This statistic shows the usage of eggs in the United States in 2020. The data has been calculated by Statista based on the U.S. Census data and Simmons National Consumer Survey (NHCS). According to this statistic, ****** million Americans used eggs in 2020.

The demand of eggs has grown in the United States over the last number of years. In 2023, consumption of eggs in the United States was estimated at 281.3 per person. This figure was projected to reach 284.4 eggs per capita by 2024. Per capita consumption is a measure of total egg production, minus exports, divided by the total U.S. population.

Cage-free and organic eggs Although the majority of laying hens in the Unites States are still caged, the production of organic and caged-free hens has increased in recent years. In cage-free production, hens are allowed to move freely outside of their cage, but this time is limited and the environment they are kept in could still be unhealthy and very crowded. In organic production however, hens are provided with free-range outdoor access.

U.S. egg industry There has been steady growth in the number of eggs produced in the United States. Additionally, the total number of laying hens in the United States has also increased in recent years. Iowa was the U.S. state with the most laying hens, with some 40.16 million laying hens as of 2022.

This statistic shows the amount of eggs used within one month in the United States in 2020. The data has been calculated by Statista based on the U.S. Census data and Simmons National Consumer Survey (NHCS). According to this statistic, ***** million Americans used * dozen or more in 2020.

Imports of Daily Products & Eggs in the United States increased to 342.79 USD Million in February from 311.05 USD Million in January of 2024. This dataset includes a chart with historical data for the United States Imports of Daily Products & Eggs.

This statistic shows the total egg production in the United States from 2001 to 2023. In 2023, around 9.1 billion dozen of eggs were produced, a slight decrease from production the previous year.

This statistic shows the amount of eggs used within ** days in the United States from 2011 to 2020. The data has been calculated by Statista based on the U.S. Census data and Simmons National Consumer Survey (NHCS). According to this statistic, ***** million Americans used * or more dozens of eggs in 2020.

Very few data exist globally regarding the use of antimicrobials in the table egg industry. Antimicrobial use data from broiler chickens and turkeys cannot be used as a surrogate of layer chickens because of the fact that table eggs for human consumption are produced daily by laying hens. To avoid the possibility of antimicrobial residues in the eggs, there are very few antimicrobials approved for use in layers in the U.S. The objective of this study was to collect on-farm antimicrobial use data from the U.S. table egg industry and to have it be representative of the national layer flock. Participation was voluntary. Data were collected for the period 2016 through 2021 and are reported on a calendar year basis. Using production statistics from USDA:NASS as a denominator, the data supplied by participating companies accounted for 3,016,183,140 dozen eggs (~40% of national egg production) in 2016 and 3,556,743,270 dozen eggs (~45% of national egg production) in 2021. All of the replacement chicks placed on pullet farms during the study period were estimated to have received 0.2 mg/chick gentamicin at the hatchery. Most of the antimicrobial administration in U.S. egg production is via the feed. The ionophores monensin and salinomycin were used in the pullets, bacitracin was used in both pullets and layers (primarily for control of necrotic enteritis), and chlortetracycline was used primarily in layers for the treatment of E. coli-related disease. In the layers, between 0.10 and 0.19% of total hen-days were exposed to chlortetracycline. Only two water-soluble administrations were recorded during the entire study period, both involving lincomycin to pullet flocks for the treatment of necrotic enteritis. Overall, antimicrobial use in the U.S. layer industry was focused mainly on controlling necrotic enteritis in the pullets and treating E. coli-related disease in the laying hens.

This statistic shows the total egg production in the United States from 2000 to 2024. In 2024, about 108.5 billion eggs were produced in the United States, a decrease from the previous year.

