Detection of Chronic Wasting Disease Prions in Prairie Soils from Endemic Regions.

Chronic wasting disease (CWD) is a contagious prion disease that affects cervids in North America, Northern Europe, and South Korea. CWD is spread through direct and indirect horizontal transmission, with both clinical and preclinical animals shedding CWD prions in saliva, urine, and feces. CWD particles can persist in the environment for years, and soils may pose a risk for transmission to susceptible animals. Our study presents a sensitive method for detecting prions in the environmental samples of prairie soils. Soils were collected from CWD-endemic regions with high (Saskatchewan, Canada) and low (North Dakota, USA) CWD prevalence. Heat extraction with SDS-buffer, a serial protein misfolding cyclic amplification assay coupled with a real-time quaking-induced conversion assay was used to detect the presence of CWD prions in soils. In the prairie area of South Saskatchewan where the CWD prevalence rate in male mule deer is greater than 70%, 75% of the soil samples tested were positive, while in the low-prevalence prairie region of North Dakota (11% prevalence in male mule deer), none of the soils contained prion seeding activity. Soil-bound CWD prion detection has the potential to improve our understanding of the environmental spread of CWD, benefiting both surveillance and mitigation approaches.

Predicting chronic wasting disease in white-tailed deer at the county scale using machine learning.

Continued spread of chronic wasting disease (CWD) through wild cervid herds negatively impacts populations, erodes wildlife conservation, drains resource dollars, and challenges wildlife management agencies. Risk factors for CWD have been investigated at state scales, but a regional model to predict locations of new infections can guide increasingly efficient surveillance efforts. We predicted CWD incidence by county using CWD surveillance data depicting white-tailed deer (Odocoileus virginianus) in 16 eastern and midwestern US states. We predicted the binary outcome of CWD-status using four machine learning models, utilized five-fold cross-validation and grid search to pinpoint the best model, then compared model predictions against the subsequent year of surveillance data. Cross validation revealed that the Light Boosting Gradient model was the most reliable predictor given the regional data. The predictive model could be helpful for surveillance planning. Predictions of false positives emphasize areas that warrant targeted CWD surveillance because of similar conditions with counties known to harbor CWD. However, disagreements in positives and negatives between the CWD Prediction Web App predictions and the on-the-ground surveillance data one year later underscore the need for state wildlife agency professionals to use a layered modeling approach to ensure robust surveillance planning. The CWD Prediction Web App is at https://cwd-predict.streamlit.app/ .

Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. More effective mitigation efforts may require expansion of the available toolkit to include new methods that provide earlier antemortem detection, higher throughput, and less expense than current immunohistochemistry (IHC) methods. The rectal mucosa near the rectoanal junction is a site of early accumulation of CWD prions and is safely sampled in living animals by pinch biopsy. A fluorescence-based, 96-well format, protein-aggregation assay-the real-time quaking-induced conversion (RT-QuIC) assay-is capable of ultra-sensitive detection of CWD prions. Notably, the recombinant protein substrate is crucial to the assay's performance and is now commercially available. In this blinded independent study, the preclinical diagnostic performance of a standardized RT-QuIC protocol using a commercially sourced substrate (MNPROtein) and a laboratory-produced substrate was studied using mock biopsy samples of the rectal mucosa from 284 white-tailed deer (Odocoileus virginianus). The samples were from a frozen archive of intact rectoanal junctions collected at depopulations of farmed herds positive for CWD in the United States. All deer were pre-clinical at the time of depopulation and infection status was established from the regulatory record, which evaluated the medial retropharyngeal lymph nodes (MRPLNs) and obex by CWD-IHC. A pre-analytic sample precipitation step was found to enhance the protocol's detection limit. Performance metrics were influenced by the choice of RT-QuIC diagnostic cut points (minimum number of positive wells and assay time) and by deer attributes (preclinical infection stage and prion protein genotype). The peak overall diagnostic sensitivities of the protocol were similar for both substrates (MNPROtein, 76.8%; laboratory-produced, 73.2%), though each was achieved at different cut points. Preclinical infection stage and prion protein genotype at codon 96 (G = glycine, S = serine) were primary predictors of sensitivity. The diagnostic sensitivities in late preclinical infections (CWD-IHC positive MPRLNs and obex) were similar, ranging from 96% in GG96 deer to 80% in xS96 deer (x = G or S). In early preclinical infections (CWD-IHC positive MRPLNs only), the diagnostic sensitivity was 64-71% in GG96 deer but only 25% in xS96 deer. These results demonstrate that this standardized RT-QuIC protocol for rectal biopsy samples using a commercial source of substrate produced stratified diagnostic sensitivities similar to or greater than those reported for CWD-IHC but in less than 30 hours of assay time and in a 96-well format. Notably, the RT-QuIC protocol used herein represents a standardization of protocols from several previous studies. Alignment of the sensitivities across these studies suggests the diagnostic performance of the assay is robust given quality reagents, optimized diagnostic criteria, and experienced staff.

