Ticks pose substantial threats to public health. Blacklegged ticks (Ixodes scapularis) are responsible for most tick-borne diseases in the US, transmitting seven human pathogens, including the etiological agent of Lyme disease, Borrelia burgdorferi. Molecular surveillance for tick-borne pathogens has been outpaced by their emergence, revealing a critical need to develop agnostic strategies that characterize expanding and reemerging pathogens. Oxford Nanopore Technology's nanopore adaptive sampling (NAS), an approach that selectively enriches or depletes for target genomes or genetic loci, provides an opportunity to generate real-time genomic insights into tick-borne pathogens. In the current study, we performed PCR and NAS on individual Borrelia burgdorferi-infected and -uninfected ticks to evaluate the capability of NAS for tick-borne pathogen surveillance. We found that NAS generates real-time genetic datasets on B. burgdorferi that can supplement infectivity status ascertained via PCR. Using a multiplexing approach consisting of whole genomic DNA from 168 total ticks multiplexed over seven sequencing experiments, our results indicated that NAS is extremely specific (0.97 [95% CI: 0.93, 1.00]) with moderate sensitivity (0.48 [95% CI: 0.41, 0.55]), suggesting a strong capacity to confirm B. burgdorferi when present at the expense of an elevated false-negative rate. We found that quality-based filtering of sequence data has a substantial influence on detection metrics, emphasizing the need to optimize our multiplexing strategy, wet-lab procedures, and bioinformatic pipelines to enhance the sensitivity of NAS for detecting tick-borne pathogens.

Prion diseases are fatal neurodegenerative disorders characterized by abnormally folded prion proteins inducing misfolding of normal prion proteins, leading to neurotoxic fibrils and plaques. Epigenetic mechanisms, particularly DNA methylation, are increasingly implicated in prion-like diseases (e.g. Alzheimer's disease), but their role in prion pathogenesis remains unclear. To investigate, we used nanopore sequencing and RNAseq to measure genome-wide methylation and gene expression in the brains of Syrian hamsters (Mesocricetus auratus) experimentally infected with a hamster-adapted murine synthetic prion strain (n = 9) and age-matched mock-infected controls (n = 9) at 80, 120, and 160 days post-infection (dpi). We identified 1,586, 1,692, and 2,429 differentially methylated regions (DMRs) at 80, 120, and 160 dpi, respectively. Early- and mid-stage prion disease (80 and 120 dpi) skewed towards hypermethylation, whereas late-stage prion disease (160 dpi) skewed towards hypomethylation. Gene ontology (GO) of DMR-associated genes at 160 dpi included neuron regulation and signalling, neurodevelopment, and cellular stress pathways. We identified 178 differentially expressed genes (DEGs) at 80 dpi, 90 at 120 dpi, and 616 at 160 dpi. The majority of DEGs were downregulated at 80 dpi, and at 120 and 160 dpi, most were upregulated. Overlap in DEGs across timepoints was limited, and GO terms were related to upregulation of disease/injury response and cell death pathways in later timepoints. Overall, we found a stage-specific transcriptional shift from immune suppression to widespread immune and inflammation activation. These findings provide time-resolved data on methylation and transcriptional changes associated with impaired neuronal structure, function, and communication during disease.

