Sample collection and postmortem evaluation
Bald eagle carcasses, and/or oropharyngeal and cloacal swabs were collected in the field and submitted to the Southeastern Cooperative Wildlife Disease Study Research and Diagnostic Service. In some cases, live bald eagles were found moribund and transported to wildlife rehabilitation clinics and either died in transit or soon after arrival. Carcasses underwent postmortem evaluation, including gross and histopathology. Tissue samples [heart, brain, kidney, spleen, lung, adrenal gland, pancreas, liver, small and large intestine, and cloacal bursa (if present)] were fixed in 10% neutral buffered formalin and routinely processed for histopathology23 at the Athens Veterinary Diagnostic Laboratory. Histopathology was assessed by a board-certified veterinary pathologist.
Additional bald eagle and waterfowl species mortality data
Data on wild bird deaths attributed to highly pathogenic influenza A viruses were retrieved from the U.S. Department of Agriculture, Animal and Plant Health Inspection Service website, at: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022/2022-hpai-wild-birds. These data are publicly available and include state, county, date detected, and species of individual birds that tested positive for HP IAV.
Immunohistochemistry
Immunohistochemistry (IHC) for avian influenza virus was performed in select cases on brain, pancreas, spleen, liver, and/or adrenal gland at the Athens Veterinary Diagnostic Laboratory. IHC was performed on an automated stainer (Nemesis 3600, Biocare Medical). Polyclonal antiserum against influenza A virus was used as the primary antibody (ab155877, Abcam), diluted 1:3000, and incubated for 60 min at 37 °C with agent-positive control. Antigen retrieval was with Target Retrieval Solution (S2367, Dako) pH (10x) at 110 °C for 15 min. Enzyme blockage was via 3% H2O2 for 20 min (H324-500, Fisher Scientific); protein blockage was with Universal Blocking Reagent (10x) Power Block diluted at 1:10 for 5 min (HK085-5 K, BioGenex); link was by biotinylated rabbit anti-goat (BA-5000, Vector) at a 1:100 dilution for 10 min with 4 + streptavidin alkaline phosphatase label for 10 min (AP605H, BioCare Medical). Staining was with warp red chromogen kit for 5 min (WR8065, BioCare Medical). Known influenza A-virus positive control tissues were tested alongside each case.
Polymerase chain reaction
Oropharyngeal and cloacal swabs from bald eagle carcasses were pooled for each individual eagle and tested by real-time reverse transcription polymerase chain reaction (rRT-PCR). Briefly, swabs samples were extracted with the KingFisher magnetic particle processer using the MagMAX-96 AI/ND Viral RNA isolation Kit (Ambion/Applied Biosystems, Foster City, CA) following a modified MagMAX-S protocol24. Resultant nucleic acids were screened against primers specific for H5 IAV in rRT-PCR; samples that yielded a cycle threshold value < 40 were submitted to the National Veterinary Services Laboratory, Ames, Iowa for confirmation of H5 highly pathogenic influenza A virus.
Bald eagle nest monitoring in Georgia, USA
From 2015 to 2017 and in 2022, the Georgia Department of Natural Resources conducted annual statewide surveys, primarily by helicopter, to assess nest success rates of bald eagles in 100% of known nest territories. From 2018–2020, 60–70% of known nest territories from the previous year’s surveys were monitored annually, including complete annual monitoring of the six coastal counties. In 2021, due to COVID-19 restrictions, only the six coastal counties were surveyed. Initial aerial surveys each nesting season were conducted between the first week of January and the first week of February, and sought to locate all active nests. Monitoring was performed according to the latitudinal gradient with regards to the timing of nesting cycles (i.e., eagles on the coast nest earlier and fledge young earlier than those in the mid-state, and much earlier than those nesting on reservoirs in the mountains). A nest was deemed occupied if it contained an adult eagle in an incubating posture, egg(s), eaglets, adult eagles engaged in nest affinity, or evidence of nest building and preparation (e.g., presence of fresh greenery, sticks with fresh breaks, or a new layer of nesting material25. These initial aerial surveys focused on nests that were active in the most previous survey, reports of potential new nest sites, and examination of sites having a high probability of supporting new nest territories.
The second round of aerial nest surveys took place from mid-March to early April, and were conducted to determine the reproductive outcome of the nests visited in January and February, as well as to visit newly reported nests. These follow-up flights aimed to maximize the probability that, if the nests in a survey route have not failed, they should contain eaglets that are approximately 80% of the age at first flight. Nests with eaglets of this age or older were considered successful as nestling mortality beyond this stage generally is low26,27. However, the timing of the commencement of nesting activities by eagle pairs within an ecoregion can vary by a few weeks. The second round of surveys confirmed that most of the nests previously determined as active had eaglets in advanced stages of development (i.e., 9–12 weeks of age), but occasionally included eaglets as young as 2–3 weeks. For these surveys, nests were recorded as successful if they contained eaglets during the follow-up flights or if eaglets were observed branching near the nests (i.e., successfully fledged), with percent nest success defined as the percentage of total occupied nests with successfully fledged eaglets. Nests were recorded as unsuccessful if eggs were still present or an adult exhibiting incubating posture was in the nest, if dead eaglets were observed, if they were empty and eaglets should have been present, or if either the nest or supporting tree had fallen. Mean annual productivity was calculated as the sum of fledglings produced divided by the sum of occupied nesting territories. Annual brood size was defined as the total count of fledglings per successful nests.
Bald eagle nest monitoring in Florida, USA
From 2016–2022, Audubon EagleWatch, a community science program, monitored bald eagle nests across the state of Florida (https://cbop.audubon.org/conservation/about-eaglewatch-program); data from four coastal counties with known incidents of early outbreak stage bald eagle HPAI mortality were used in this study. Trained volunteers visited nests a minimum of every two weeks during the peak bald eagle nesting period (October 1-May 15) with a minimum monitoring period of 20 min at each nest per site visit. One to four volunteers monitor each nest to ensure a robust dataset ideally spanning a range of temporal observation periods.
Nests that remained inactive or failed by May 15 of each year were no longer monitored, while occupied nests were monitored until young were fully fledged. Observed nests were either viewable from publicly accessible sites or via private property with appropriate permission. Thus, efforts were most concentrated within urban and suburban areas, and monitored nests can vary each survey year. Nest observers recorded data from the ground beneath the nest and from a distance that does not disturb the eagles (approximately 100 m, with some variation based on the nesting eagle’s tolerance of human activity). Nesting data were submitted via a custom Survey123 form and includes nest identifier and location, nest status, date and time, count of observed adults and young, and number of young fledged and/or perished noted during each site visit. If available, additional information on nest relocation, substrate changes, disturbance issues, and any observed leg bands is included. Nest status was inactive if no eagles were observed, only one adult was consistently observed over repeated visits, or two non-breeding adults were observed but displayed no interest in utilizing the nest. Nests were deemed occupied if a pair of eagles was consistently seen at the nest and showed interest in using the nest.
At the end of each nesting season, the EagleWatch Program Manager reviewed, validated, and summarized all submitted observations. Metrics reported for the 2022 season were extracted from institutional database version 2022–05-19. Data quality control includes case-by-case exclusion of nests with insufficient monitoring efforts. Eaglets between 8–10 weeks of age were assumed to have successfully fledged if the nest was empty after this period28. If no young were successfully produced, occupied nests were given a final status of failed, while nests that fledged at least one chick were categorized as successful. As with Georgia, post-fledging mortality is not accounted for in these analyses. However, unlike for Georgia eagle surveys, any sick or injured eaglets that were collected from monitored sites and successfully rehabilitated (i.e., released back into the wild) were considered successfully fledged. If a rescued eaglet was unable to be released, the young was categorized as perished (i.e., removed from the wild population). As with Georgia, percent nest success in Florida was defined as the percentage of total occupied nests that successfully fledged an eaglet. Summary nesting metrics of annual brood size (mean fledglings per successful nests) and productivity (fledglings per occupied nests) similarly aligned with definitions used in the Georgia methods.
All methods were carried out in accordance with relevant guidelines and regulations, and were done so with approval of the University of Georgia Institutional Animal Care and Use Committee (AUP# A2020 11–010-A6) and USFWS Eagle Scientific Collecting Permit number MB49069B-0.
Source: Ecology - nature.com