Predicting the odds of chronic wasting disease with Habitat Risk software.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that was first detected in captive cervids in Colorado, United States (US) in 1967, but has since spread into free-ranging white-tailed deer (Odocoileus virginianus) across the US and Canada as well as to Scandinavia and South Korea. In some areas, the disease is considered endemic in wild deer populations, and governmental wildlife agencies have employed epidemiological models to understand long-term environmental risk. However, continued rapid spread of CWD into new regions of the continent has underscored the need for extension of these models into broader tools applicable for wide use by wildlife agencies. Additionally, efforts to semi-automate models will facilitate access of technical scientific methods to broader users. We introduce software (Habitat Risk) designed to link a previously published epidemiological model with spatially referenced environmental and disease testing data to enable agency personnel to make up-to-date, localized, data-driven predictions regarding the odds of CWD detection in surrounding areas after an outbreak is discovered. Habitat Risk requires pre-processing publicly available environmental datasets and standardization of disease testing (surveillance) data, after which an autonomous computational workflow terminates in a user interface that displays an interactive map of disease risk. We demonstrated the use of the Habitat Risk software with surveillance data of white-tailed deer from Tennessee, USA.

Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Prion forensics: a multidisciplinary approach to investigate CWD at an illegal deer carcass disposal site.

Infectious prions are resistant to degradation and remain infectious in the environment for several years. Chronic wasting disease (CWD) has been detected in cervids inhabiting North America, the Nordic countries, and South Korea. CWD-prion spread is partially attributed to carcass transport and disposal. We employed a forensic approach to investigate an illegal carcass dump site connected with a CWD-positive herd. We integrated anatomic, genetic, and prion amplification methods to discover CWD-positive remains from six white-tailed deer (Odocoileus virginianus) and, using microsatellite markers, confirmed a portion originated from the CWD-infected herd. This approach provides a foundation for future studies of carcass prion transmission risk.

RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. RESULTS: A total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10-2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10-3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. CONCLUSIONS: Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

A review of chronic wasting disease (CWD) spread, surveillance, and control in the United States captive cervid industry.

Chronic wasting disease (CWD) is a fatal prion disease of the family Cervidae that circulates in both wild and captive cervid populations. This disease threatens the health and economic viability of the captive cervid industry, which raises cervids in contained spaces for purposes such as hunting and breeding. Given the high transmissibility and long incubation period of CWD, the introduction and propagation of the infectious prion protein within and between captive cervid farms could be devastating to individual facilities and to the industry as a whole. Despite this risk, there does not yet exist a literature review that summarizes the scientific knowledge, to date, about CWD spread, surveillance, or control measures. Our review, which focused on peer reviewed, primary research conducted in the United States, sought to address this need by searching Google Scholar, Scopus, and Web of Science with a five-term keyword string containing terms related to the (1) location, (2) species affected, (3) disease, (4) captive cervid industry, and (5) topic of focus. Between the three databases, there were 190 articles that were selected for further examination. Those articles were then read to determine if they were about CWD spread, surveillance, and/or control in captive cervid facilities. The 22 articles that met these inclusion criteria were evaluated in detail and discussed, with recommendations for future collaborative work between captive cervid owners, government agencies, and researchers. This work will help to address, inform, and mitigate the rising problem of CWD spread and establishment.