Infectious prion adsorption on metal, minerals, wood, and plastic is well documented, raising the specter of food safety hazards for meat packing workers, sport hunters, and consumers. We previously demonstrated that sodium hypochlorite, and to a lesser extent, potassium peroxymonosulfate, and hypochlorous acid can decontaminate prion-contaminated nonporous surfaces. However, the extent to which chemical aging of surfaces affects subsequent recoverable prion seeding activity is unknown. In this study, we investigated the potential for four chemical decontaminants known for their anti-prion activity (sodium hypochlorite [bleach], hypochlorous acid [Briotech], potassium peroxymonosulfate [Virkon-S], and Wex-Cide-128) to alter the surfaces of steel knives and the subsequent prion decontamination efficacy of each. We found that hypochlorous acid, sodium hypochlorite, and potassium peroxymonosulfate corrode the surfaces of steel knives, resulting in significant physical alterations. Knives exposed to hypochlorous acid exhibited the most substantial corrosion (rust), which is consistent with its oxidizing effects. Oxidation of the knife surface was corroborated by complementary energy-dispersive X-ray spectroscopy data trends. Scanning electron microscopy data indicate corrosion is apparent after minimal exposure to oxidizing agents. Finally, we used the real-time quaking-induced conversion assay on swabs collected from chemically aged knife surfaces to evaluate recoverable surface-associated CWD-prion seeding activity detected by RT-QuIC after prion exposure and decontamination. Our results indicate decreased recoverable prion seeding activity from knife surfaces aged with 40% bleach. We also observed some recoverable seeding activity post-decontamination on knives chemically aged with 10% bleach and Wex-Cide-128, but largely similar efficacy to prior studies. This implies that existing chemical prion decontaminants are likely effective after repeated use on steel surfaces.

Nanopore sequencing is a powerful tool for real-time pathogen detection and genomic characterization; however, its application to individual ticks is limited by abundant host-derived nucleic acids and low viral RNA levels. In this study, we applied nanopore adaptive sampling (NAS) to sequence viral RNA from individual Haemaphysalis (H.) ticks collected in the Republic of Korea (ROK). By combining NAS with long-read sequencing, high-resolution genome assembly can be achieved from samples containing low-abundance viral RNA and relatively short complementary DNA (cDNA) fragments generated during library preparation. These results indicate that NAS remains effective under suboptimal fragment-size conditions and improves genome assembly compared to conventional nanopore workflows. Phylogenetic analyses revealed that the detected Dabieshan tick virus (DTV) sequences were clustered with isolates from China and Japan, suggesting regional circulation facilitated by the widespread distribution of H. longicornis. Unlike previous studies relying on pooled samples without selective sequencing, NAS allowed high-resolution viral genome assembly from single ticks. These findings confirm the presence and genotypes of DTV for the first time in the ROK and demonstrate NAS as a practical, scalable approach for tick-borne RNA virus surveillance in single ticks, improving genomic assembly and supporting the monitoring of emerging tick-borne viruses in endemic regions.

The role of the Common Vampire Bat (Desmodus rotundus) as a vector for chronic wasting disease (CWD) remains uninvestigated, and the effects of prion exposure in vampire bats are unknown. Desmodus feeds on the blood of various animals including deer, livestock, and humans across its expansive distribution. Given the continued southward spread of CWD in North American cervid populations and potential for the disease to already be circulating in Mexico, where it may overlap with established Desmodus populations, it is critical to assess potential risks at the interface between vampire bats and prion-infected hosts. Desmodus is also predicted to expand its range northward, potentially establishing populations in CWD-endemic regions of the southern United States, further underscoring the need for proactive surveillance and research on the ecological and epidemiological implications of this emerging interface. We explore aspects of prion biology and the natural history of Desmodus, highlighting factors that may contribute to prion exposure events among vampire bats and sympatric mammals. In light of Desmodus feeding behaviors, vampire bats could experience elevated prion exposures over time if they encounter CWD-positive prey. We recommend risk assessments and surveillance to evaluate vampire bat-prion transmission pathways that could impact mammalian wildlife, livestock, and human health.

