\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0000\u0026ndash;1.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum ID Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.9933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9898\u0026ndash;0.9968\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum IDEXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.7419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01743\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7077\u0026ndash;0.7761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk ID Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.9517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9309 to 0.9724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk IDEXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5111 to 0.6208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01942\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eKappa coefficients (κ) further evaluated the agreement beyond chance: VN and IDvet ELISA showed substantial agreement (κ\u0026thinsp;=\u0026thinsp;0.695, p-value\u0026thinsp;=\u0026thinsp;0), VN and IDEXX ELISA showed slight agreement (κ\u0026thinsp;=\u0026thinsp;0.1, p-value\u0026thinsp;=\u0026thinsp;1.49e-08), and agreement between the two ELISAs was also slight (κ\u0026thinsp;=\u0026thinsp;0.124, p-value\u0026thinsp;=\u0026thinsp;3.15e-11) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCohen's Kappa coefficient comparison between serological assays performed in serum and milk samples.\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgreement (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKappa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVN vs. IDEXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSlight agreement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.49 \u0026times; 10⁻⁸\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVN vs. ID.Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstantial agreement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIDEXX vs. ID.Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSlight agreement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.15 \u0026times; 10⁻\u0026sup1;\u0026sup1;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMilk\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVN vs. IDEXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFair\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVN vs. ID.Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlmost perfect agreement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIDEXX vs. ID.Vet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFair\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRennet IDEXX vs. IDEXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0755\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRennet IDVet vs. IDVet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e92.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00395\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRennet IDEXX vs. VN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSlight agreement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.619\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRennet IDVet vs. VN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e89.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFair\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of H5N1 antibodies in milk\u003c/h2\u003e \u003cp\u003eFor detection of antibodies in milk we first evaluated the impact of rennet treatment, as this was required prior to VN testing due to toxicity of untreated milk samples to the indicator cells. We evaluated whether this treatment influenced the performance of the two ELISAs by testing subsets of 23 and 27 milk samples with each assay, both with and without rennet treatment.\u003c/p\u003e \u003cp\u003eFor the VN comparison, among rennet-treated milk samples, the IDvet ELISA showed high concordance with VN (89.3% agreement; κ\u0026thinsp;=\u0026thinsp;0.364; p\u0026thinsp;=\u0026thinsp;0.0126), indicating fair agreement. In contrast, the IDEXX ELISA exhibited much lower concordance with VN (30.8% agreement; κ\u0026thinsp;=\u0026thinsp;0.029; p\u0026thinsp;=\u0026thinsp;0.619), indicating slight agreement (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen assessing the impact of rennet treatment alone, the IDvet ELISA performance was largely unaffected, showing high agreement between treated and untreated samples (92.6% agreement; κ\u0026thinsp;=\u0026thinsp;0.471; p\u0026thinsp;=\u0026thinsp;0.00395). The IDEXX ELISA, however, showed poor stability following rennet treatment, with low agreement between treated and untreated milk samples (34.8% agreement; κ = \u0026minus;\u0026thinsp;0.332; p\u0026thinsp;=\u0026thinsp;0.0755) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall milk sample screening results further reflected these performance trends. Using VN, 868 (78.9%) samples tested negative and 233 (21.18%) tested positive. The IDvet ELISA identified 837 (76.09%) negatives, 236 (21.45%) positives, and 28 (2.54%) suspect samples. In comparison, the IDEXX ELISA classified 1,049 (95.36%) negatives, 39 (3.54%) positives, and 12 (1.09%) suspects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDetecting specific