Prospective fecal microbiomic biomarkers for chronic wasting disease.

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the United States. Here, we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the USA compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer. Through machine-learning, these taxa and taxa pairs at each rank level could facilitate identification of around 70% of both the CWD-negative and the CWD-positive samples. Our results provide a potential tool for diagnostics and surveillance of CWD in the wild, as well as conceptual advances in our understanding of the disease.IMPORTANCEThis is a comprehensive study that tests the connection between the composition of the gut microbiome in deer in response to chronic wasting disease (CWD). We analyzed 50 fecal samples obtained from CWD-positive animals compared to 50 CWD-negative controls to identify CWD-dependent changes in the gut microbiome, matched with the analysis of fecal metabolites. Our results show promising trends suggesting that fecal microbial composition can directly correspond to CWD disease status. These results point to the microbial composition of the feces as a potential tool for diagnostics and surveillance of CWD in the wild, including non-invasive CWD detection in asymptomatic deer and deer habitats, and enable conceptual advances in our understanding of the disease.

Detection of prions from spiked and free-ranging carnivore feces.

Chronic wasting disease (CWD) is a highly contagious, fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting wild and captive cervids. Although experimental feeding studies have demonstrated prions in feces of crows (Corvus brachyrhynchos), coyotes (Canis latrans), and cougars (Puma concolor), the role of scavengers and predators in CWD epidemiology remains poorly understood. Here we applied the real-time quaking-induced conversion (RT-QuIC) assay to detect PrPCWD in feces from cervid consumers, to advance surveillance approaches, which could be used to improve disease research and adaptive management of CWD. We assessed recovery and detection of PrPCWD by experimental spiking of PrPCWD into carnivore feces from 9 species sourced from CWD-free populations or captive facilities. We then applied this technique to detect PrPCWD from feces of predators and scavengers in free-ranging populations. Our results demonstrate that spiked PrPCWD is detectable from feces of free-ranging mammalian and avian carnivores using RT-QuIC. Results show that PrPCWD acquired in natural settings is detectable in feces from free-ranging carnivores, and that PrPCWD rates of detection in carnivore feces reflect relative prevalence estimates observed in the corresponding cervid populations. This study adapts an important diagnostic tool for CWD, allowing investigation of the epidemiology of CWD at the community-level.

Ecology and Chronic Wasting Disease Epidemiology Shape Prion Protein Gene Variation in Rocky Mountain Elk (Cervus elaphus nelsoni).

As chronic wasting disease (CWD) continues to spread across North America, the relationship between CWD and host genetics has become of interest. In Rocky Mountain elk (Cervus elaphus nelsoni), one or two copies of a leucine allele at codon 132 of the prion protein gene (132L*) has been shown to prolong the incubation period of CWD. Our study examined the relationship between CWD epidemiology and codon 132 evolution in elk from Wyoming, USA, from 2011 to 2018. Using PCR and Sanger sequencing, we genotyped 997 elk and assessed the relationship between genotype and CWD prevalence estimated from surveillance data. Using logistic regression, we showed that each 1% increase in CWD prevalence is associated with a 9.6% increase in the odds that an elk would have at least one copy of leucine at codon 132. In some regions, however, 132L* variants were found in the absence of CWD, indicating that evolutionary and epidemiologic patterns can be heterogeneous across space and time. We also provide evidence that naturally occurring CWD is not rare in 132L* elk, which merits the study of shedding kinetics in 132L* elk and the influence of genotype on CWD strain diversity. The management implications of cervid adaptations to CWD are difficult to predict. Studies that investigate the degree to which evolutionary outcomes are shaped by host spatial structure can provide useful epidemiologic insight, which can in turn aid management by informing scale and extent of mitigation actions.