Across the Neotropics, the common vampire bat ( Desmodus rotundus ) is an obligate hematophage, feeding on wildlife, domestic animals, and occasionally humans. While Desmodus is ecologically significant (e.g., nutrient cycling, complex sociality, parasitism), its role as a pathogen reservoir and vector (i.e., rabies virus) poses risks to animal and human health and local economies. Traditional single-gene and mitochondrial markers suggest high intra-species diversity within Desmodus ; however, next-generation sequencing and bioinformatic advancements enable comprehensive whole-genome analyses to resolve complex evolutionary histories. Here, we employed nanopore-based whole-genome skimming (~ 0.35X coverage) of 67 D. rotundus individuals across nine countries to characterize both nuclear and mitochondrial genomic diversity. Approximately 43 million mapped reads and 41 million loci informed our genotype likelihood analysis with the ANGSD: Analysis of Next Generation Sequencing Data software. We additionally assembled 28 complete mitogenomes along with 61 COI and 60 CYTB consensus genes. Our analysis revealed mitonuclear discordance patterns across the Neotropics. Genome-wide F ST values ranged from 0.114 to 0.358, with nuclear PCA and ancestry population structure modeling clustering populations along a continuous geographic gradient and mapping a latitudinal genetic transition zone through Panama. Conversely, mitogenomic phylogenies and maternal population structure models resolved highly structured, isolated regional lineages, including maternal divergence across the Ecuadorian Andes and localized matrilineal sorting in Guyana. This mitonuclear discordance suggests male-mediated gene flow and identifies Panama as a critical corridor and the Andes as a primary barrier to gene flow, supporting population isolation in Pleistocene refugia. Our findings refine the evolutionary history of Desmodus and demonstrate the utility of genome skimming for resolving wildlife lineages.

Infectious prions (PrPSc) are largely resistant to proteolytic digestion, including proteinase K (PK) digestion. While nucleic acid extracts are generally considered non-infectious from a classical microbiology context (i.e. free of intact bacteria and viruses), we investigated whether standard DNA purification methods co-purify PrPSc, posing an unrecognized biosafety risk. Commercial DNA extraction kits can eliminate conventional pathogens but are likely ineffective against PrPSc due to resistance to kit reagents and enzymatic degradation. Two laboratories, the University of Minnesota Center for Prion Research and Outreach (MNPRO) and the Canadian Food Inspection Agency (CFIA), independently tested filter-based and magnetic bead-based DNA extraction kits using tissues from chronic wasting disease (CWD)-positive and -negative white-tailed deer (WTD; Odocoileus virginianus), as well as prion-infected and control Syrian hamster (Mesocricetus auratus) brains. CFIA used two filter-based kits (one automated, one manual), while MNPRO tested two manual kits (filter- and magnetic bead-based). PrPSc seeding activity was measured in extracted DNA and source tissues using real-time quaking-induced conversion (RT-QuIC). MNPRO found substantial to almost perfect agreement between RT-QuIC seeding activity of DNA eluates from both extraction methods and that of the source WTD tissue homogenate. CFIA optimized RT-QuIC to a 30-hour runtime, achieving 74% sensitivity and 94% specificity in 88 archived WTD DNA samples. Both laboratories concluded that commercial DNA extraction kits do not eliminate PrPSc, enabling carry-over into DNA eluates. Until infectivity is resolved by animal bioassay, DNA from PrPSc-positive tissues should be handled under biosafety protocols appropriate for the originating prion disease, with decontamination and containment procedures.

Across the globe, anthropogenic environmental changes are threatening animal biodiversity and contributing to the emergence of vector-borne and zoonotic pathogens through host range shifts. To combat these challenges, accurate and timely biodiversity assessments and molecular species monitoring efforts are critical. Here, we document how the implementation of a portable laboratory in combination with targeted long-read nanopore sequencing can facilitate in situ genomic and systematic analyses across several animal taxa. Working at two ecologically divergent field sites in Guyana, South America, we collected small mammals and blood-feeding insects, including bats, rodents, a marsupial, mosquitoes, and a phlebotomine sand fly. For each specimen sampled, genomic DNA was extracted in the field and used for the preparation of nanopore sequencing libraries. For field sequencing, we utilized a novel software-based targeted sequencing approach-nanopore adaptive sampling (NAS)-that enabled the selective sequencing of mitochondrial reads using mitogenome assemblies of related taxa as enrichment targets. Basecalled reads from our field sequencing experiments were used to assemble complete mitogenomes and to generate mitochondrial biomarker consensus gene sequences for all nine small mammals and four blood-feeding insects sequenced. Confirmatory molecular identifications were made with a combination of local nucleotide BLAST queries and maximum likelihood analyses using biomarker consensus sequences. Importantly, the mitogenome-based targeted sequencing strategies outlined here are amplification-free and allowed us to bypass time-consuming and potentially troublesome PCR-based methods in the field, streamlining library preparation, sequencing experiments, and on-site analyses. Our findings describe targeted sequencing with NAS as an effective tool for implementation into portable laboratories to widely enhance field-based biodiversity monitoring and rapid molecular species assessments across vertebrate and invertebrate hosts of consequential emerging pathogens.

