Metagenomics for pathogen detection during a wildlife mortality event in songbirds

Abstract

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria comprising the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds, and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Birds affected by a 2021 avian mortality event are strongly associated with supplemental feeding and ground foraging behaviors

“…Coupled with potential immunosuppression from even small quantities of mycotoxin or other contaminants that were potentially below the limit of detection of the toxicological tests (e.g., originating from spoiled feed or pesticide sprays), these would likely have had more adverse health impacts in younger birds [25,[99][100][101][102]. Such outbreaks are often multifactorial, and one possibility is that these events could have initiated a cascading dysbiosis that allowed common avian pathogens, such as those reported by Mwakibete et al 2024 [1] to overwhelm the affected birds.…”

“…All analyses utilized data from bird carcasses that were submitted to the New Hampshire Veterinary Diagnostic Laboratory, the Wildlife Futures Program at the University of Pennsylvania, the Southeastern Cooperative Wildlife Disease Study at the University of Georgia, or the Ohio State University [1, 4]. Prior to these analyses, a large subset of these birds underwent either RNA or DNA sequencing for pathogen metagenomics [1]. Each bird categorized as a clinical case in this study demonstrated clinical signs and/or gross lesions consistent with the mortality event prior to death or euthanasia [1].…”

“…Prior to these analyses, a large subset of these birds underwent either RNA or DNA sequencing for pathogen metagenomics [1]. Each bird categorized as a clinical case in this study demonstrated clinical signs and/or gross lesions consistent with the mortality event prior to death or euthanasia [1]. Carcasses were identified to species by wildlife rehabilitators, wildlife veterinarians, and state wildlife agency biologists who submitted the birds, and confirmed at laboratories where necropsied/sampled.…”

“…Given these circumstances, we undertook a data science approach to incorporating knowledge from bird ecology and natural history [8] to generate and test hypotheses for the cause of the mortality event. We utilized the same sample of birds that underwent pathogen metagenomics sampling in our data science approach [1]. Our specific objective was to identify ecological traits that linked the affected bird species to potentially inform hypotheses regarding their susceptibility to exposure to potential etiologies [8][9][10].…”

“…During the spring and summer of 2021, the eastern coast of the United States experienced a large-scale die-off of passerines and near-passerines [1][2][3][4][5]. Thousands of primarily nestling and fledgling songbirds with neurological signs and periocular lesions were reported by the public to wildlife rehabilitators and state agencies [4].…”