Sporadic cases of chronic wasting disease in old moose - an epidemiological study.

Transmissible spongiform encephalopathies or prion diseases comprise diseases with different levels of contagiousness under natural conditions. The hypothesis has been raised that the chronic wasting disease (CWD) cases detected in Nordic moose (Alces alces) may be less contagious, or not contagious between live animals under field conditions. This study aims to investigate the epidemiology of CWD cases detected in moose in Norway, Sweden and Finland using surveillance data from 2016 to 2022.In total, 18 CWD cases were detected in Nordic moose. All moose were positive for prion (PrPres) detection in the brain, but negative in lymph nodes, all were old (mean 16 years; range 12-20) and all except one, were female. Age appeared to be a strong risk factor, and the sex difference may be explained by few males reaching high age due to hunting targeting calves, yearlings and males.The cases were geographically scattered, distributed over 15 municipalities. However, three cases were detected in each of two areas, Selbu in Norway and Arjeplog-Arvidsjaur in Sweden. A Monte Carlo simulation approach was applied to investigate the likelihood of such clustering occurring by chance, given the assumption of a non-contagious disease. The empirical P-value for obtaining three cases in one Norwegian municipality was less than 0.05, indicating clustering. However, the moose in Selbu were affected by different CWD strains, and over a 6 year period with intensive surveillance, the apparent prevalence decreased, which would not be expected for an ongoing outbreak of CWD. Likewise, the three cases in Arjeplog-Arvidsjaur could also indicate clustering, but management practices promotes a larger proportion of old females and the detection of the first CWD case contributed to increased awareness and sampling.The results of our study show that the CWD cases detected so far in Nordic moose have a different epidemiology compared to CWD cases reported from North America and in Norwegian reindeer (Rangifer tarandus tarandus). The results support the hypothesis that these cases are less contagious or not contagious between live animals under field conditions. To enable differentiation from other types of CWD, we support the use of sporadic CWD (sCWD) among the names already in use.

Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

Dynamics of CWD prion detection in feces and blood from naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Confirmatory testing of CWD is currently performed postmortem in obex and lymphoid tissues. Extensive evidence demonstrates the presence of infectious prions in feces of CWD-infected deer using in vitro prion-amplification techniques and bioassays. In experimental conditions, this has been achieved as soon as 6-month post-inoculation, suggesting this sample type is a candidate for antemortem diagnosis. In the present study, we optimized the detection of CWD-prions in fecal samples from naturally infected, pre-clinical white-tailed deer by comparing protocols aiming to concentrate CWD-prions with direct spiking of the sample into the PMCA reactions. Results of this screening were compared with similar analyses made in blood. Our data shows that CWD-prion detection in feces using PMCA is best in the absence of sample pre-treatments. We performed a screening of 169 fecal samples, detecting CWD-prions with diagnostic sensitivity and specificity of 54.81% and 98.46%, respectively. In addition, the PMCA seeding activity of 76 fecal samples was compared with that on blood of matched deer. Our findings, demonstrate that CWD-prions in feces and blood are increased at late pre-clinical stages, exhibiting similar detection in both sample types (> 90% sensitivity) when PrP96GG animals are tested. Our findings contribute to understand prion distribution across different biological samples and polymorphic variants in white-tailed deer. This information is also relevant for the current efforts to identify platforms to diagnose CWD.

PMCA screening of retropharyngeal lymph nodes in white-tailed deer and comparisons with ELISA and IHC.