Bats are extraordinary mammals. They have evolved to consume various dietary sources, such as insects, fruits, nectar, blood, and meat. This diversity has generated considerable interest in the scientific community, resulting in efforts to leverage bats as model organisms to study the correlation between diet and gut microbiome community. Although such studies now commonly use Next Generation Sequencing (NGS), similar studies are early in their development in Southeast Asia, especially in Malaysia, which harbours an incredibly diverse bat fauna. This study provides pioneering NGS metabarcoding information on Bornean bats. By using a high-throughput Nanopore-based 16S rRNA gene sequencing method, Bacillota, Pseudomonadota, and Campylobacterota were found in insectivorous bats and phytophagous bats. Both insectivorous and phytophagous groups harboured no dominant taxon (D = 0.076; D = 0.085). A comparative analysis of gut bacteria functional groups identified eight major groups in both phytophagous and insectivorous bats, with fermentation being the predominant group. The correlation network analysis revealed a negative correlation between the ‘good bacteria’ Lactobacillus and various pathogenic bacteria genera, such as Salmonella (-0.4124) and Yersinia (-0.4654), demonstrating its prebiotic characteristics. This study broadens our understanding of the bat gut microbiome from various diets, with emphasis on new data from Borneo.

Sensitive and specific antemortem diagnostic tests are a prerequisite for effective management of chronic wasting disease (CWD). Paired with readily accessible samples that accurately reflect CWD status, the real-time quaking-induced conversion (RT-QuIC) assay has the potential to enable more effective CWD surveillance and interventions. We evaluated the feasibility of RT-QuIC as a CWD diagnostic test using 6-mm ear tissue biopsies from elk (Cervus canadensis). First, we evaluated the effect of ear spatial location on seeding activity. We observed an effect of ear punch spatial location on the amyloid formation rate (AFR): Samples collected from the periphery of the ear evidenced a statistically significant increase in AFR relative to ear punches from the ventral midline. Gross microdissection of an ear pinna suggested that there was more small nerve innervation around the periphery of the ear. Second, we evaluated the diagnostic sensitivity, specificity, and predictive value of RT-QuIC using ear punches from elk that had been previously diagnosed via ELISA testing. We evaluated the impact of nonstatistical and statistical approaches on diagnostic accuracy. Specificity and positive predictive value were perfect when statistical analyses were used to evaluate the binomial distribution (CWD positive versus CWD negative) of the data. Conversely, sensitivity and negative predictive value were modest, independent of the application of statistical analysis, indicating that RT-QuIC may be susceptible to false-negative data in this context. Taken together, our data support the idea that RT-QuIC, when paired with US Department of Agriculture-approved diagnostic tests, may provide more time to stakeholders for making major management decisions.

Seed Amplification Assays (SAAs) detect misfolded proteins associated with neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, ALS, and prion diseases. However, current data analysis methods rely on manual, time-consuming, and potentially inconsistent processes. We introduce AI-QuIC, an artificial intelligence platform that automates analyzing data from Real-Time Quaking-Induced Conversion (RT-QuIC) assays. Using a well-labeled RT-QuIC dataset comprising over 8000 wells, the largest curated dataset of its kind for chronic wasting disease prion seeding activity detection, we applied various AI models to distinguish true positive, false positive, and negative reactions. Notably, the deep learning-based1 Multilayer Perceptrons (MLP) model achieved a classification sensitivity of over 98% and specificity of over 97%. By learning directly from raw fluorescence data, the MLP approach simplifies the data analytic workflow for SAAs. By automating and standardizing the interpretation of SAA data, AI-QuIC holds the potential to offer robust, scalable, and consistent diagnostic solutions for neurodegenerative diseases.

Chronic wasting disease (CWD) is a fatal neurodegenerative disease affecting cervids. CWD is caused by infectious prions, which can enter the environment through bodily fluids or the carcasses of infected animals. Prions can be stored, remain infectious in both soil and water for many years, and transported hydrologically, possibly expanding the geographic range of CWD transmission. In order to better predict hydrological prion transport, we investigated how CWD prion protein (PrPCWD) partitions and persists in environmental waters. We performed PrPCWD spike experiments with water samples containing fine sediments from two locations within a CWD-contaminated site, at which contamination sources were removed one year prior. Samples were filtered after spiking, and filtrates and sediments were tested separately for PrPCWD using real-time quaking-induced conversion (RT-QuIC). Unspiked filtrates tested negative for PrPCWD, while unspiked sediments were positive, indicating PrPCWD persistence in environmental sediments for at least one year. Spiked sediments were positive immediately after spiking and throughout 28 days of incubation. Spiked filtrates were largely negative immediately after spiking and remained negative for 28 days, with some inconsistent positives from one sampling location. Our results indicate that PrPCWD readily partitions to the sediment fraction of environmental waters, suggesting that hydrological prion transport is sediment-facilitated.

As chronic wasting disease (CWD) continues to spread, researchers have studied stakeholder attitudes, behaviors, and risk perceptions of deer hunting associated with CWD. Findings inform natural resource agencies' methods to educate the public, address concerns, and implement management plans. However, little work has been done with underrepresented populations, such as the Amish. We conducted semi-structured interviews with male members of Amish communities in southeastern Minnesota, an area where CWD has been present in wild white-tailed deer (Odocoileus virginianus) for over a decade. Participants were asked about their knowledge of CWD, attitudes and behaviors regarding CWD, and sources of CWD information. Thematic analysis revealed that participants had limited CWD knowledge, perceived CWD as a low risk, and disliked culling as a management strategy, instead preferring to "let nature take its course." The knowledge shared and gathered in this study provides information for Amish community-focused, CWD-related educational materials.

Prion diseases, including chronic wasting disease (CWD), are caused by prions, which are misfolded aggregates of normal cellular prion protein. Prions possess many characteristics that distinguish them from conventional pathogens, in particular, an extraordinary recalcitrance to inactivation and a propensity to avidly bind to surfaces. In middle to late stages of CWD, prions begin accumulating in cervid muscle tissues. Those features collectively create scenarios in which occupational hazards arise for workers processing venison and pose risks to consumers through direct prion exposure through ingestion and cross-contamination of food products. In this study, we demonstrate that steel and plastic surfaces used in venison processing can be directly contaminated with CWD prions and that cross-contamination of CWD-negative venison can occur from equipment that had previously been used with CWD-positive venison. We also show that several decontaminant solutions (commercial bleach and potassium peroxymonosulfate) are efficacious for prion inactivation on those same surfaces.