antibodies against the highly pathogenic avian influenza (HPAI) H5N1 virus in the serum of dairy cows represents a critical epidemiological tool, particularly considering the incursion and continuous circulation of clade 2.3.4.4b in this mammalian host. The findings of this study revealed substantial differences in diagnostic sensitivity and specificity between an in-house VNT and two commercial ELISA kits evaluated. The IDvet assay demonstrated markedly superior performance and stronger concordance with the virus neutralization test, a recognized gold standard for detecting neutralizing antibodies. These results underscore the need for targeted validation of diagnostic assays in non-conventional species such as cattle. Ensuring high diagnostic accuracy is essential for effective surveillance, timely outbreak detection, and the implementation of appropriate control measures.\u003c/p\u003e \u003cp\u003eResults from our comparative analysis indicate that the IDvet ELISA exhibits higher sensitivity than the IDEXX ELISA, during both the acute and convalescent phases of infection. A likely explanation for the low sensitivity observed with the IDEXX assay could be that the assay was developed and validated for use in avian species (e.g., chickens, ducks, geese, turkeys), and assay parameters such as target antigen and antibody concentrations were chosen based on the immune responses of a different non-mammalian species, potentially resulting in lower sensitivity to detect bovine antibodies (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Nevertheless, it is important to note that both ELISAs achieved high specificity during the acute phase, approaching or equaling 100%, a critical feature for minimizing false-positive results. However, the sensitivity of the VNT and ID.vet ELISA\u0026rsquo;s is a strong performance feature in favor of the use of these assays.\u003c/p\u003e \u003cp\u003eThis study also used Kappa analysis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) to assess the performance of the two ELISA assays in terms of specificity, sensitivity, percentage agreement, and concordance. The virus neutralization (VN) test showed good alignment with the hemagglutination inhibition (HI) assay (data not shown). The VN is a reliable and highly sensitive method, capable of serving as a strong comparator.\u003c/p\u003e \u003cp\u003eBuilding on the Kappa analysis, VNT was subsequently used as the reference standard for evaluating the ELISAs. The IDvet ELISA demonstrated substantial agreement with VNT (κ\u0026thinsp;=\u0026thinsp;0.695), indicating good diagnostic performance. In contrast, the IDEXX ELISA showed only slight agreement with VN (κ\u0026thinsp;=\u0026thinsp;0.1), reflecting poor alignment and raising concerns about the use of this kit in bovine samples due to is low sensitivity - particularly in samples with low antibody titers (\u0026lt;\u0026thinsp;128). Agreement between the two ELISA assays was also low (κ\u0026thinsp;=\u0026thinsp;0.124), despite both targeting anti-NP antibodies. These results highlight the markedly different diagnostic capabilities of the assays, with VNT showing greater reliability for diagnostics followed by the IDvet ELISA.\u003c/p\u003e \u003cp\u003eReceiver operating characteristic (ROC) analysis (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e) of the IDEXX ELISA indicated that the assay is highly specific - approximately 100% - across the evaluated range, with virtually no false positives. The positive likelihood ratio (LR) also increased alongside specificity, reinforcing that a positive result from this assay is a strong indicator of infection/previous exposure (Supplementary Table\u0026nbsp;1). However, the assay\u0026rsquo;s sensitivity was notably low. Using the manufacturer\u0026rsquo;s recommended cut-off (\u0026lt;\u0026thinsp;0.51, positive), sensitivity increased modestly to 34% without compromising specificity (Supplementary Table\u0026nbsp;1), yet this level still risks missing a substantial number of true positive cases. Furthermore, adjusting the cut-off to achieve a sensitivity of approximately 80% resulted in a dramatic reduction in specificity to 41.50% (\u0026lt;\u0026thinsp;0.92, positive), significantly increasing the potential for false positives (Supplementary Table\u0026nbsp;1). These findings highlight the trade-off between sensitivity and specificity in the IDEXX ELISA, particularly when applied to bovine samples.\u003c/p\u003e \u003cp\u003eIn milk samples, VNT detected antibody-positive cases at rates of approximately 21\u0026ndash;22%, serving as the benchmark for performance comparison. The IDvet ELISA showed close agreement with VNT, producing similar positivity rates and a comparable qualitative distribution of results. The IDEXX ELISA detected only 3.5% positives, which was lower than VNT, indicating reduced alignment in this matrix. This difference in milk samples was consistent with patterns observed in serum testing. Overall, these findings highlight VNT\u0026rsquo;s central role in accurately identifying H5N1 antibody\u0026ndash;positive samples, while illustrating how ELISA performance can vary depending on assay design and potentially, species-specific validation.\u003c/p\u003e \u003cp\u003eWhen examining the impact of rennet treatment, VNT results provided a robust baseline for comparison. The IDvet ELISA maintained high agreement with VNT regardless of treatment, suggesting stable performance in milk matrices. The IDEXX ELISA showed a decline in agreement following rennet treatment; however, this may reflect a combination of assay characteristics and matrix effects rather than solely the clarification step. Maintaining strong concordance with VNT under varying sample conditions is an important attribute for assays intended for field surveillance.\u003c/p\u003e \u003cp\u003eFrom a broader surveillance standpoint, assays that align closely with VNT can provide more accurate prevalence estimates and aid in detecting antibodies in animals with subclinical infections, especially after an outbreak. While differences in sensitivity and specificity were observed between the ELISAs, both demonstrated high specificity during acute infection, minimizing false positives. Accurate detection is particularly important given the zoonotic nature of the H5N1 virus, which poses potential risks to rural workers and those in the milk production chain. These findings emphasize the value of validated serological tools that perform well against standard methods like VNT to ensure reliable assessment of herd exposure and human infection risk.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the producer and veterinarian Dr. William Leone for allowing sample collection in his farm. The authors also thank Dr. Dennis Summers and his staff and veterinarians, and Lauren E. Meyer for their help with sample collection. The study was funded in part by the Cornell University Animal Health Diagnostic Center Virology Laboratory activity funds.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCaserta LC, Frye EA, Butt SL, Laverack M, Nooruzzaman M, Covaleda LM, Thompson AC, Prarat Koscielny M, Cronk B, Johnson A, Kleinhenz K, Edwards EE, Gomez G, Hitchener G, Martins M, Kapczynski DR, Suarez DL, Alexander Morris ER, Hensley T, Beeby JS, Lejeune M, Swinford AK, Elvinger F, Dimitrov KM, Diel DG (2024) Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. 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Nature 634:669\u0026ndash;676\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen J (1960) A Coefficient of Agreement for Nominal Scales. Educ Psychol Meas 20:37\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcHugh ML (2012) Interrater reliability: the kappa statistic. Biochem Med (Zagreb) 22:276\u0026ndash;282\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLudi AB, Morris A, Gubbins S, Asfor A, Afzal M, Browning CF, Grazioli S, Foglia EA, Wilsden G, Burman A, Brocchi E, Paton DJ, King DP (2022) Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus. Viruses 14:1495\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Cornell University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9163114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9163114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSince the emergence of highly pathogenic avian influenza (HPAI) H5N1 virus in the United States in 2022, it has caused the deaths of millions of wild and domestic birds. In early 2024, HPAI H5N1 spilled over into dairy cattle in Texas and has since been detected in multiple states, raising concerns about cross-species transmission and zoonotic risk. We assessed the feasibility of serological assays for antibody responses to HPAI H5N1 in dairy cattle by comparing two competitive commercial ELISA assays with a virus neutralization (VN) assay. Samples, including serum and milk matrices, were collected during the acute phase of confirmed outbreaks and 60 days post-outbreak. Results were analyzed for sensitivity, specificity, concordance, and agreement using Kappa statistics and receiver operating characteristic (ROC) analysis. Comparisons to VN revealed substantial agreement for the IDvet ELISA in both serum and milk, while the IDEXX ELISA showed high specificity but lower sensitivity, particularly for low-titer samples. Rennet treatment of milk, required for the VN test, did not affect IDvet performance but reduced agreement for IDEXX. Seroprevalence estimates based on VN and IDvet were similar, indicating consistent detection of exposed animals. In contrast, IDEXX underestimated the number of positives, which may limit surveillance accuracy. These findings emphasize the importance of assay validation in non-conventional species and highlight VN and high-performing ELISAs, as valuable tools for post-outbreak surveillance in cattle. Given the zoonotic potential of H5N1, reliable serological testing is essential for monitoring livestock exposure and assessing risks to human health.\u003c/p\u003e","manuscriptTitle":"Comparison of serological assays to monitor exposure to highly pathogenic avian influenza H5N1 virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 06:43:11","doi":"10.21203/rs.3.rs-9163114/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"154735ab-411e-4ba0-9db5-562deb68edb0","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-20T06:43:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 06:43:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9163114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9163114","identity":"rs-9163114","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}