The Fluvial Egg Drift Simulator (FluEgg) estimates bighead, silver, and grass carp egg and larval drift in rivers using species-specific egg developmental data combined with user-supplied hydraulic inputs (Garcia and others, 2013, Domanski, 2020). This data release contains results from 240 FluEgg 4.1.0 simulations of bighead carp eggs in the Illinois River under steady flow conditions. The data release also contains the hydraulic inputs used in the FluEgg simulations and a KML file of the centerline that represents the model domain. FluEgg simulations were run for all combinations of four spawning locations, six water temperatures, and ten steady flow conditions. Each simulation included 5,000 bighead carp eggs, which develop and eventually hatch into larvae. The simulations end when the larvae reach the gas bladder inflation stage. The four spawning locations were just downstream of the lock and dam structures at Marseilles, Starved Rock, Peoria, and LaGrange. For each of these spawning locations, the eggs were assumed to have been spawned at the water surface and at the midpoint of the channel. The six water temperatures were 18, 20, 22, 24, 26, and 28 degrees Celsius. The ten steady flow conditions ranged from half the annual mean flow to the 500-year peak flow and are discussed in more detail below. Note that in the streamwise coordinate system used by FluEgg, the streamwise coordinate of the Mississippi River confluence is 396,639 meters. Any drift distances greater than this value should be excluded from any further analysis of this data. The hydraulic inputs for the FluEgg simulations were generated using a one-dimensional steady Hydrologic Engineering Center-River Analysis System (HEC-RAS) 5.0.7 model for the Illinois River between Marseilles Lock and Dam and the Mississippi River confluence near Grafton, Illinois (HEC-RAS, 2019). The HEC-RAS model was developed by combining four individual HEC-RAS models obtained from the U.S. Army Corps of Engineers Rock Island District (U.S. Army Corps of Engineers Rock Island District, 2003). The model was run for the following ten flow profiles: half the annual mean flow, annual mean flow, annual mean flood, 2-year peak flow, 5-year peak flow, 10-year peak flow, 25-year peak flow, 50-year peak flow, 100-year peak flow, and 500-year peak flow. The flow rates for each of the profiles were obtained for the following U.S. Geological survey (USGS) streamgaging stations from USGS StreamStats: 5543500 Illinois River at Marseilles, Illinois, 5558300 Illinois River at Henry, Illinois, 5560000 Illinois River at Peoria, Illinois, 5568500 Illinois River at Kingston Mines, Illinois, 5570500 Illinois River near Havana, Illinois, 5585500 Illinois River at Meredosia, Illinois, 5586100 Illinois River at Valley City, Illinois (Soong and others, 2004; Granato and others, 2017). Garcia, T., Jackson, P.R., Murphy, E.A., Valocchi, A.J., Garcia, M.H., 2013. Development of a Fluvial Egg Drift Simulator to evaluate the transport and dispersion of Asian carp eggs in rivers. Ecol. Model. 263, 211–222, https://doi.org/10.1016/j.ecolmodel.2013.05.005. Granato G.E., Ries, K.G., III, and Steeves, P.A., 2017, Compilation of streamflow statistics calculated from daily mean streamflow data collected during water years 1901–2015 for selected U.S. Geological Survey streamgages: U.S. Geological Survey Open-File Report 2017–1108, 17 p., https://doi.org/10.3133/ofr20171108. Domanski, M.M., Berutti, M.C., 2020, FluEgg, U.S. Geological Survey software release, https://doi.org/10.5066/P93UCQR2. Hydrologic Engineering Center-River Analysis System (HEC-RAS), 2019, accessed August 20, 2020, at http://www.hec.usace.army.mil/software/hec-ras/. Soong, D.T., Ishii, A.L., Sharpe, J.B., and Avery, C.F., 2004, Estimating flood-peak discharge magnitudes and frequencies for rural streams in Illinois: U.S. Geological Survey Scientific Investigations Report 2004–5103, 147 p., https://doi.org/10.3133/sir20045103. U.S. Army Corps of Engineers Rock Island District, 2004, Upper Mississippi River System Flow Frequency Study, Hydrology and Hydraulics, Appendix C, Illinois River, accessed August 20, 2020, at https://www.mvr.usace.army.mil/Portals/48/docs/FRM/UpperMissFlowFreq/App.%20C%20Rock%20Island%20Dist.%20Illinois%20River%20Hydrology_Hydraulics.pdf.

During 2010-2014, tree swallow (Tachycineta bicolor) reproductive success was monitored at 68 sites across all 5 Great Lakes, including 58 sites located within Great Lakes Areas of concern (AOCs) and 10 non-AOCs. Sample eggs were collected from tree swallow clutches and analyzed for contaminants including polychlorinated biphenyls (PCBs), dioxin and furans, polybrominated diphenyl ethers, and 34 other organic compounds. Contaminant data were available for 360 of the 1249 clutches monitored. Markov chain multistate modeling was used to assess the importance of 5 ecological and 11 of the dominant contaminants in explaining the pattern of egg and nestling failure rates. Four of 5 ecological variables (female Age, Date within season, Year, and Site) were important explanatory variables. Of the 11 contaminants, only total dioxin and furan toxic equivalents (TEQs) explained a significant amount of the egg failure probabilities. Neither total PCBs nor PCB TEQs explained the variation in egg failure rates. In a separate analysis, polycyclic aromatic hydrocarbon exposure in nestling diet was significantly correlated with the daily probability of egg failure. The eight sites within AOCs which had poorer reproduction when compared to 9 non-AOC sites, the measure of impaired reproduction as define by the Great Lakes Restoration Initiative, were associated with exposure to dioxins and furans, PAHs, or depredation. Only 2 sites had poorer reproduction than the poorest performing non-AOC. Using a classic (non-modeling) approach to estimating reproductive success, 82% of nests hatched at least 1 egg, and 75% of eggs hatched.

Pheno Forecast maps predict key life cycle stages in a range of species to improve conservation and management outcomes. For insect pest species, Pheno Forecasts are based on growing degree day (GDD) thresholds for key points in species life cycles. These key points typically represent life cycle stages when management actions are most effective. Emerald ash borer is a beetle that causes significant harm to ash trees throughout the eastern United States.The adult emergence and egg hatch forecasts for emerald ash borer was developed by Oregon State University, using the Degree-Days, Risk, and Phenological event mapping (DDRP) platform. The adult emergence model predicts the earliest date that overwintering individuals are predicted to emerge as adults. This event is predicted at 391 growing degree days (F) (lower threshold: 54F, upper threshold: 97F, method: single sine, start date: Jan 1). The egg hatch model predicts the earliest date that overwintering eggs are predicted to hatch. This event is predicted at 830 growing degree days (F) (lower threshold: 54F, upper threshold: 97F, method: single sine, start date: Jan 1). The forecast is available for the full calendar year. Temperature inputs are drawn from 3 data sources, as follows: PRISM data are used from Jan 1 through the current day; North American Multi-Model Ensemble (NMME) data are used from the current day through 7 months in the future and the most recent PRISM 10 year normal data are used for dates more than 7 months in the future. The model excludes climatically unsuitable locations for EAB using the current year's temperature data described above. Further information is available at USPest.org/CAPS. Forecasts are available for the current and prior year in the USA-NPN Visualization Tool (https://www.usanpn.org/data/visualizations). Emerald ash borer is a beetle that causes significant harm to ash trees throughout the eastern United States.For all inquiries regarding this dataset, please contact the USA-NPN. This data is subject to the USA-NPN's Data Use Policy.

This metadata record represents passive immunity components of six songbird species (Passeriformes) in a single taxonomic family, the New World blackbirds (Icteridae). Six immune elements were compared among blackbird eggs collected in North Dakota, South Dakota, and Puerto Rico, including immunoglobulins (Ig) in both yolk and albumen, lipopolysaccharide-specific immunoglobulins in both yolk and albumen, and ovotransferrin and lysozyme in albumen. The data were summarized and used in the analysis for a peer-reviewed journal publication entitled: "Do life history traits influence patterns of maternal immune elements in New World blackbirds (Icteridae)?" The data consist of two data sets. The first data set includes day, year, and location (county or municipality, U.S. State or Territory) of egg collection, a species code, a ratio between positive and negative values of immunoglobulin in egg yolk, and a ratio between positive and negative values of lipopolysaccharide-specific immunoglobulins in egg yolk. The second data set includes day, year, and location (county or municipality, U.S. State or Territory) of egg collection, a species code, a ratio between positive and negative values of immunoglobulin in egg albumen, a ratio between positive and negative values of lipopolysaccharide-specific immunoglobulins in egg albumen, concentration of lysozyme (an antimicrobial enzymen) in egg albumen, and concentration of ovotransferrin (an antimicrobial enzymen) in egg albumen. Both data sets also include information on parasitism rates in the northern Great Plains between 1990 to 2015 (L.D. Igl, unpublished data) and average incubation periods for blackbird species from the literature.

This dataset comprises transcripts of interviews generated through the Curious Connections study, which explores the impact of donating egg or sperm for donors and their relatives. The research focusses on meanings and experiences of donating in the context of increased openness in the context of UK donor conception, including the removal of donor anonymity for new donors since 2005. As part of this project, we interviewed 52 donors (half men, half women) and 23 relatives of donors (partners, parents and siblings). In addition, we analysed UK laws and policies which impact donors and interviewed 18 members of staff who work with donors in UK clinics. The project was approved by the University of Manchester Research Ethics Committee.In a culture that emphasises the importance of genetic connectedness and which holds that vital and enduring family relationships pass through genetic reproduction, the decision to give away one's eggs or sperm is radical indeed. This is however something that is becoming increasingly common as more people struggle with issues of infertility, and the fertility industry is growing. Questions arise about how donors experience the process of donating and how that process impacts on their everyday lives and relationships. Whereas, in the past, donors were anonymous and so could choose to keep their donation a secret from close kin, recent legal changes mean that donors are now identifiable. In 2023, the first children born through 'identity release' egg or sperm donors will be able to seek contact. Donors nowadays are therefore likely to deliberate on how to manage such openness and contact within the context of their own relationships with partners, parents and their own children. Being open may not be a straightforward task. This is a sister project to our previous study Relative Strangers (funded by the ESRC 2010-2013, C Smart PI, P Nordqvist, CI) which explored family life after receiving donor egg and sperm. This showed the impact of donor conception on family relationships and raised unforeseen questions about donors, suggesting that giving away (as opposed to receiving) egg and sperm may impact on family relationships in significant ways. Proposed study and research focus: The Curious Connections study explored how donating impacts on donors' everyday lives and relationships. The study: 1) analysed the policy on donation and the rights and obligations of donors 2) examined how egg and sperm donors negotiate donation within the context of their everyday lives and relationships, and also if and how they share information with close kin 3) explored how close kin react to and experience the existence of donor offspring 4) investigated the similarities and difference between male and female donors' experiences 5) considers what the practice of sperm/egg donation as a practice can tell us about contemporary kinship and family cultures Approach and methods: This study took the donor as a starting point, but in order to better