Eliminating supplemental feeding is a common regulatory action within chronic wasting disease (CWD) management zones. These regulations target the potential for increased animal-animal contact and environmental contamination with CWD prions. Prions, the causative agent of CWD, have been detected on feeder surfaces in CWD-positive, captive deer facilities but not among free-ranging populations, and information on the relative risk of transmission at anthropogenic and natural food sources is limited. In this study, we established and maintained 13 gravity feeders from September 2022 to March 2023 in a CWD zone in northern Mississippi, USA (apparent prevalence similar to 30%). We set up feeders up in 3 ways: no exclusion (deer feeders, n = 7), exclusion of deer using fencing with holes cut at the ground-level to permit smaller wildlife to enter (raccoon feeders, n = 3), and environmental control feeders, which were fully fenced and not filled with feed (control feeders, n = 3). We swabbed feeder spouts at setup and at 4 intervals approximately 6 weeks apart to test for prion contamination via real-time quaking-induced conversion (RT-QuIC). We detected prions 12 weeks after setup on all deer and raccoon feeders. We compared relative transmission risk using camera traps at these feeders, 6 agronomic plantings for wildlife forage (i.e., food plots), and 7 oak mast trees. Weekly visitation rate by white-tailed deer (Odocoileus virginianus; hereafter: deer) differed (P = 0.02) among deer feeders (median = 24.5 deer/week, range = 15.6-65.7), food plots (median = 12.7, range = 3.8-24.7), and mast trees (median = 2.0, range = 0.4-5.1). Contact rates between individual deer also differed between site types (P < 0.01): deer feeders (median = 2.1 deer-to-deer contacts/week, range = 0-10.1), food plots (median = 0.1, range = 0-4.0), and mast trees (median = 0, range = 0-0.3). Raccoons also visited feeders at greater rates than food plots and mast trees (P < 0.04). Finally, we swabbed 19 feeders in 2 areas where CWD was newly detected, finding prion contamination on swabs from 4 feeders. We show that deer feeders in free-ranging populations with high CWD prevalence become contaminated with CWD prions quickly, becoming a potential site of exposure of deer to CWD prions. Our results also demonstrate the ability to find evidence of prion contamination on deer feeders, even in areas where CWD is newly detected.

Real-time quaking induced conversion (RT-QuIC) has become a valuable diagnostic tool for protein misfolding disorders such as Creutzfeldt–Jakob disease and Parkinson’s disease. Given that the technology is relatively new, academic and industry standards for quality filtering data and high throughput analysis of results have yet to be fully established. The open source R library, quicR, was developed to provide a standardized approach to RT-QuIC data analysis. quicR provides functions, which can be easily integrated into existing R workflows, for data curation, analysis, and visualization.

Prion diseases are fatal neurodegenerative disorders that affect mammals, including Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids, and bovine spongiform encephalopathy in cattle. During the disease, abnormally folded prion proteins induce misfolding of normal prion proteins, leading to neurotoxic fibrils and plaques. Epigenetic mechanisms, particularly DNA methylation, are increasingly implicated in prion-like diseases (e.g., Alzheimer’s disease), but their role in prion pathogenesis remains unclear. To investigate, we used nanopore sequencing and RNAseq to measure genome-wide methylation and gene expression in the brains of Syrian hamsters (Mesocricetus auratus) experimentally infected with a hamster-adapted murine synthetic prion strain (n = 9) and age-matched mock-infected controls (n = 9) at 80, 120, and 160 days post-infection (dpi). We identified 1,586, 1,692, and 2,429 differentially methylated regions (DMRs) at 80, 120, and 160 dpi, respectively. Early and mid-stage prion disease (80 and 120 dpi) were skewed toward hypermethylation, whereas late-stage prion disease (160 dpi) was skewed toward hypomethylation. Gene ontology (GO) of nearest genes to DMRs at 160 dpi included terms related to neuron regulation and signaling, neurodevelopment, and cellular stress pathways. We identified 178 differentially expressed genes (DEGs) at 80 dpi, 90 at 120 dpi, and 616 at 160 dpi. The majority of DEGs were downregulated at 80 dpi, and at 120 and 160 dpi, most DEGs were upregulated. Overlap in DEGs across timepoints was limited, and GO terms were related to upregulation of disease/injury response and cell death pathways in later timepoints. Overall, we found stage-specific responses to infection with a transcriptional shift from suppression of immune pathways to widespread immune and inflammation pathway activation. These findings indicate dynamic epigenetic and transcriptional changes marked by progressive and heterogeneous disruption of neuronal structure, function, and communication.

Aim:One of the most common patient-reported complaints following intramedullary nailing (IMN) of tibial shaft fractures is anterior knee pain reported by 10% to 80% of patients. The present study aimed to compare the 12-month Knee injury and Osteoarthritis Outcome Score (KOOS) sport and recreation activities subscale (sport/rec) scores after IMN with external ring fixation (RF) to adult patients with tibial shaft fractures. Methods:This study was a pragmatic multicentre randomized, non-blinded trial, with two-group parallel design. Included were adult patients (aged ≥ 18 years) presenting with an acute tibial shaft fracture deemed operable with an intramedullary nail. The primary outcome was the KOOS sport/rec, ranging from 0 (worst score) to 100 (best score) at 12-month follow-up. Secondary outcomes included the Foot and Ankle Outcome Score (FAOS), health-related quality of life assessed by EuroQol five-dimension five-level health questionnaire, and pain scores. Results:A total of 67 patients were included in the study. In all, 33 patients were randomized to standard IMN and 34 patients to RF. The mean age of the patients was 47.7 years (SD 19.2; 18 to 84) and 34% were female (n = 23). The primary analysis revealed no statistically significant difference in KOOS sport/rec between the IMN and RF groups at the 12-month follow-up (adjusted mean difference -18.1 (95 % CI -43.4 to 7.2); favouring RF). Conclusion:No statistically significant differences in the KOOS sport/rec were observed between RF and IMN at 12-month follow-up. However, these results should be interpreted with caution, due to high risk of a type II error.

Chronic wasting disease (CWD) is a contagious prion disorder affecting cervids such as deer, elk, caribou, and moose, causing progressive and severe neurological degeneration followed by eventual death. As CWD prions (PrPSc) accumulate in the body, they are shed through excreta and secreta, as well as through decomposing carcasses. Prions can persist in the environment for years, posing significant concerns for ongoing transmission to susceptible cervids and pose an unknown risk to sympatric species. We used a validated protocol for real-time quaking-induced conversion (RT-QuIC) in vitro prion amplification assay to detect prions in soil collected within and around an illegal white-tailed deer (Odocoileus virginianus, WTD) carcass disposal site and associated captive WTD farm in Beltrami County, Minnesota. We detected PrPSc in 26 of 201 soil samples across 15 locations within the illegal disposal site and one on the farm that housed the cervids. Importantly, a subset of RT-QuIC positive soil samples was collected from soils where carcasses were recovered, providing direct evidence that environmental contamination resulted from this illegal activity. These findings reveal that improper cervid carcass disposal practices may have important implications for ongoing CWD transmission through the environment.

Wildlife disease surveillance has received considerable attention following recent emergence of high-consequence zoonotic pathogens in humans. Increased portability and affordability of sequencing technologies over the last decade have made real-time sequencing of wild animals and their pathogens a reality. Wildlife samples screened for pathogens, however, are rarely permanently archived in museum biorepositories, which limits potential for scientific validation and prevents extension by related disciplines (e.g., ecology, evolution, conservation). To better connect biodiversity and biomedical sciences, the Museums and Emerging Pathogens in the Americas (MEPA) network developed the Field+Genomics Workshop to build capacity for surveillance of wildlife and their pathogens in biodiverse countries. Here, we share workshop resources, in English and Spanish, to facilitate reproducibility and expansion of the workshop into the future. The workshop lasted 10 days, 6 days of fieldwork and 4 days of molecular lab and bioinformatic techniques. The field component emphasized the importance of holistic collecting-that is, permanently preserving many parts and symbionts from each sampled organism-as a critical step in wildlife and pathogen surveillance and to build foundational scientific infrastructure. The molecular component of the workshop used samples collected during the field portion to identify hosts and pathogens in real-time. For this component, we trained participants in methods of DNA extraction, library preparation, and Nanopore Adaptive Sampling (a software feature for real-time selective enrichment or depletion of target sequences). Bioinformatic training consisted of a basic introduction to computational genomics, a worked example to analyze a small sequence dataset, and an exercise using data generated from samples collected during the workshop. In total, the workshop cost similar to$37K (similar to$3K per participant), however, similar to 25% of those funds are invested in basic equipment and infrastructure that is reusable in future workshops (e.g., sequencer, computer, etc.). This workshop highlights the effort and expertise required to conduct voucher-backed surveillance of wildlife and their pathogens and the many benefits of uniting biodiversity and biomedical sciences to build local capacity.