Avian Influenza Infection and Antibodies Not Identified in Eastern Wild Turkeys (Meleagris gallopavo silvestris) Sampled on the Delmarva Peninsula Open Access

Highly pathogenic avian influenza (HPAI) presents a notable threat to domestic turkeys (Meleagris gallopavo domesticus), with previous research demonstrating that domestic turkeys are significantly more likely to experience an outbreak than domestic chickens (1,2,3). Additionally, when domestic turkeys are exposed to HPAI they shed high levels of virus, spread the disease easily to contact birds, and generally suffer complete mortality (4,5). This combination of high susceptibility and mortality means that HPAI outbreaks can have large-scale impacts on turkey farms, as seen in the 2014–2015 HPAI outbreak (6).

Despite the clear susceptibility of domestic turkeys to HPAI, there have been few observations of this pathogen in wild turkeys (Meleagris gallopavo). Historically, there was a single reported instance of a wild turkey testing weakly positive for any influenza A virus (IAV) in North America (7), with other available literature reporting no detections (8,9,10,11). The lack of detected infections is likely the result of differential habitat needs resulting in minimal overlap with waterfowl that serve as the primary reservoirs of naturally circulating low pathogenic avian influenza (LPAI) viruses and vectors of HPAI (12,13).

The introduction of clade 2.3.4.4b HP H5N1 into North America in the winter of 2021–2022 changed the nature of HPAI in this region, however, with many novel species being impacted with both lethal and sublethal infections (14). From December 2021 (beginning of current outbreak) to July 2024 there have been 18 individual wild turkeys reported as HP H5N1 positive by the U.S. Department of Agriculture (USDA) (15), from 5 separate mortality events. It should be noted that the total number of individuals testing positive likely underrepresents the actual number of individuals impacted, as only a small number of samples are typically submitted from a larger mortality event. For instance, 11 birds were tested and confirmed positive for HPAI infection from a larger mortality event (41 wild turkeys) in Johnson County, Wyoming, all of which were HP H5N1 positive (16). However, instead of the observed infections in wild turkeys resulting from either direct or environmental transmission of viruses caried by other wild birds, these infections may be the result of spillback from domestic turkeys (16).

This study presents avian influenza sampling data for Eastern wild turkeys (Meleagris gallopavo silvestris) across the Maryland portion of the Delmarva Peninsula in the winter of 2023–2024. Our objectives were to identify any circulating IAVs as well as determine the amount of previous exposure to IAVs in this population.

We conducted this study in Dorchester, Wicomico, and Worcester counties of Maryland (Fig. 1). These counties are located on the Delmarva Peninsula (containing Delaware and the eastern shores of Maryland and Virginia). The Delmarva Peninsula was chosen for this study because of the combination of high poultry production in the region (17) and heavy use by wintering waterfowl (18,19). This convergence of poultry and wild waterfowl puts the Delmarva Peninsula at elevated risk for IAV transmission across the wild bird–domestic poultry interface (20), and thus, also at risk for spillback into wild turkey populations such as seen in Wyoming (16). For instance, there have been 10 confirmed HPAI outbreaks in poultry identified by USDA on Delmarva since 2022, with the most recent occurring in Caroline County, which borders our study area, in November of 2023 (21). Additionally, outbreaks in backyard poultry flocks are likely underreported (22). Similarly, evidence of HPAI infection in wild birds has been documented in several waterfowl species and bald eagles (Haliaeetus leucocephalus) across Delmarva, including Dorchester and Wicomico counties, during and surrounding our study period (15). This region also has LPAI viruses regularly detected in wild waterfowl populations (18,23).