Chronic wasting disease (CWD) is a prion disease affecting cervids. CWD diagnosis is conducted through enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in retropharyngeal lymph nodes. Unfortunately, these techniques have limited sensitivity against the biomarker (CWD-prions). Two in vitro prion amplification techniques, real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA), have shown promise in detecting CWD-prions in tissues and bodily fluids. Recent studies have demonstrated that RT-QuIC yields similar results compared to ELISA and IHC. Here, we analyzed 1003 retropharyngeal lymph nodes (RPLNs) from Texas white-tailed deer. PMCA detected CWD at a higher rate compared to ELISA/IHC, identified different prion strains, and revealed the presence of CWD-prions in places with no previous history. These findings suggest that PMCA exhibits greater sensitivity than current standard techniques and could be valuable for rapid and strain-specific CWD detection.

Identification of chronic wasting disease prions in decaying tongue tissues from exhumed white-tailed deer.

Chronic wasting disease (CWD) prions cause fatal neuropathies in farmed and free-ranging cervids. The deposition of prions in natural and humanmade environmental components has been implicated as a major mechanism mediating CWD spread in wild and captive populations. Prions can be deposited in the environment through excreta, tissues, and carcasses from pre-clinical and clinical animals. Furthermore, burial of CWD-positive animals may reduce but not completely mitigate prion spread from carcasses into the surrounding environment. Here, we analyzed exhumed, decaying deer carcasses for the presence of CWD prions. By analyzing tongue tissues through the protein misfolding cyclic amplification (PMCA) technique, we were able to identify seven out of 95 exhumed white-tailed deer carcasses as CWD prions carriers. Confirmatory analyses were performed using the real-time quaking-induced conversion (RT-QuIC) technique. In addition, we evaluated the potential contamination of the pens that housed these animals by swabbing feeders and waterers. PMCA analyses of swabs confirmed CWD contamination on farming equipment. This work demonstrates the usefulness of PMCA to detect CWD prions in a variety of contexts, including exhumed/decaying tissues. In addition, this is the first report demonstrating swabbing coupled with PMCA as a method for the detection of prion seeding activity on naturally exposed surfaces. Considering that this study was focused on a single site, further studies should confirm whether prion amplification assays are useful to identify CWD prions not only in animals but also in the environment that contains them. IMPORTANCE Environmental contamination is thought to be a major player in the spread of chronic wasting disease (CWD), a fatal prion disease affecting a wide variety of cervid species. At present, there are no officially approved methods allowing for the detection of prion infectivity in environmental components. Importantly, animal as well as anthropogenic activities are thought to contribute to prion environmental contamination. Here, we detected CWD prions in exhumed white-tailed deer carcasses by using the protein misfolding cyclic amplification (PMCA) assay. In addition, we identified CWD prions in feeders used within the infected facility. These results highlight the potential role of PMCA in identifying prion infectivity in a variety of scenarios, ranging from decaying tissues to farming equipment.

Heterogeneity of pathological prion protein accumulation in the brain of moose (Alces alces) from Norway, Sweden and Finland with chronic wasting disease.

Prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. They are characterized by the conformational conversion of the cellular prion protein (PrPC) into the pathological prion protein (PrPSc). In 2016, chronic wasting disease (CWD) gained great importance at European level due to the first disease detection in a wild reindeer (Rangifer tarandus) in Norway. The subsequent intensive CWD surveillance launched in cervids resulted in the detection of CWD in moose (Alces alces), with 11 cases in Norway, 3 in Finland and 4 in Sweden. These moose cases differ considerably from CWD cases in North American and reindeer in Norway, as PrPSc was detectable in the brain but not in lymphoid tissues. These facts suggest the occurrence of a new type of CWD. Here, we show some immunohistochemical features that are clearly different from CWD cases in North American and Norwegian reindeer. Further, the different types of PrPSc deposits found among moose demonstrate strong variations between the cases, supporting the postulation that these cases could carry multiple strains of CWD.

Inter- and intra-species conversion efficacies of Norwegian prion isolates estimated by serial protein misfolding cyclic amplification.

Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease.