Abstract
Arthropod-borne viruses (arboviruses) are a taxonomically varied group of viruses that affect the health of many avian species, including the ruffed grouse (Bonasa umbellus), a popular upland game bird whose numbers are in decline in portions of its range. Hunter-harvested ruffed grouse tissue samples were tested for arboviruses during the 2018–2022 hunting seasons in Michigan, Minnesota, and Wisconsin, USA. A low percentage of harvested ruffed grouse were infected with West Nile virus (8/1892; 0.4%), eastern equine encephalitis virus (18/1892; 1.0%), and Highlands J virus (4/1892; 0.2%), and approximately half (16/30) of those infected had histologic cardiac lesions consistent with arboviral infection. Some ruffed grouse may be adversely affected following infection with these viruses, highlighting the need for increased awareness and continued surveillance, particularly in the face of additional stressors such as climate change, which may alter virus-vector-host dynamics, host susceptibility to arbovirus infections, and geographical distributions.
Introduction
Arthropod-borne viruses (arboviruses) are widely distributed, diverse, and pathogenic in numerous vertebrates, including birds. Multiple arboviruses circulate in the upper Midwest USA, including West Nile virus (WNV; Flavivirus), eastern equine encephalitis virus (EEEV; Alphavirus), and Highlands J virus (HJV; Alphavirus), but our understanding of sylvatic transmission cycles and wildlife susceptibility to disease varies, particularly for forest-dwelling species (Anderson et al., 2021; Cilnis et al., 1996).
The ruffed grouse (Bonasa umbellus), a forest-dwelling upland game bird, has experienced population declines in portions of its range since the early 2000s, notably in the east (Stauffer et al., 2018). West Nile virus has been implicated in contributing to these declines in the eastern USA (Nemeth et al., 2017; Stauffer et al., 2018). In addition, EEEV-associated disease was recently documented in ruffed grouse carcasses during routine disease monitoring in the upper Midwest USA (Anderson et al., 2021). Although considered to have similar distribution and transmission cycles as EEEV, published research, including grouse susceptibility, is lacking for HJV (Cilnis et al., 1996). We screened for three arboviruses, WNV, EEEV, and HJV, in hunter-harvested ruffed grouse from three states in the upper Midwest USA, with an emphasis on WNV due to evidence suggesting its role in population declines (Stauffer et al., 2018). In addition, heart was histologically evaluated in all grouse from which an arbovirus was detected, based on documented arbovirus-attributed disease in grouse (Anderson et al., 2021; Kunkel et al., 2022; Nemeth et al., 2017).
Materials and Methods
Hunter-harvested ruffed grouse samples were collected from September to January, 2018–2022, in Michigan (MI), Minnesota (MN), and Wisconsin (WI). Field sample kits, including a Whirl-Pak, sample collection instructions, and an envelope or Ziploc® bag, were distributed to hunters prior to and during each hunting season. Hunters (MN, WI) and state wildlife agency personnel (MI) voluntarily and opportunistically collected hearts (or brain and/or kidney instead of heart) and feathers from harvested ruffed grouse (Roy et al., 2022). Feathers were stored dry at ambient temperature; hunters were encouraged to chill organs (on freezer packs) as soon as possible until returned to state wildlife agencies, where tissues were most often stored frozen (−18°C to −20°C) for 2–3 months until overnight shipment on ice to the Southeastern Cooperative Wildlife Disease Study (SCWDS, University of Georgia; Athens, Georgia), where all samples were frozen at −80°C until processing. Hearts received by SCWDS were either whole or halved longitudinally. Sex and age were estimated based on feather structure, wear, and replacement (MN, WI) or reproductive organ and cloacal bursa presence/absence (MI), either as subadult or adult (Roy et al., 2022).
Approximately 0.5 cm3 heart samples (brain and/or kidney when heart was unavailable) were processed and underwent virus isolation, as described (Kunkel et al., 2021; Roy et al., 2022). Viral RNA was extracted from all homogenized tissue samples and from harvested virus isolation samples that exhibited cytopathic effects using the QiaAmp Viral RNA Mini Kit (Qiagen, Valencia, California, USA). These viral RNA extracts were tested for WNV RNA, as described, due to heightened concern over potential WNV impacts (Roy et al., 2022). Viral RNA from all virus isolation samples that exhibited cytopathic effects was also tested for EEEV and HJV RNA via RT-PCRs using modified thermocycler conditions (Huang et al., 2001; Monroy et al., 1996; Whitehouse et al., 2001). Routine histology was completed on the remaining heart samples from which an arbovirus was detected; lesion severity was subjectively graded, as described (Kunkel et al., 2021).
Results
1892 grouse tissue samples (1834 hearts and 58 brain and/or kidneys) were screened for arboviruses by virus isolation and for WNV RNA by real-time RT-PCR (MN WNV results previously published in Roy et al. [2022]). Cumulatively, 8 (0.4%) samples had WNV RNA (Ct value range: 21.2–35.0). Of the arboviruses isolated from 22 samples, 18 were EEEV (1.0%), and 4 (0.2%) were HJV (Table 1; Supplementary Fig. S1). All arbovirus detections were from hearts, and the majority were from grouse harvested in September and October (Supplementary Table S1), corresponding to late summer-early fall. The majority (6/8; 75.0%) of WNV detections were subadults, whereas subadults accounted for 38.9% (7/18) and 50.0% (2/4) of EEEV and HJV detections, respectively.
WNV, West Nile virus; RT-PCR, reverse transcription PCR; EEEV, eastern equine encephalitis virus; HJV, Highlands J virus; n, sample size; MI, Michigan; MN, Minnesota; WI, Wisconsin; ND, not detected.
All samples underwent virus isolation and were screened for WNV RNA by real time RT-PCR test. Those samples that exhibited cytopathic effects during virus isolation were subsequently tested for EEEV and HJV by RT-PCR and for WNV by real time RT-PCR tests.
A subset of MN WNV real time RT-PCR test results previously reported (Roy et al., 2022).
Lymphoplasmacytic inflammation was in 50.0% (4/8) of the hearts in which WNV RNA was detected, 55.6% (10/18) of those with EEEV isolated (concurrent with mineralization in two cases), and 50.0% (2/4) with HJV isolated (Supplementary Table S2; Supplementary Fig. S2). Myocardial degeneration and/or necrosis were associated with inflammation in two hearts with WNV RNA detected, five with EEEV isolated, and one HJV isolation (Supplementary Table S3).
Discussion
A low percentage of hunter-harvested ruffed grouse in the upper Midwest USA were infected with WNV, EEEV, and HJV (all documented in this region), among which cardiac damage was evident in approximately half infected with each virus. The pattern of lymphoplasmacytic myocarditis occasionally associated with myocardial degeneration/necrosis was consistent among arboviruses, but lesions generally were most severe for EEEV and least for HJV. Histopathologic findings are consistent with those observed in clinically-affected, WNV- and EEEV-infected ruffed grouse (Anderson et al., 2021; Kunkel et al., 2022; Nemeth et al., 2017), although lack of immunohistochemistry precluded our ability to directly correlate lesions with virus.
Harvested grouse may not represent the whole population but rather a biased, convenience sample. Decomposition and freeze-thaw tissue artifacts may have decreased test sensitivity. While the true prevalence and health impacts of arbovirus infections in ruffed grouse populations remain unclear, results from previous studies and our study suggest that some grouse are adversely affected, potentially resulting in higher susceptibility to harvest and/or hunting dog retrieval (Anderson et al., 2021; Kunkel et al., 2022; Nemeth et al., 2017). Future studies that encompass surveillance, serology, diagnostic evaluation of mortalities, and survival analysis with incorporation of vector data would provide a more holistic understanding of the impacts of these arboviruses on ruffed grouse populations and heighten awareness regarding circulating zoonotic viruses. This is particularly important in the face of stressors such as anthropogenic landscape alterations and climate change, which may alter geographical distributions, virus-vector-host dynamics, and host susceptibility to arbovirus infections.
Footnotes
Acknowledgments
The authors thank the participating state agencies, including Michigan Department of Natural Resources, Wisconsin Department of Natural Resources, and Minnesota Department of Natural Resources, and hunters that collected ruffed grouse samples. Charbel E. Gerges, Sydney Burke, Annie Vizurraga, and Alex Dhom of the SCWDS Virology Laboratory provided technical support with sample processing. The University of Georgia Histopathology Laboratory staff performed the histopathology processing.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
Funding was provided by participating state agencies and the member state wildlife management agencies of SCWDS provided by the Federal Aid to Wildlife Restoration Act (50 Stat. 917), with further support from the US Fish and Wildlife Service and US Geological Survey Ecosystems Mission Area.
Supplementary Material
Supplementary Figure S1
Supplementary Figure S2
Supplementary Table S1
Supplementary Table S2
Supplementary Table S3
References
Supplementary Material
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