From 3 January to 14 March 2024 personnel from the Maryland Department of Natural Resources captured Eastern wild turkeys via rocket nets set over six separate sites baited with corn (24) as part of a larger study focused on movement ecology and survival (capture and marking conducted via state authority). During processing, measurements of weight, body condition, and age (adult versus juvenile) were recorded. Although transmitters were applied to sampled individuals as part of the larger project, movement data are not included here, and as such descriptions of marking methods are precluded. It should be noted that only females were sampled for IAV (as only females were receiving transmitters), and all individuals appeared healthy at the time of sampling. Oropharyngeal (OP) and cloacal (CL) swabs were collected and placed in a single vial per bird containing 2 ml of brain heart infusion media (Becton Dickinson and Co., Sparks, MD) supplemented with penicillin G (1000 units/ml), streptomycin (1 mg/ml), kanamycin (0.5 mg/ml), gentamicin (0.25 mg/ml), and amphotericin B (0.025 mg/ml) (Sigma Chemical Company, St. Louis, MO) and kept on ice packs for <24 hr until they were placed in storage at −70 C until they were shipped on dry ice and processed in the laboratory. Where possible, approximately 1 ml of whole blood was collected from the medial metatarsal vein and kept on ice packs for <4 hr until they were centrifuged at 2500 rpm for 20 min. Sera were then stored at −70 C until they were shipped on dry ice and processed in the laboratory. Sampling was approved by the U.S. Geological Survey, Eastern Ecological Science Center ACUC (2022-11P).

Nucleic acids were extracted from all OP/CL samples with the MagMax AI/ND Viral RNA extraction kit (Ambion Inc., Austin, TX) as described (25) and screened for the IAV matrix gene using real-time reverse transcription polymerase chain reaction (RRT-PCR) (26). Samples with cycle threshold (Ct) values ≤45.0 were considered positive for IAV viral RNA. All sera were tested using a commercial biomimetic enzyme-linked immunosorbent assay (bELISA; IDEXX AI MultiS-Screen AB test, IDEXX Laboratories, Westbrook, ME) for antibodies to the IAV nucleoprotein (NP) according to the manufacturer’s instructions. Sera were reported as positive for antibodies to IAV if the serum-sample-to-negative-control (S/N) absorbance value was less than 0.7 (27,28,29).

We collected OP/CL swab samples from 36 female eastern wild turkeys across Dorchester, Wicomico, and Worcester counties, Maryland. Influenza A viral RNA was not detected in any swab based on rRT-PCR. We also collected sera from 31 of the 36 sampled individuals, all of which had S/N values >0.7 (range: 0.79–1.29) indicating a lack of antibodies to IAVs. All data supporting these results are published openly (30).

These results indicate no evidence of current or detectable previous avian influenza infection in Eastern wild turkeys on the Delmarva Peninsula portion of Maryland. However, there are some important limitations to consider when interpreting these results. For instance, this study had a limited sample size aimed at leveraging ongoing turkey capture events for an independent ecology-based study. Given the infrequency with which wild turkeys are exposed to wild waterfowl (11,12) and the relative rarity of poultry farm outbreaks (21), larger sample sizes may be needed to identify exposure to IAVs. Similarly, based on observations in wild turkeys (16) and experimental infection studies in domestic turkeys (4), it is reasonable to expect that any wild turkey exposure to HPAI will result in mortality rather than seroconversion and recovery. However, the expanded host range observed during the ongoing HPAI outbreak (14), the observation of seroconversion in species that traditionally experience high mortality from HPAI (31,32), and the ability of domestic turkeys to seroconvert following exposure to some LPAI strains (33) demonstrate the need for assessing this potential in areas of possible exposure such as the Delmarva Peninsula.

Taken as a whole, our data, along with the lack of observed mortality events for this species in this region (15) suggest that both HPAI and LPAI infections have either not occurred in wild turkeys on the Delmarva Peninsula or occurrence is exceptionally rare. Indeed, our data provide further support that despite the ever-expanding list of impacted species (15,34), wild turkeys continue to be unexposed to IAVs in this region outside of likely spillbacks from domestic poultry. However, given the susceptibility of domestic turkeys to HPAI (4,5), continuing to monitor for exposure of wild populations to IAVs is likely important for proper population management, especially considering the difficulty in detection and assessment of wildlife mortality events (35).

Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Funding for project execution was provided by the U.S. Fish and Wildlife Service Zoonotic Disease Initiative (K00B4600075). Partial funding was also provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract 75N93021C00016. Partial support for JDS was provided by the U.S. Geological Survey, Ecosystems and Environmental Health Mission Areas, as well as the Chesapeake Bay Studies program. We thank Deborah Carter and Lyndon Sullivan Brügger for laboratory support.

CL =

cloacal;

Ct =

cycle threshold;

HPAI =

highly pathogenic avian influenza;

IAV =

influenza A virus;

NP =

nucleoprotein;

OP =

oropharyngeal;

RRT-PCR =

real-time reverse transcriptase PCR;

S/N =

serum-sample-to-negative-control