understand the process and family dynamics that underscore donors' experience, it looked beyond the donor and situates donating within the context of their close relationships. We utilised a qualitative approach, enabling us to explore donors' experiences and understandings. We analysed existing policy on donors and conducted 88 qualitative semi-structured interviews with 52 donors (26 with egg donors, ,26 sperm donors and 1 embryo donor), 23 close kin (partners, parents and siblings) and 18 fertility counsellors or donor coordinators. Beneficiaries and impact: A range of policy makers, practitioners, user groups, users and the general public stand to benefit from this study. Sought outcomes include increasing the effectiveness of policy and better targeted practical support and information offered to donors and families by donor conception. The impact will be realised through a range of pathways, e.g. a conference, a public debate, a podcast, blogs, films and through the production of findings-based user group leaflets. Academic communities also stand to benefit from this research, including those in the field of reproductive technologies and reproduction more broadly, sociologists of family life and intimacy, and medical sociologists. An exploratory qualitative methodology was used with the aim of understanding the practices and processes through which donation is made meaningful in people’s lives. This document details the methods used in relation to interviews with donors, donors’ relatives and clinic staff. Please note that, due to the limitations of anonymization in relation to unusual cases and non-consent from some participants, some transcripts produced as part of this project have not been archived with the UKDS. In addition, a number of the transcripts and further details of the cases are available only following request, and after review by, the PI. This report provides information about the whole sample with the aim of providing some context to the archived data. Interviews with donors took place during 2018 and 2019. They usually lasted between 90 and 120 minutes (ranging from 35 to 160 minutes of audio recorded conversation) and took place either in participants’ homes, a public place (such as a café) or an office (usually at the donor’s workplace but on two occasions at the University). We took an in-depth, loosely structured approach to interviewing donors, beginning with a variation on the request, ‘tell me how you became a donor?’ Interviewers then encouraged participants to tell their ‘donation stories’ in their own words, focussing on the topics they considered most important. A topic guide was used to probe for areas of particular interest and ensure topics of interest, not spontaneously raised, were covered. Such topics included: talking to others about their donation, thoughts about the possibility of future (or ongoing) contact with recipient families, experiences of the process of donation, finding out (or not) about the outcome of the donation. Interviews with donor relatives and with clinic staff (counsellors and donor coordinators) were often shorter (averaging approximately an hour in both cases) and slightly more structured. For practical reasons, a minority of the interviews with clinic staff and donor relatives were conducted via telephone, at the request of participants. Interviews with family members began by asking how they first found out their relative was considering donating/had donated, how that had been presented to them and what their reaction had been. As well as asking relatives to recount their relative’s ‘donation story’, we also asked who they had told about the donation and their thoughts about the future. Interviews with fertility counsellors and donor coordinators focussed on their work with donors. We asked about their aims and approach in the work they (and their organisations) did with donors and tried to establish the kinds of topics that these professionals covered in their conversations with them and the reasons these were considered important. In addition, we asked questions about their impressions of donors they had worked with e.g. what kinds of people came forward to donate, what were their reasons for doing so, how did they respond to the possibility of being traced and what kinds of issues or questions did they generally raise. In total we conducted 88 interviews including 52 interviews with donors, 23 with donors’ relatives and 18 with infertility counsellors or donor coordinators. Five donors were also partners of donors, hence the numbers for each group do not total 88. Further details on our sample and recruitment are included in the supporting documents.

Data were collected during July-September in 1981 through 1985 and during June-September 2002 through 2004. Mark-release-recapture was done for adult butterflies, and daily survival rate estimated for females using Jolly-Seber methods. Egg clusters were counted, along with number of eggs per cluster. Data were collected each year on egg cluster survival, and in 2004 on larval survival conditional on egg cluster survival. The data were used to construct yearly vital rates, including daily probability of laying an egg cluster, cluster size, survival from egg to diapause, survival over-winter to adult stage, and female daily survival probability.

Population sizes were decreasing during nearly all years included in the data set, although a 1-2 order of magnitude increase in size occured ca. 2000-2, which is not included here.

United States Imports from Canada of Dairy products, eggs, honey, edible products was US$324.96 Million during 2024, according to the United Nations COMTRADE database on international trade. United States Imports from Canada of Dairy products, eggs, honey, edible products - data, historical chart and statistics - was last updated on September of 2025.

Viruses. All procedures using infectious material were reviewed and approved by the Institutional Biosafety Committee of US National Poultry Research Center (USNPRC), US Department of Agriculture-Agricultural Research Service, Athens, GA. The HPAI virus isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) was provided by Dr. Mia Torchetti, National Veterinary Services Laboratories, US Department of Agriculture-Animal and Plant Health Inspection Service, Ames, IA. The A/Vietnam/1203/2004 H5N1 HPAI virus (Viet/04), A/Whooper Swan/Mongolia/244/2005 H5N1 (WS/Mongolia/05) HPAI virus, and A/Flycatcher/CA/14875-1/1994 H7N1 low pathogenic avian influenza virus isolates were provided by the repository at the USNPRC. Virus isolates were propagated and titrated in SPF embryonating chicken eggs using standard procedures [1]. Titers were determined using the Reed-Muench method [2].Vaccines. Two commercial rHVT-H5 vaccines were selected because they are licensed in the US (and may be licensed elsewhere) and were supplied by the manufacturers: 2.2-HVT (Vectormune HVT AIV, Ceva Animal Health LLC, Lenexa, KS) (serial 395-134); and COBRA-HVT (Vaxxitek HVT+IBD+H5, Boehringer-Ingelheim Animal Health USA, Ridgefield, CT) (serial EW003). The amino acid similarity between the vaccine antigens and the challenge virus HA1 was 91.7% (COBRA-HVT) and 91.2% (2.2-HVT).Challenge study design. All animal work was reviewed and approved by the USNPRC Institutional Animal Care and Use Committee. Mixed sex, SPF WL chickens (Gallus gallus domesticus) were obtained at hatch from in-house flocks. Broiler chicken eggs were obtained from a commercial hatchery at 18 days of incubation prior to administration of any in ovo vaccines and were hatched on-site. All birds were randomly assigned to vaccine groups based on breed. Vaccine groups are shown in Table 1. All vaccines were prepared and administered on the day of hatch by the subcutaneous route at the nape of the neck in accordance with the manufacturer’s instructions (0.2ml per chicken). Serum was collected from all chickens 25 days post vaccination to evaluate the antibody response to the vaccines.Four weeks post vaccination (four weeks of age) chickens were challenged with a target dose 6.0log10 50% egg infectious doses (EID50) per bird of TK/IN/22 in 0.1ml by the intrachoanal route (titration of the challenge virus after dilution confirmed the challenge dose to be 6.7log10 EID50 per bird). Oropharyngeal and CL swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized birds.To evaluate antibody-based DIVA-VI tests, serum was collected at 7-, 10- and 14DPC. Mortality and morbidity were recorded for 14DPC. Surviving birds were euthanized at 14DPC. If birds were severely lethargic or presented with neurological signs, they were euthanized and were counted as mortality at the next observation time for mean death time calculations. Euthanasia was performed by cervical dislocation in accordance with American Veterinary Medical Association guidelines.Quantitative rRT-PCR (qRRT-PCR). RNA was extracted from OP and CL swabs using the MagMax magnetic bead extraction kit (Thermo Fisher Scientific, Waltham, MA) with the wash modifications as described by Das et al., [3]. Quantitative real-time RT-PCR was conducted as described previously [4] on a QuantStudio 5 (Thermo Fisher Scientific) instrument. A standard curve was generated from a titrated stock of TK/IN/22 and was used to calculate titer equivalents using the real time PCR instrument’s software.Hemagglutination inhibition assay. Hemagglutination inhibition (HI) assays were run in accordance with standard procedures [5]. All pre-challenge sera collected at 25 days post vaccination were tested against the challenge virus and the closest isolates available to the vaccine antigens. The serum from the 2.2-HVT group was tested against WS/Mongolia/05 (99.3% similarity) and the serum from the COBRA-HVT group was tested against Viet/04 (98.2% similarity). Titers of eight or below were considered negative.Commercial ELISA. A commercial AIV antibody ELISA (AI Ab Test, IDEXX laboratories, Westbrook, ME) was used in accordance with the manufacturer’s instructions. Sera were tested to detect anti-NP antibodies pre-challenge (25days pos-vaccination) and at 7-, 10- and 14DPC.Enzyme-linked lectin assay (ELLA) for detection of neuraminidase inhibition (NI) antibody. The ELLA was performed as previously described with minor modifications [6, 7]. Briefly, the NA activity of a beta-propiolactone inactivated H7N1 virus (A/Flycatcher/CA/14875-1/1994) was quantified to determine the effective concentration (EC) of antigen. The 98% EC (EC98) of antigen was subsequently used for the ELLA-NI assays. For ELLA-NI assay, the antigen and serum mixture was incubated overnight (approximately18hr) at 37°C and the NA activity was determined following the procedure as described in Spackman et al. [7]. The average background absorbance value was subtracted from the sample absorbance value then that value was divided by the average values of wells with only NA antigen. This value was multiplied by a factor of 100 to calculate the percent NA activity. The percent NI activity of individual serum samples was determined by subtracting the percent NA activity from 100%. A cut-off value for positive NI activity was determined by adding three standard deviations to the mean NI activity of pre-challenge sera (i.e., NA antibody negative sera) of each corresponding group of chickens at 7-, 10- and 14DPC. Each serum was tested at dilutions of 1:20 and 1:40.

Molecular genetics makes it possible to measure basic, but long elusive parameters of the breeding biology of the Brown-headed Cowbird (Molothrus ater). Cowbird fecundity and host selection behavior were examined using a combination of molecular genetic techniques to link female cowbirds to the eggs they lay, radio-telemetry techniques to track female cowbirds' daily movements, and geographic information systems (GIS) to integrate these genetic and spatial data. The study site lies within a forested 1300 ha landscape in New York composed primarily of mature forest with adjacent old fields. It was found that female cowbirds used their home ranges as principal egg-laying areas. Individual females used characteristic individual home ranges throughout the breeding season, and they returned to the same home range every breeding season. Over one-half (54%) of females laid all their eggs in host nests inside or close to their home range. Proximity to a females' home range was the only significant ecological or biological feature affecting a cowbird's host.

Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both breeds in the COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken breeds. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both breeds of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.MethodsViruses. All procedures using infectious material were reviewed and approved by the Institutional Biosafety Committee of US National Poultry Research Center (USNPRC), US Department of Agriculture-Agricultural Research Service, Athens, GA. The HPAI virus isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) was provided by Dr. Mia Torchetti, National Veterinary Services Laboratories, US Department of Agriculture-Animal and Plant Health Inspection Service, Ames, IA. The A/Vietnam/1203/2004 H5N1 HPAI virus (Viet/04), A/Whooper Swan/Mongolia/244/2005 H5N1 (WS/Mongolia/05) HPAI virus, and A/Flycatcher/CA/14875-1/1994 H7N1 low pathogenic avian influenza virus isolates were provided by the repository at the USNPRC. Virus isolates were propagated and titrated in SPF embryonating chicken eggs using standard procedures [1]. Titers were determined using the Reed-Muench method [2].Vaccines. Two commercial rHVT-H5 vaccines were selected because they are licensed in the US (and may be licensed elsewhere) and were supplied by the manufacturers: 2.2-HVT (Vectormune HVT AIV, Ceva Animal Health LLC, Lenexa, KS) (serial 395-134); and COBRA-HVT (Vaxxitek HVT+IBD+H5, Boehringer-Ingelheim Animal Health USA, Ridgefield, CT) (serial EW003). The amino acid similarity between the vaccine antigens and the challenge virus HA1 was 91.7% (COBRA-HVT) and 91.2% (2.2-HVT).Challenge study design. All animal work was reviewed and approved by the USNPRC Institutional Animal Care and Use Committee. Mixed sex, SPF WL chickens (Gallus gallus domesticus) were obtained at hatch from in-house flocks. Broiler chicken eggs were obtained from a commercial hatchery at 18 days of incubation prior to administration of any in ovo vaccines and were hatched on-site. All birds were randomly assigned to vaccine groups based on breed. Vaccine groups are shown in Table 1. All vaccines were prepared and administered on the day of hatch by the subcutaneous route at the nape of the neck in accordance with the manufacturer’s instructions (0.2ml per chicken). Serum was collected from all chickens 25 days post vaccination to evaluate the antibody response to the vaccines.Four weeks post vaccination (four weeks of age) chickens were challenged with a target dose 6.0log10 50% egg infectious doses (EID50) per bird of TK/IN/22 in 0.1ml by the intrachoanal route (titration of the challenge virus after dilution confirmed the challenge dose to be 6.7log10 EID50 per bird). Oropharyngeal and CL swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized birds.To evaluate antibody-based DIVA-VI tests, serum was collected at 7-, 10- and 14DPC. Mortality and morbidity were recorded for 14DPC. Surviving birds were euthanized at 14DPC. If birds were severely lethargic or presented with neurological signs, they were euthanized and were counted as mortality at the next observation time for mean death time calculations. Euthanasia was performed by cervical dislocation in accordance with American Veterinary Medical Association guidelines.Quantitative rRT-PCR (qRRT-PCR). RNA was extracted from OP and CL swabs using the MagMax magnetic bead extraction kit (Thermo Fisher Scientific, Waltham, MA) with the wash modifications as described by Das et al., [3]. Quantitative real-time RT-PCR was conducted as described previously [4] on a QuantStudio 5 (Thermo Fisher Scientific) instrument. A standard curve was generated from a titrated stock of TK/IN/22 and was used to calculate titer equivalents using the real time PCR instrument’s software.Hemagglutination inhibition assay. Hemagglutination inhibition (HI) assays were run in accordance with standard procedures [5]. All pre-challenge sera collected at 25 days post vaccination were tested against the challenge virus and the closest isolates available to the vaccine antigens. The serum from the 2.2-HVT group was tested against WS/Mongolia/05 (99.3% similarity) and the serum from the COBRA-HVT group was tested against Viet/04 (98.2% similarity). Titers of eight or below were considered negative.Commercial ELISA. A commercial AIV antibody ELISA (AI Ab Test, IDEXX laboratories, Westbrook, ME) was used in accordance with the manufacturer’s instructions. Sera were tested to detect anti-NP antibodies pre-challenge (25days pos-vaccination) and at 7-, 10- and 14DPC.Enzyme-linked lectin assay (ELLA) for detection of neuraminidase inhibition (NI) antibody. The ELLA was performed as previously described with minor modifications [6, 7]. Briefly, the NA activity of a beta-propiolactone inactivated H7N1 virus (A/Flycatcher/CA/14875-1/1994) was quantified to determine the effective concentration (EC) of antigen. The 98% EC (EC98) of antigen was subsequently used for the ELLA-NI assays. For ELLA-NI assay, the antigen and serum mixture was incubated overnight (approximately18hr) at 37°C and the NA activity was determined following the procedure as described in Spackman et al. [7]. The average background absorbance value was subtracted from the sample absorbance value then that value was divided by the average values of wells with only NA antigen. This value was multiplied by a factor of 100 to calculate the percent NA activity. The percent NI activity of individual serum samples was determined by subtracting the percent NA activity from 100%. A cut-off value for positive NI activity was determined by adding three standard deviations to the mean NI activity of pre-challenge sera (i.e., NA antibody negative sera) of each corresponding group of chickens at 7-, 10- and 14DPC. Each serum was tested at dilutions of 1:20 and 1:40.References.1. Spackman E, Killian ML. Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs. Methods Mol Biol. 2020;2123:149-64. Epub 2020/03/15.2. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. American Journal of Hygiene. 1938;27:493-7.3. Das A, Spackman E, Pantin-Jackwood MJ, Suarez DL. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR. Journal of Veterinary Diagnostic Investigation. 2009;21(6):771-8.4. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Journal of Clinical Microbiology. 2002;40(9):3256-60.5. Spackman E, Sitaras I. Hemagglutination Inhibition Assay. Methods Mol Biol. 2020;2123:11-28. Epub 2020/03/15.6. Bernard MC, Waldock J, Commandeur S, Strauss L, Trombetta CM, Marchi S, et al. Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study. Front Immunol. 2022;13:909297. Epub 20220617.7. Spackman E, Suarez DL, Lee CW, Pantin-Jackwood MJ, Lee SA, Youk S, Ibrahim S. Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens. Vaccine. 2023. Epub 20231104.