Methods and materials for detecting prion diseases

Abstract

Provided herein are methods and materials for detecting misfolded polypeptides. For example, provided are devices (e.g., feeding devices) designed to collect samples from one or more mammals (e.g., one or more cervids). In some cases, provided are methods and materials that can be used to detect one or more prion diseases (e.g., chronic wasting disease (CWD)) in a mammalian population (e.g., a cervid herd) based at least in part on the presence or absence of misfolded polypeptides in samples from one or more mammals obtained from the feeding devices.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 197,822, filed June 7, 2021. The disclosure of the prior application is considered part of (and incorporated by reference into) the disclosure of this application.

[0002] Federal Funding Statement This invention was made with government support under AI077774 awarded by the National Institutes of Health and under CBET-1149424 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] 1.Technical Field The present specification relates to methods and materials for detecting misfolded polypeptides. For example, the present specification provides a device (e.g., a feeding device) designed to collect samples from one or more mammals (e.g., one or more cervids). In some cases, the methods and materials provided herein can be used to assess the presence or absence of one or more prion diseases (e.g., chronic wasting disease (CWD)) in a mammalian population (e.g., a cervid herd) based at least in part on the presence or absence of one or more misfolded polypeptides in samples from one or more mammals obtained from the feeding device provided herein. [Background technology]

[0004] 2. Background information CWD is a newly emerged prion disease in North America and Europe. It has been detected in 30 states in the United States (USGS National Wildlife Health Center, "Distribution of Chronic Wasting Disease in North America", usgs.gov / centers / nwhc / science / expanding-distribution-chronic-wasting-disease?qt-science_center_objects=0#qt-science_center_objects) and 3 provinces in Canada (Canadian Food Inspection Agency, "Herds infected with chronic wasting disease in Canada", inspection.canada.ca / animal-health / terrestrial-animals / diseases / reportable / cwd / herds-infected / eng / 1554298564449 / 1554298564710). Recent discoveries of CWD in reindeer and moose in Norway, Finland, and Sweden (Benestad et al., Vet. Res., 47:88 (2016); and Osterholm et al., mBio, 10(4):e01091-19 (2019)) have raised concerns about the spread and emergence of CWD worldwide. In CWD endemic areas, disease incidence in free-ranging deer herds can exceed 50%, potentially negatively impacting cervid populations (Miller et al., J. Wildl. Dis., 40(2):320-327 (2004)).Although the zoonotic potential of CWD is poorly understood, there is evidence that CWD can be transmitted to sympatric species, such as cattle, cats, pigs, sheep, and, as has been recently suggested, to humans (Wolfe et al., J. Wildl. Dis., 58(1):40-49(2021); Hamir et al., Vet. Pathol., 44:487-493(2007); Hamir et al., J. Vet. Diagn. Invest., 18:558-565(2006); Hamir et al., J. Vet. Diagn. Invest., 17:276-281(2005); Bartz et al., Virology, 251:297-301(1998); Moore et al., J. Virology, 91:e00926-00917 (2017); and Hannaoui et al., bioRxiv, 2022.2004.2019.488833, doi:10.1101 / 2022.04.19.488833 (2022)). Summary of the Invention

[0005] Prion diseases affect a wide range of host species, including humans (Creutzfeldt-Jakob disease, CJD), cattle (bovine spongiform encephalopathy, BSE or "mad cow disease"), deer (CWD), dromedaries, and sheep and goats (scrapie). Prions are expressed as a prion polypeptide (PrP C ) misfolded self-propagating form (PrP Sc Currently, the most widely accepted method for diagnosing prion diseases, such as CWD, is the detection of PrP Sc The first step is to autopsy the brain and lymphatic tissue for the presence of

[0006] The present disclosure relates to misfolded polypeptides (e.g., PrP ScFor example, the present specification provides a device (e.g., a feeding device having at least one sample collection surface) designed to collect a sample (e.g., a biological sample, e.g., saliva and / or mucus) from one or more mammals (e.g., one or more non-human mammals, e.g., cervidae). The one or more samples attached to the device provided herein can be used to detect one or more misfolded polypeptides (e.g., PrP Sc In some cases, the methods and materials provided herein can be used to assess the presence or absence of one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals obtained from a device provided herein. Sc The present invention can be used to detect, and optionally monitor, one or more prion diseases (e.g., CWD) in a mammalian population (e.g., a deer herd) based, at least in part, on the presence or absence of a prion gene (e.g., CWD) detected in a mammalian population (e.g., a deer herd).

[0007] As demonstrated herein, misfolded polypeptides (e.g., PrP Sc ) can be recovered from the sample collection surface using a swab and identified. For example, a feeding device having at least one sample collection surface can be used to obtain a sample from the sample collection surface (e.g., via a swab) and identify one or more misfolded polypeptides (e.g., PrP Sc The samples may be located in specific geographic regions and / or farms so that the samples can be evaluated for the presence or absence of one or more misfolded polypeptides (e.g., PrP Sc The presence of prion diseases can be used to detect the presence of one or more prion diseases in a mammalian population (e.g., a cervid herd).

[0008] By having the ability to detect one or more prion diseases in a mammalian population (e.g., a cervid herd) as described herein, one or more misfolded polypeptides (e.g., PrP) attached to a sample collection surface of a feeding device provided herein can be detected. Sc ), provides a unique and unrealized opportunity for environmental monitoring of prion disease herd levels. For example, monitoring of prion disease herd levels in wild and captive mammal populations (e.g., cervid herds) can be used to monitor and potentially control disease transmission.

[0009] In general, one aspect of the present disclosure features a method of assessing a population of non-human mammals for prion disease. The method may include, or consist essentially of, swabbing a sample collection surface of a feeding device to obtain a swab containing a sample, the feeding device including a reservoir and a sample collection surface, the reservoir containing bait, and the bait being able to pass from the reservoir to the sample collection surface; extracting a polypeptide from the sample to obtain an extract; concentrating the extract to obtain a concentrated extract; detecting the presence of a misfolded polypeptide in the concentrated extract; identifying the population of non-human mammals as having a prion disease if the presence of the misfolded polypeptide is detected; and identifying the population of non-human mammals as not having a prion disease if the presence of the misfolded polypeptide is not detected. The sample collection surface may be stainless steel, mica, slate, aluminum, ceramic, or glass. The bait may be corn, soy, oat, or commercial feed pellets. The sample may be saliva, mucus, or tongue epithelial cells. The population of non-human mammals may include moose, fallow deer, marsh deer, mule deer, barking deer, moose, pampas deer, red deer, reindeer, roe deer, sambar, sika deer, white-tailed deer, antelope, goat, camel, mink, cat, cow, sheep, mouse, rat, hamster, red brocket deer, axis deer, macaque, lemur, spider monkey, and chimpanzee. The feeding apparatus may be located in a wilderness area. The feeding apparatus may be located in an urban, suburban, or rural environment. The feeding apparatus may be unattended for about 1 to about 14 days. The feeding apparatus may be located on a farm. Samples may be taken daily. Samples may be taken weekly. The swabs may be cotton or foam swabs. The swabs may be stored in a solution prior to extracting the polypeptide from the sample to obtain the extract. The solution may be a phosphate buffered saline (PBS) solution. The swabs may be stored at a temperature of about -80° C. to about 4° C. The extracting step may include sonication. The concentrating step may include vacuum concentration.Misfolded polypeptides can be detected using real-time vibration-induced conversion (RT-QuIC), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), protein misfolding cyclic amplification (PMCA) or western blotting. Misfolded polypeptides include scrapie-associated misfolded polypeptide (PrP. Sc ), chronic wasting disease-associated misfolded polypeptide (PrP CWD ), bovine spongiform encephalopathy-associated misfolded polypeptide (PrP BSE ), Creutzfeldt-Jakob disease-associated misfolded polypeptide (PrP CJD ), feline spongiform encephalopathy-associated misfolded polypeptide (PrP FSE ), transmissible mink encephalopathy-associated misfolded polypeptide (PrP TME ) or camel spongiform encephalopathy-associated misfolded polypeptide (PrP CSE The prion disease may be chronic wasting disease (CWD), transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE), scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy or camilid spongiform encephalopathy.

[0010] In another aspect, the document features a method of detecting misfolded polypeptides. The method can include, or consist essentially of, swabbing a sample collection surface to obtain a swab containing the sample, extracting the polypeptide from the sample to obtain an extract, concentrating the extract to obtain a concentrated extract, and detecting the presence of the misfolded polypeptide in the concentrated extract. The sample collection surface can be stainless steel, mica, slate, aluminum, ceramic, or glass. The collection surface can be in a food processing facility, a water treatment facility, or a hospital. The sample can be blood, urine, feces, saliva, or mucus. The sample can be obtained daily. The sample can be obtained weekly. The swab can be a cotton swab or a foam swab. The swab can be stored in a solution prior to extracting the polypeptide from the sample to obtain the extract. The solution can be a PBS solution. The swab can be stored at a temperature of about -80°C to about 4°C. The extraction can include sonication. The concentrating can include vacuum concentration. The misfolded polypeptide may be detected using RT-QuIC, ELISA, IHC, PMCA or Western blotting. The misfolded polypeptide may be a misfolded tau polypeptide, a misfolded alpha-synuclein polypeptide or a misfolded amyloid beta polypeptide. The misfolded polypeptide may be associated with a prion disease. The prion disease may be Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), Alzheimer's disease or Parkinson's disease.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials to those described herein can be used to practice the present invention, and suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0012] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0013] [Figure 1-1] Figures 1A-1H show the recovery of the Hyper (HY) strain of transmissible mink encephalopathy (TME) PrPSc from foam or cotton swabs using shaking. Representative 96-well immunoblots (Figures 1A, 1C, 1E, and 1G) and quantification (bar graphs in Figures 1B, 1D, 1F, and 1H) of HY PrPSc recovered from three consecutive extractions (1st, 2nd, and 3rd) of both foam and cotton swabs dried at room temperature for various lengths of time. Line graphs represent the moisture content of swabs moistened with HY TME brain homogenate (solid line) or ultrapure water (dashed line) after drying at room temperature. PrPSc recovery results are expressed as the mean ± SEM of total recovery, n=4, and data points on the line graphs represent the mean moisture content, n=3. * indicates significant differences (P<0.05) between samples and the respective undried controls (0 h). [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 2-1]Figures 2A-2D show the recovery of HY TME PrPSc extracted from foam swabs extracted using sonication. Representative 96-well immunoblots (Figures 2A and 2C) and quantification (Figures 2B and 2D) of HY TME PrPSc recovered from three consecutive extractions (1st, 2nd, 3rd) of foam swabs that were either undried (0 h) or dried at room temperature for 24 h. Sonication times consisted of 1, 3, 6 or 12 cycles of pulse treatment with 5 s of sonication followed by 5 s of incubation. Prion recovery results are expressed as mean ± SEM of total recovery, n=4. [Figure 2-2] Continued from Figure 2-1. [Figure 3-1] Figures 3A-3F show the recovery of CWD PrPSc extracted from foam swabs by sonication. Representative 96-well immunoblots (Figures 3A, 3C, and 3E) and quantification (Figures 3B, 3D, and 3F) of CWD PrPSc recovered from three consecutive extractions (1st, 2nd, and 3rd) of foam swabs that were either undried (0 h) or dried at room temperature for 24 h. Sonication for 15 s (3 cycles of pulse treatment) was used for each extraction. Prion recovery results are expressed as mean ± SEM of total recovery, n = 4. * indicates significant difference (P < 0.05) between samples dried for 24 h and the respective undried control (0 h). [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 4-1]Figures 4A-4L show the recovery of CWD PrPSc from glass, stainless steel or wood. Representative 96-well immunoblots (Figures 4A-4C) and quantification (Figures 4D-4L) of CWD PrPSc recovered from foam swabs (combined extracts of two successive extractions for each swab) applied to surfaces that were either undried (control) or dried at room temperature for 24 h. Sonication for 15 s (3 cycles of pulse treatment) was used for each extraction. As a control, the highest contaminating level of CWD PrPSc was applied. Prion recovery results are expressed as mean ± SEM of total recovery, n = 3. * indicates significant difference (p < 0.05) between samples dried for 24 h and the respective undried control (0 h). [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Figure 5-1] Figures 5A-5D show prion recovery from a swab-free process under various processing conditions. HY or CWD PrPSc spiked into 200 μL, 400 μL or 600 μL of DPBS buffer was subjected to sonication for 15, 30 or 60 seconds (consisting of 3, 6 or 12 cycles of 5 seconds of sonication followed by 5 seconds of incubation). Samples were then vacuum concentrated for 3, 4 or 5 hours. To test the effect of sonication time, the buffer volume was fixed at 200 μL and the lyophilization time was fixed at 3 hours. To test the effect of buffer volume, the sonication time was fixed at 15 seconds and the vacuum concentration time was fixed at 5 hours. To test the effect of concentration time, the sonication time was fixed at 15 seconds and the buffer volume was fixed at 200 μL. * indicates significant difference compared to control (p<0.05, n=4). # indicates significant difference (p<0.05, n=4) compared to the respective control (i.e., 15 sec, 200 μL or 3 h). [Figure 5-2] Continued from Figure 5-1. [Figure 6-1]6A-6C are diagrams illustrating an exemplary feeding device with a sample collection surface. FIG. 6A. Side view of a pole-mounted feeding device for monitoring misfolded polypeptides (e.g., PrPSc). FIG. 6B. Close-up of the boxed area in FIG. 6A. Grains can flow from a central reservoir, pass through a hinged stainless steel plate, and land on a stainless steel sample collection surface. FIG. 6C. Schematic images of a swab to collect a sample of CWD prions from the stainless steel surface of the feeding device, transfer the sample to a tube (left panel), and use real-time vibration-induced transduction (RT-QuIC) to identify the presence of misfolded polypeptides (e.g., PrPSc) in the sample (right panel). [Figure 6-2] Continued from Figure 6-1. [Figure 7] 7A-7B are diagrams illustrating an exemplary feeding device with multiple sample collection surfaces. FIG. 7A. Side view of a feeding bank for monitoring misfolded polypeptides (e.g., PrPSc). Grains can flow from a central reservoir through a discharge outlet onto a stainless steel sample collection surface. FIG. 7B. Top view of the feeding bank shown in FIG. 7A. The central grain reservoir can be equipped with four discharge outlets, each of which discharges grains onto a separate stainless steel sample collection surface. [Figure 8-1]8A-8C: RT-QuIC detection of CWD prions from swabs and stainless steel - first round extraction as maxpoint ratio. Figure 8A. RT-QuIC detection methodology for CWD PrPSc. Figure 8B. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied directly to swabs and immediately extracted. Figure 8C. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied to a stainless steel surface and wiped after drying for 24 hours at 22 °C, followed by immediate swab extraction. Swab extracts were analyzed for the presence of PrPSc that may misfold recHaPrP to amyloid in the RT-QuIC reaction and expressed as maxpoint ratio (MPR, mean ± standard deviation) determined as the ratio of maximum thioflavin T (ThT) fluorescence over the entire RT-QuIC run to ThT fluorescence at the initiation cycle of the RT-QuIC reaction. The threshold for positive signal was set at 2 (dashed line). [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 9-1] Figures 9A-9B: RT-QuIC detection of CWD prions from stainless steel - second extraction as maximum point ratio. The RT-QuIC detection methodology for CWD PrPSc is as shown in Figure 8A. Figure 9A. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied directly to a swab and immediately extracted. Figure 9B. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied to a stainless steel surface, wiped after drying for 24 hours at 22 °C, followed by immediate swab extraction. Swab extracts were analyzed for the presence of PrPSc that may misfold recHaPrP to amyloid in the RT-QuIC reaction and expressed as MPR (mean ± standard deviation) determined as the ratio of maximum ThT fluorescence in the entire RT-QuIC run to ThT fluorescence of the initiation cycle of the RT-QuIC reaction. The threshold for positive signal was set at 2 (dashed line). [Figure 9-2] Continued from Figure 9-1. [Figure 10-1]Figures 10A-10B: RT-QuIC detection of CWD prions from swabs and stainless steel - 1st extraction as amyloid formation rate. The RT-QuIC detection methodology for CWD PrPSc is as shown in Figure 8A. Figure 10A. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied directly to a swab and immediately extracted. Figure 10B. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied to a stainless steel surface, wiped after drying for 24 hours at 22 °C, followed by immediate swab extraction. Swab extracts were analyzed for the presence of PrPSc that may misfold recHaPrP into amyloid in the RT-QuIC reaction and expressed as amyloid formation rate (RAF, mean ± standard deviation) determined as the reciprocal of the RT-QuIC reaction time to reach the fluorescence threshold (MPR = 2). [Figure 10-2] Continued from Figure 10-1. [Figure 11-1] 11A-11B. RT-QuIC detection of CWD prions from swabs and stainless steel - second extraction as amyloid formation rate. The RT-QuIC detection methodology for CWD PrPSc is as shown in FIG. 8A. FIG. 11A. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied directly to swabs and immediately extracted. FIG. 11B. RT-QuIC detection of serial 10-fold dilutions of CWD PrPSc applied to a stainless steel surface, dried at 22°C for 24 hours, then wiped and immediately swab extracted. Swab extracts were analyzed for the presence of PrPSc that may misfold recHaPrP into amyloid in the RT-QuIC reaction and expressed as the RAF (mean ± standard deviation) determined as the reciprocal of the RT-QuIC reaction time to reach the fluorescence threshold (MPR=2). [Figure 11-2] Continued from Figure 11-1. [Figure 12-1]Figures 12A-12E show RT-QuIC detection of samples collected from DPBS-moistened swabs and uncontaminated stainless steel. Figure 12A. RT-QuIC detection methodology for samples collected from negative swabs and surface controls. Figures 12B and 12C. RT-QuIC detection of samples collected from DPBS-moistened swabs and immediately extracted. Figures 12D and 12E. RT-QuIC detection of samples collected from stainless steel and immediately extracted without swabbing. Swab extracts were analyzed for the presence of PrPSc, which may misfold recHaPrP to amyloid in the RT-QuIC reaction, and expressed as (1) the MPR (mean ± standard deviation), determined as the ratio of maximum ThT fluorescence over the entire RT-QuIC run to the ThT fluorescence of the initiation cycle of the RT-QuIC reaction (Figures 12B and 12D), and (2) the RAF (mean ± standard deviation), determined as the reciprocal of the RT-QuIC reaction time to reach the fluorescence threshold (MPR = 2, Figures 12C and 12E). [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 13]Figure 1 shows RT-QuIC detection of misfolded polypeptides swabbed from a stainless steel surface as maximum point ratio (MPR). All misfolded polypeptides of interest diluted to the levels specified in Table 5 were applied to a stainless steel surface for 24 h drying at room temperature, followed by immediate swab extraction and concentration. Swab extracts were analyzed for the presence of PrPSc, which may misfold recHaPrP to amyloid in the RT-QuIC reaction, and expressed as maximum point ratio (MPR, mean ± standard deviation) determined as the ratio of maximum thioflavin T (ThT) fluorescence in the entire RT-QuIC run to ThT fluorescence at the initiation cycle of the RT-QuIC reaction. The threshold for positive signal was set at 2 (red dashed line). RT-QuIC detection of all samples was for their respective dilutions listed in Table 5, except for scrapie at log-3 dilution. On each RT-QuIC plate, samples without misfolded protein (blank), normal prion protein (negative) and / or known misfolded protein (positive) were tested as controls. Brain homogenates (all diluted log-3, log-4 for bovine adapted TME) were added directly to RT-QuIC (307CL, scrapie, bovine TME, TME and squirrel monkey) as detection controls. Numbers 1-27 were sample IDs referring to samples listed in Table 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure relates to misfolded polypeptides (e.g., PrP Sc For example, the present specification provides a device (e.g., a feeding device having at least one sample collection surface) for detecting one or more samples (e.g., biological samples, e.g., saliva and / or mucus) from one or more mammals (e.g., one or more non-human mammals, e.g., cervids). In some cases, one or more samples from one or more mammals (e.g., one or more cervids) can be deposited on a sample collection surface of a device provided herein, the sample can be obtained from the sample collection surface, and one or more misfolded polypeptides (e.g., PrP ScAs described herein, the presence or absence of a misfolded polypeptide (e.g., PrP Sc ) can be recovered from the sample collection surface and identified. For example, one or more feeding devices, each having at least one sample collection surface, can be deployed in a particular geographic area and / or farm to collect saliva and / or mucus samples from a population of mammals (e.g., a herd of cervids) that feed from the feeding devices. The present specification also provides methods for identifying one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals (e.g., one or more cervids) obtained from a device provided herein. Sc Also provided are methods and materials for detecting, and optionally monitoring, one or more prion diseases (e.g., CWD) in a population of mammals (e.g., a cervid herd) based, at least in part, on the presence or absence of one or more misfolded polypeptides (e.g., PrP Sc In some cases, the presence or absence of one or more misfolded polypeptides (e.g., PrP) in a sample obtained from one or more mammals (e.g., one or more cervids) in a population of mammals (e.g., a herd of cervids) can be assessed. Sc The presence of one or more misfolded polypeptides (e.g., PrP) in a sample obtained from one or more mammals (e.g., one or more cervids) in a population of mammals (e.g., a herd of cervids) can be used to determine that at least one mammal (e.g., at least one cervid) in a population of mammals (e.g., a herd of cervids) has one or more prion diseases. In some cases, the presence of one or more misfolded polypeptides (e.g., PrP) in a sample obtained from one or more mammals (e.g., one or more cervids) in a population of mammals (e.g., a herd of cervids) can be used to determine that at least one mammal (e.g., at least one cervid) in a population of mammals (e.g., a herd of cervids) has one or more prion diseases. Sc ) can be used to determine that a mammalian population (e.g., a cervid herd) is free of prion disease.

[0015] The devices provided herein (e.g., devices such as feeding devices having at least one sample collection surface) can include any number of sample collection surfaces. In some cases, the feeding devices provided herein can include at least one (e.g., 1, 2, 3, 4, 5 or more) sample collection surface. For example, the feeding devices provided herein can include 1 to about 10 sample collection surfaces (e.g., 1 to about 7, 1 to about 5, 1 to about 4, 1 to about 3, 2 to about 10, 5 to about 10, 1 to about 10, 7 to about 10, 2 to about 8, 3 to about 5, 2 to about 4, 5 to about 7, or 6 to about 8 sample collection surfaces). In some cases, the feeding devices provided herein can include multiple (e.g., 2, 3, 4, 5 or more) sample collection surfaces. In some cases, the feeding devices provided herein can include a single sample collection surface.

[0016] The sample collection surface of the device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) can be made of any suitable material. Examples of materials that can be used as the sample collection surface of the feeding device provided herein include, but are not limited to, stainless steel, mica, slate, aluminum, ceramic and glass. When the feeding device provided herein includes multiple sample collection surfaces, the sample collection surfaces can be made of the same material or different materials.

[0017] The devices provided herein (e.g., devices such as feeding devices having at least one sample collection surface) can be placed in any suitable environment. When the devices provided herein are feeding devices, the feeding devices can be placed in a location where one or more mammals (e.g., one or more non-humans, e.g., cervids) can access the feeding device. Examples of environments in which the devices provided herein can be placed include, but are not limited to, environments in which domestic (e.g., livestock) herds live (e.g., farms, ranches, fenced hunting preserves, zoos, environments in which wild herds live (e.g., forests, grasslands, agricultural lands, e.g., alfalfa, corn, soybean, potato, wheat, and barley fields), environments in which humans live (e.g., rural, suburban, and urban areas), food processing facilities (e.g., meat processing facilities), water treatment facilities, and hospitals (e.g., human and veterinary hospitals).

[0018] When the device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) is a feeding device, the feeding device can provide any suitable type of feed. In some cases, the feeding device provided herein can be used to provide grains. In some cases, the feeding device provided herein can be used to provide pellets. Examples of feed that can be provided using the feeding device provided herein include, but are not limited to, corn, soybeans, oats, commercially available feed pellets (e.g., commercially available deer feed pellets), and any combination thereof.

[0019] In some cases, a feeding apparatus provided herein (e.g., a feeding apparatus having at least one sample collection surface) can include a reservoir (e.g., a food reservoir). For example, the reservoir can be used to store and supply food as the food passes through the sample collection surface of a feeding apparatus provided herein. In some cases, food passing from a reservoir of a feeding apparatus provided herein to a sample collection surface of a feeding apparatus provided herein can exit through an outlet. In some cases, food passing from a reservoir of a feeding apparatus provided herein to a sample collection surface of a feeding apparatus provided herein can exit through a plate (e.g., a hinged plate). For example, food passing from a reservoir of a feeding apparatus provided herein to a sample collection surface of a feeding apparatus provided herein can exit through a plate (e.g., a hinged plate) that can be a feed collection surface. In some cases, bait can pass from a reservoir of a feeding device provided herein to a sample collection surface of a feeding device provided herein to increase interaction between one or more mammals (e.g., one or more non-humans, e.g., cervids) and the sample collection surface. For example, bait can pass from a reservoir of a feeding device provided herein and accumulate at a point adjacent to a sample collection surface of a feeding device provided herein such that one or more mammals (e.g., one or more non-humans, e.g., cervids) leave a sample on the sample collection surface while reaching across the sample collection surface to reach the bait.

[0020] In some cases, a feeding apparatus provided herein (eg, a feeding apparatus having at least one sample collection surface) may be as shown in FIG. 6.

[0021] In some cases, a feeding apparatus provided herein (eg, a feeding apparatus having at least one sample collection surface) may be as shown in FIG.

[0022] The devices provided herein (e.g., devices such as feeding devices having at least one sample collection surface) can be used to collect any suitable type of sample. Examples of samples that can be obtained from the sample collection surface of the devices provided herein include, but are not limited to, biological fluids (e.g., blood, urine, and feces), saliva, and mucus. When the devices provided herein are feeding devices, the feeding devices can be used to collect any suitable type of sample from one or more mammals (e.g., one or more non-humans, e.g., cervids). In some cases, when a mammal (e.g., cervids) feeds from a feeding device provided herein, the mammal will deposit one or more samples on the sample collection surface of the feeding device. Examples of samples that can be deposited by the mammal on the sample collection surface of a feeding device provided herein when the mammal feeds from the feeding device include, but are not limited to, saliva, mucus, and epithelial cells (e.g., tongue epithelial cells).

[0023] When a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) is a feeding device, any suitable mammal (e.g., any suitable non-human mammal) can feed from the feeding device and deposit one or more samples on the sample collection surface of the feeding device. In some cases, the non-human mammal can be a cervid (e.g., a member of the Cervidae family). In some cases, the non-human mammal can be a wild non-human mammal (e.g., a wild animal). In some cases, the non-human mammal can be a domesticated non-human animal (e.g., a livestock animal). Examples of non-human mammals for which samples can be left on the feeding devices provided herein include, but are not limited to, moose, fallow deer, marsh deer, mule deer, barking deer, moose, pampas deer, red deer, reindeer, roe deer, sambar, sika deer, white-tailed deer, antelope, goat, camel, mink, cat, cow, sheep, mouse, rat, hamster, brocket deer, chital deer, and non-human primates (e.g., macaques, lemurs, spider monkeys, and chimpanzees).

[0024] The present specification also provides methods for detecting one or more misfolded polypeptides (e.g., PrP) in one or more samples from one or more mammals (e.g., one or more cervids) obtained from a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). Sc Also provided are methods for detecting the presence or absence of one or more misfolded polypeptides (e.g., PrP). For example, samples from one or more mammals (e.g., one or more cervids) can be obtained from a sample collection surface of a feeding device provided herein, and one or more misfolded polypeptides (e.g., PrP Sc In some cases, the presence or absence of one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals (e.g., one or more cervids) obtained from a sample collection surface of a feeding device provided herein can be assessed. Sc The presence of a misfolded polypeptide, such as PrP Sc For example, the method can be used to identify a population of mammals (e.g., a cervid herd) as having one or more prion diseases based at least in part on the presence of one or more misfolded polypeptides (e.g., PrP Sc A population of mammals (e.g., a herd of cervids) in an area in which the feeding apparatus is placed can be identified as having one or more prion diseases if one or more misfolded polypeptides (e.g., PrP) are detected in a sample obtained from a sample collection surface of a feeding apparatus provided herein. In some cases, one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals (e.g., one or more cervids) obtained from a sample collection surface of a feeding apparatus provided herein can be identified as having one or more prion diseases. Sc ) indicates the absence of a misfolded polypeptide (e.g., PrP Sc For example, the present invention can be used to identify a population of mammals (e.g., a cervid herd) as free of one or more prion diseases based at least in part on the absence of one or more misfolded polypeptides (e.g., PrP ScIf no prion disease is detected in a sample obtained from the sample collection surface of a feeding device provided herein, a mammalian population (e.g., a cervid herd) in the area in which the feeding device is located can be identified as being free of one or more prion diseases.

[0025] Any suitable method can be used to collect a sample from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). In some cases, a swab can be used to collect a sample from a sample collection surface of a feeding device provided herein. Any suitable swab can be used to collect a sample from a sample collection surface of a feeding device provided herein. Examples of swabs that can be used to collect a sample from a sample collection surface of a feeding device provided herein include, but are not limited to, cotton swabs (e.g., cotton-bearing swabs) and foam swabs (e.g., foam-bearing swabs).

[0026] Samples can be taken from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) at any suitable time. When a feeding device provided herein is installed in a wilderness area (e.g., forest or grassland), farmland, urban, suburban, or rural environment), a sample can be taken after the feeding device is left unattended for more than about 1 day. For example, a sample can be taken from a feeding device provided herein installed in a wilderness area after about 1 day to about 14 days after being left unattended. When a feeding device provided herein is installed in an urban, suburban, or rural environment, a sample can be taken after the feeding device is left unattended for more than about 1 day. For example, a sample can be taken from a feeding device provided herein installed in an urban, suburban, or rural environment after about 1 day to about 14 days after being left unattended. When a feeding device provided herein is installed in a farm, a sample can be taken daily. When a feeding device provided herein is installed in a farm, a sample can be taken weekly.

[0027] In some cases, the sample obtained from the sample collection surface of the device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) can be extracted from the swab used to obtain the sample. Any suitable method can be used to extract the sample from the swab. In some cases, shaking can be used to extract the sample from the swab. For example, the swab used to obtain the sample from the sample collection surface of the feeding device provided herein can be shaken in a microtube mixer (Tomy MT-360, speed 5) (e.g., in a buffer solution, e.g., PBS solution). The swab used to obtain the sample from the sample collection surface of the feeding device provided herein can be shaken for any period of time. For example, a swab used to obtain a sample from a sample collection surface of a feeding device provided herein can be shaken for about 5 minutes to about 40 minutes (e.g., about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 40 minutes, about 20 minutes to about 40 minutes, about 30 minutes to about 40 minutes, about 10 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 10 minutes to about 20 minutes, or about 20 minutes to about 30 minutes). In some cases, a swab used to obtain a sample from a sample collection surface of a feeding device provided herein can be shaken for about 30 minutes. The shaking step can be performed any number of times. For example, a swab used to obtain a sample from a sample collection surface of a feeding device provided herein can be shaken about 1 time to about 5 times (e.g., about 3 times). The shaking step can be performed at any temperature. In some cases, the swabs used to obtain samples from the sample collection surfaces of the feeding devices provided herein can be shaken at room temperature.

[0028] In some cases, sonication can be used to extract samples from swabs used to obtain samples from sample collection surfaces of devices provided herein (e.g., devices such as feeding devices having at least one sample collection surface). For example, swabs used to obtain samples from sample collection surfaces of feeding devices provided herein can be sonicated at any suitable speed (e.g., about speed 5 on a Tomy MT-360) (e.g., can be sonicated in a buffer solution, e.g., a PBS solution). Swabs used to obtain samples from sample collection surfaces of feeding devices provided herein can be sonicated at any suitable amplitude. For example, swabs used to obtain samples from sample collection surfaces of feeding devices provided herein can be sonicated at about amplitude 15 to about amplitude 20 (e.g., about amplitude 17). For example, swabs used to obtain samples from sample collection surfaces of feeding devices provided herein can be sonicated at about 0.7 watts / cm 2 ~ approx. 7 watts / cm 2 (For example, about 0.7 watts / cm 2 ~ approx. 6 watts / cm 2 , about 0.7 watts / cm 2 ~ approx. 5 watts / cm 2 , about 0.7 watts / cm 2 ~ approx. 4 watts / cm 2 , about 0.7 watts / cm 2 ~ approx. 3 watts / cm 2 , about 0.7 watts / cm 2 ~ approx. 2 watts / cm 2 , about 0.7 watts / cm 2 ~ approx. 1 watt / cm 2 , approximately 1 watt / cm 2 ~ approx. 7 watts / cm 2 , about 2 watts / cm 2 ~ approx. 7 watts / cm 2 , about 3 watts / cm 2 ~ approx. 7 watts / cm 2 , about 4 watts / cm 2 ~ approx. 7 watts / cm 2 , about 5 watts / cm 2 ~ approx. 7 watts / cm 2 , about 6 watts / cm 2~ approx. 7 watts / cm 2 , approximately 1 watt / cm 2 ~ approx. 6 watts / cm 2 , about 2 watts / cm 2 ~ approx. 5 watts / cm 2 , about 3 watts / cm 2 ~ approx. 4 watts / cm 2 , approximately 1 watt / cm 2 ~ approx. 3 watts / cm 2 , about 2 watts / cm 2 ~ approx. 4 watts / cm 2 , about 3 watts / cm 2 ~ approx. 5 watts / cm 2 , or about 4 watts / cm 2 ~ approx. 6 watts / cm 2 ) ultrasonic intensity. The swabs used to obtain samples from the sample collection surface of the feeding apparatus provided herein can be sonicated for any length of time. For example, the swabs used to obtain samples from the sample collection surface of the feeding apparatus provided herein can be sonicated for about 15 seconds to about 60 seconds (e.g., about 15 seconds to about 60 seconds, about 15 seconds to about 45 seconds, about 15 seconds to about 30 seconds, about 30 seconds to about 60 seconds, about 45 seconds to about 60 seconds, about 20 seconds to about 40 seconds, about 20 seconds to about 30 seconds, about 30 seconds to about 40 seconds, or about 40 seconds to about 50 seconds). In some cases, the swabs used to obtain samples from the sample collection surface of the feeding apparatus provided herein can be sonicated for about 15 seconds. The sonication step can be performed any number of times. For example, the swabs used to obtain samples from the sample collection surface of the feeding apparatus provided herein can be sonicated for about 1 to about 12 times. In some cases, the sonication step can be performed 1, 3, 6, or 12 times. The sonication step can be performed at any temperature. In some cases, the swabs used to obtain samples from the sample collection surfaces of the feeding devices provided herein can be sonicated at about 37° C. If more than one sonication step is performed, the swabs can be maintained at about 37° C. between each sonication step.

[0029] In some cases, a sample obtained from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) contains one or more misfolded polypeptides (e.g., PrP Sc The sample may be concentrated (e.g., vacuum concentrated) before being evaluated for the presence or absence of one or more misfolded polypeptides (e.g., PrP). For example, the extract obtained from the swab used to obtain the sample from the sample collection surface of the feeding device provided herein may be concentrated (e.g., vacuum concentrated) before being evaluated for the presence or absence of one or more misfolded polypeptides (e.g., PrP ScThe sample may be concentrated (e.g., vacuum concentrated) before being evaluated for the presence or absence of . In some cases, the concentration step (e.g., vacuum concentrated) may include centrifugation. In some cases, the concentration step (e.g., vacuum concentrated) may include evaporation (e.g., solvent evaporation). In some cases, the concentration step (e.g., vacuum concentrated) may be performed in the absence of an eluent (e.g., such that the concentration step is not dependent on the chemical composition of the eluent). In some cases, the concentration step (e.g., vacuum concentrated) may remove the liquid phase of the sample and retain the solid phase of the sample. The concentration step (e.g., vacuum concentrated) may be performed for any length of time. For example, an extract obtained from a swab used to obtain a sample from a sample collection surface of a feeding device provided herein may be vacuum concentrated for about 3 hours to about 5 hours. The concentration step (e.g., vacuum concentrated) may be performed at any temperature. For example, an extract obtained from a swab used to obtain a sample from a sample collection surface of a feeding device provided herein may be vacuum concentrated at 45°C. For example, extracts obtained from a swab used to obtain a sample from a sample collection surface of a feeding device provided herein can be concentrated under reduced pressure at about 65° C. In some cases, the concentration step (e.g., vacuum concentration) can be performed by concentrating the sample at a concentration of about 10-fold to about 100-fold (e.g., about 10-fold to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-fold, about 10-fold to about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold, about 10-fold to about 30-fold, about 10-fold to about 20-fold, about 20-fold to about 100-fold, about 30-fold to about 100-fold, about 40-fold to about 100-fold, about 50-fold to about 100-fold, or about 60-fold to about 100-fold). fold, about 60-fold to about 100-fold, about 70-fold to about 100-fold, about 80-fold to about 100-fold, about 90-fold to about 100-fold, about 20-fold to about 90-fold, about 30-fold to about 80-fold, about 40-fold to about 70-fold, about 50-fold to about 60-fold, about 20-fold to about 40-fold, about 30-fold to about 50-fold, about 40-fold to about 60-fold, about 50-fold to about 70-fold, about 60-fold to about 80-fold, or about 70-fold to about 90-fold).

[0030] In some cases, a sample obtained from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface) contains one or more misfolded polypeptides (e.g., PrP Sc The sample can be digested (e.g., digested with an enzyme, e.g., proteinase K) before being evaluated for the presence or absence of .

[0031] In some cases, a swab used to obtain a sample from a sample collection surface of a feeding device provided herein (e.g., a device such as a feeding device having at least one sample collection surface), or a sample extracted from a swab used to obtain a sample from a sample collection surface of a feeding device provided herein (e.g., a device such as a feeding device having at least one sample collection surface), contains one or more misfolded polypeptides (e.g., PrP Sc For example, a swab may be stored prior to evaluation of the sample for the presence or absence of one or more misfolded polypeptides (e.g., PrP Sc The sample can be stored moist (e.g., in a buffer solution, such as a phosphate buffered saline (PBS) solution) prior to evaluating the sample for the presence or absence of one or more misfolded polypeptides (e.g., PrP Sc The sample may be stored at a temperature of about -80°C to about 4°C (e.g., about -80°C, about -20°C, and about 4°C) prior to evaluating the sample for the presence or absence of one or more misfolded polypeptides (e.g., PrP Sc The sample may be stored at a temperature of about -80°C to about 4°C for any period of time before being evaluated for the presence or absence of .

[0032] Any suitable method can be used to detect one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals (e.g., one or more cervids) obtained from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). ScFor example, RT-QuIC, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), protein misfolding cyclic amplification (PMCA) and / or Western blot testing can be used to detect the presence or absence of one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals (e.g., one or more cervids) obtained from a sample collection surface of a feeding device provided herein. Sc ) can be used to detect the presence or absence of

[0033] The methods and materials provided herein can be used to detect the presence or absence of any misfolded polypeptide in a sample from one or more mammals (e.g., one or more cervids) obtained from a sample collection surface of a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). In some cases, the misfolded polypeptide may be associated with disease (e.g., prion disease). An example of a polypeptide that may be misfolded, where the misfolded polypeptide may be detected using the methods and materials described herein, is PrP. Sc , transmissible spongiform encephalopathy-associated misfolded polypeptide (PrP TSE ), chronic wasting disease-associated misfolded polypeptide (PrP CWD ), bovine spongiform encephalopathy-associated misfolded polypeptide (PrP BSE ), Creutzfeldt-Jakob disease-associated misfolded polypeptide (PrP CJD ), feline spongiform encephalopathy-associated misfolded polypeptide (PrP FSE ), transmissible mink encephalopathy-associated misfolded polypeptide (PrP TME ), camel spongiform encephalopathy-associated misfolded polypeptide (PrP CSE ), misfolded tau polypeptides, misfolded alpha-synuclein polypeptides and misfolded amyloid beta polypeptides.

[0034] The methods described herein (e.g., methods for detecting the presence or absence of a misfolded polypeptide) can be used to detect the presence or absence of a misfolded polypeptide associated with any prion disease. As used herein, a prion disease is characterized by misfolding and, optionally, one or more aggregates of misfolded polypeptides (e.g., PrP Sc Examples of prion diseases associated with polypeptides that can be misfolded, where the misfolded polypeptides can be detected as described herein, include, but are not limited to, CWD, TME, BSE, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, camelid spongiform encephalopathy, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), Alzheimer's disease, and Parkinson's disease.

[0035] The present specification also provides methods for detecting one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals obtained from a device provided herein. Sc Also provided are methods and materials for monitoring a mammalian population (e.g., a cervid herd) for the presence or absence of one or more prion diseases described herein (based at least in part on the presence or absence of PrP in a sample obtained from a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). Sc If a mammal is identified as having one or more prion diseases based at least in part on the presence of one or more misfolded polypeptides (e.g., PrP), a population of mammals (e.g., a herd of deer) that feeds from the feeding device can be monitored more frequently for the presence or absence of one or more prion diseases. For example, one or more misfolded polypeptides (e.g., PrP) in samples from one or more mammals obtained from the devices provided herein can be monitored more frequently for the presence or absence of one or more prion diseases. ScA population of mammals (e.g., a cervid herd) identified as having one or more prion diseases described herein (based at least in part on the presence or absence of a prion gene), can be assessed for the presence or absence of one or more prion diseases on about a daily to about a monthly basis (e.g., once a day, once a week, or once a month).

[0036] A population of mammals (e.g., a herd of cervids) is subjected to detection of one or more misfolded polypeptides (e.g., PrP) in a sample obtained from a device provided herein (e.g., a device such as a feeding device having at least one sample collection surface). Sc In some cases where a mammal has been identified as having one or more prion diseases based at least in part on the presence of one or more misfolded polypeptides (e.g., PrP), one or more additional mammalian populations living nearby (e.g., one or more additional cervid herds) can be evaluated for the presence or absence of one or more prion diseases to monitor any spread of the one or more prion diseases. For example, one or more misfolded polypeptides (e.g., PrP) in a sample from one or more mammals obtained from a device provided herein can be evaluated for the presence or absence of one or more prion diseases to monitor any spread of the one or more prion diseases. ScOne or more mammal populations (e.g., one or more cervid herds) that reside about 1 mile to about 60 miles (e.g., about 1 mile to about 50 miles, about 1 mile to about 40 miles, about 1 mile to about 30 miles, about 1 mile to about 20 miles, about 1 mile to about 10 miles, about 10 miles to about 60 miles, about 20 miles to about 60 miles, about 30 miles to about 60 miles, about 40 miles to about 60 miles, about 50 miles to about 60 miles, about 10 miles to about 50 miles, about 20 miles to about 40 miles, about 10 miles to about 20 miles, about 20 miles to about 30 miles, about 30 miles to about 40 miles, or about 40 miles to about 50 miles) from a mammal population (e.g., one or more cervid herds) identified as having one or more prion diseases described herein (based at least in part on the presence or absence of a prion disease vector or gene(s) present in the mammal population (e.g., one or more cervid herds) can be evaluated for the presence or absence of one or more prion diseases. For example, one or more mammalian populations (e.g., one or more cervid herds) that live adjacent (e.g., adjacent to and separated by a barrier, e.g., a fence) to a mammalian population (e.g., a cervid herd) that has been identified as having one or more prion diseases described herein (e.g., based at least in part on the presence or absence of one or more misfolded polypeptides (e.g., PrPSc) in samples from one or more mammals obtained from a device provided herein) can be evaluated for the presence or absence of one or more prion diseases.

[0037] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0038] [Example 1] Quantitative measurement of chronic wasting disease prions recovered from swab samples and environmentally relevant surfaces This example describes a rapid method to obtain prions by swabbing different types of surfaces, extracting prions from the swabs, and quantifying CWD prions recovered on the swabs.

[0039] Materials and Methods Preparation of prion sources and tissues Brain tissue was collected from a hamster infected with the Hyper (HY) strain of transmissible mink encephalopathy (TME) and from three moose infected with CWD, designated isolates CWD t1821, CWD JB R296, CWD JB B188, and CWD 307 CL. Brain tissue was homogenized with Ca using a strain-specific Tenbroeck tissue grinder (Kontes, Vineland, NJ) or a Beadblaster 24 microtube homogenizer (D2400, Benchmark Scientific, Inc., Sayreville, NJ, US). 2+ Or Mg 2+ The cells were homogenized in 10 or 20% (w / v) Dulbecco's Phosphate Buffered Saline (DPBS) (Mediatech, Herndon, VA) or PBS. Samples were stored at -80°C until use.

[0040] Swab Contamination Cotton-backed swabs (3M™ Quick Swab, 3M, Saint Paul, MN, US) and foam-backed swabs (Fisherbrand™ PurSwab Foam Swabs, Catalog No.: 14-960-3E, Thermo Fisher Scientific, Waltham, MA, US) were used. To contaminate the swabs with prions, 500 (HY TME, CWD JB B188 and CWD JB R296) or 1000 (CWD t1821) μg brain equivalent (BE) of brain homogenate (BH) was applied to the surface of the swab by pipette. Triplicate contaminated swabs were incubated at room temperature and allowed to dry for 0 hours (undried control), 0.25 hours, 0.5 hours, 1 hour, 6 hours, 12 hours or 24 hours. After drying, the swabs were immediately placed in 1.7 mL microcentrifuge tubes containing 300 μL (for foam swabs) or 500 μL (for cotton swabs) of DPBS to completely cover the swab tips, and the swab handles were cut to fit into the tubes with the caps closed.

[0041] Surface contamination Ten-fold serial dilutions of brain homogenates ranging from 500 to 0.5 (HY TME, CWD JB B188, and CWD JB R296) or 1000 to 1 (CWD t1821) μg BE were applied by pipette to glass slides (Fisherbrand Superfrost Plus Microscope slides, catalog number: 12-550-15), stainless steel (316L grade, Millard Metal Services, La Vista, NE, US), or oak wood coupons (Lowe's, Omaha, NE, US). Samples were taken with foam-tipped swabs after the contaminated surfaces were allowed to dry for 24 h at room temperature. The swabs were moistened with ultrapure water and then applied 10 times to the surface with rotation to maximize swab surface exposure. To prepare positive controls, surfaces contaminated with the highest levels of prions (500 μg BE for CWD JB B188 and CWD JB R296, 1000 μg BE for CWD t1821) were swabbed with dry swabs immediately after contamination in a manner that absorbed all liquid. Surfaces without prion contamination (negative controls) were swabbed as described for prion-contaminated surfaces. To prepare prion-contaminated surfaces for RT-QuIC detection, 50 μL from each dilution (log-2 to -6) of CWD 307 CL was applied to stainless steel and dried for 24 h at 22 °C before wiping with foam-tipped swabs as described above. Surfaces were prepared in triplicate for each sample, including positive and negative controls. According to previous experiments, subsequent swabs failed to recover detectable prions, so one swab was used for each area (Table 1). The tips of the swabs, cut to fit the tubes, were each placed into a 1.7 mL microcentrifuge tube containing 300 μL of DPBS, covering the foam tips, and immediately extracted.

[0042] [Table 1]

[0043] Swab Extraction The swabs in the microcentrifuge tubes were incubated with shaking or sonication for prion extraction. Four replicates of swabs were prepared for the swab drying experiments. For shaking extraction, wet or dried swabs were incubated in 300 μL (for foam swabs) or 500 μL (for cotton swabs) of DPBS and agitated on a microtube mixer (Tomy MT-360, speed 5) at room temperature for 30 minutes for the first extraction. This was followed by two successive extractions in which the swabs were incubated in different microcentrifuge tubes containing 200 μL (for foam swabs) or 300 μL (for cotton swabs) of DPBS for 30 minutes for each extraction. For sonication extraction, the swabs stored in the microcentrifuge tubes were placed in a QSonica sonicator (model Q700) with the amplitude set at level 17, producing an average power output of approximately 170 W during sonication. Sonication was performed at 37°C and consisted of a different number (1, 3, 6 or 12) of treatment cycles (5 seconds of sonication followed by 5 seconds of incubation) depending on the desired length of total treatment time for each extraction (5 seconds, 15 seconds, 30 seconds or 60 seconds). For each swab, three consecutive extractions with the same length of sonication time were applied. The buffer usage for sonication extraction was also the same as for shaking. Extracts (approximately 200 μL each for foam swabs and approximately 300 μL each for cotton swabs) were stored at -80°C until ready for concentration. Swabs used for surface sampling were extracted twice (one time for negative surface control swabs) using 15 seconds of sonication for each extraction (three treatment cycles). Extracts for each swab were combined (approximately 400 μL) and stored at -80°C.

[0044] Swabs used for surface sampling were extracted twice (one time for negative surface control swabs for immunodetection) using 15 seconds of sonication for each extraction (three treatment cycles). Extracts were either combined (approximately 400 μL for swabs for immunodetection) or stored separately (approximately 200 μL for swabs for RT-QuIC). All extracts were stored at -80°C.

[0045] Extract concentration All extracts were concentrated under vacuum using a Savant Speed-Vac concentrator equipped with a Savant refrigerated vapor trap (RVT4104). Samples were evaporated at high speed (65°C in chamber) for 3, 4 or 5 hours for 200 μL, 300 μL or 400 μL extracts, respectively. Extracts for RT-QuIC detection were concentrated under vacuum using a SpeedVac (SPD 1030, Thermo Fisher Scientific, Waltham, MA, US). Samples were evaporated at 10 Torr vacuum at 45°C for 2 hours and rehydrated with 50 μL of ultrapure water. Concentrated extracts were rehydrated with 10 μL, 15 μL or 20 μL of ultrapure water, respectively, and stored at -80°C before analysis.

[0046] Detection and Quantification Samples were digested with 23.25 μg / mL (HY TME, CWD JB B188, and CWD JB R296) or 46.5 μg / mL (CWD t1821) proteinase K (PK) (Roche Diagnostics Corporation, Indianapolis, IN) for 30 min at 37° C. with constant agitation. 96-well immunoblot assays were performed with primary monoclonal antibodies 3F4 (Sigma-Aldrich, St. Louis, MO, US; 0.1 μg / mL, 37° C. 1 h) for HY TME or 8H4 (Sigma-Aldrich, St. Louis, MO, US; 0.17 μg / mL, 37° C. 1 h) for CWD, and secondary antibodies (horseradish peroxidase-conjugated anti-mouse IgG, Invitrogen, Carlsbad, CA, US; 0.01 μg / mL, 37° C. 30 min). Well plate membranes were developed with Supersignal West Femto maximum sensitivity substrate according to the manufacturer's instructions (Pierce, Rockford, IL, US), imaged on a 4000R imaging station (Kodak, Rochester, NY), and analyzed using Kodak (New Haven, CT) molecular imaging software (v.5.0.1.27), which outputs the net intensity of each well. For each plate, a 2-fold dilution control was prepared to measure PrP levels used to quantify sample intensities. Sc A linear regression (standard curve) of the abundance of PrP recovered from the swabs or surfaces was generated. Sc Load an aliquot of 100 μl (e.g., 1 / 10 to generate a signal within the linear range) into the wells and measure PrP according to the standard curve. Sc The amount of recovered PrP was then calculated. Sc The total amount of PrP used in this study was adjusted for aliquots. Sc The units are PrP in μg of wet weight of brain tissue. Sc The content was expressed in μg brain equivalent (BE). The recovery rate was calculated by the amount of recovered PrP Sc Total amount of PrP initially loaded ScStatistical analysis was performed using GraphPad Prism 8 with an unpaired parametric Welch t test.

[0047] result Surface drying reduced prion recovery from swabs extracted under gentle shaking HY PrP from each extraction and / or three consecutive extractions of swabs dried for different times Sc (Figures 1A, 1B, 1C, and 1D) and CWD t1821 PrP Sc (Figures 1E, 1F, 1G and 1H) Detection and quantification of HY PrP from non-dried (0 hours) foam swabs. Sc The recovery rate of HY PrP from the foam swabs was 49% ± 1% (mean ± standard error of the mean) (Figure 1B). Sc The recovery rate was similarly maintained at 50% ± 4% (p>0.05) and significantly decreased to 36% ± 2% after 30 min of drying (0.5 h) (p<0.05) (Figure 1B). After 1 h of drying, the extracted HY PrP Sc PrP Sc The PrP concentration was close to the detection limit (Fig. 1A), and the recovery rate was significantly reduced to almost 1% (p<0.05) (Fig. 1B). When HY was applied to the cotton swab (Fig. 1C and 1D), the PrP concentration was significantly reduced without drying (0 h). Sc PrP was recovered at 51% ± 12% and significantly decreased to less than 6% after 15 min of drying (0.25 h) (p < 0.05). Similar results were observed for CWD. From foam swabs, CWD PrP was recovered at 0, 0.25, and 0.5 h of drying. Sc The total recoveries of CWD PrP were 68% ± 7%, 58% ± 3%, and 59% ± 7%, respectively (Figures 1E and 1F). After drying for 1 h, the extracted CWD PrP Sc The CWD PrP concentration in the cotton swabs was significantly reduced to less than 5% (p<0.05) (Figures 1E and 1F). Sc were recovered, and the total CWD recovery rate subsequently decreased significantly to below 12% (p<0.05) (Figures 1G and 1H).

[0048] To investigate whether differences between foam and cotton swabs are related to drying dynamics, we performed a 10-mL swab analysis of HY or CWD PrP Sc The moisture content of both types of swabs was measured, either without or with the same volume of ultrapure water. The overall trend in swab moisture content was similar between all conditions, with the moisture content decreasing to nearly 10% after 30 min of surface drying and decreasing further through 1 h of drying (Figure 1), suggesting that the difference in moisture content between the cotton and foam swabs did not affect prion recovery.

[0049] Enhanced HY recovery from dried foam swabs using ultrasonic treatment Higher energy mechanical forces dried the surface of PrP Sc To investigate whether ultrasonication could improve the extraction efficiency of HY, foam swabs were sonicated for 5, 15, 30, and 60 seconds immediately after application of HY (Figures 2A and 2B). Sc The recoveries were 66% ± 16%, 58% ± 4%, 51% ± 4% and 52% ± 5%, respectively. Sc The recoveries of HY PrP were similar between the sonication times tested and from undried foam swabs extracted with gentle shaking (p>0.05) (Figures 1A and 1B). After drying on the foam swabs for 24 hours, total HY PrP Sc The recoveries were 22% ± 6%, 46% ± 14%, 57% ± 21%, and 41% ± 18% for sonication times tested ranging from short (5 s) to long (60 s). PrP from sonicated and dried foam swabs Sc The recoveries of PrP from dried foam swabs extracted with gentle shaking were generally significantly (p<0.05) greater than those from dried swabs extracted with gentle shaking (>1 h) (Figures 1A and 1B). Overall, sonication reduced PrP from dried foam swabs. Sc The recovery rate increased.

[0050] Recovery of surface-dried CWD from foam swabs by sonication extraction varied among isolates To examine the efficiency of sonication extraction of surface-dried CWD from foam swabs, CWD-contaminated swabs were extracted with 15 s of sonication either immediately (0 h) or after 24 h of drying (24 h). After three successive extractions, total CWD PrP from undried foam swabs was Sc The recoveries were 57% ± 4% for CWD t1821, 50% ± 5% for CWD JB R296, and 34% ± 3% for CWD JB B188 (Figure 3). After 24 h of drying, total CWD PrP Sc The recoveries were significantly (p<0.05) reduced to 13% ± 2%, 32% ± 3% and 10% ± 2%, respectively. PrP from wet foam swabs contaminated with either CWD t1821 or CWD JB R296 Sc The total recovery of CWD JB R296 from the foam swabs dried for 24 hours was significantly (p<0.05) higher compared to the foam swabs contaminated with CWD JB B188, whereas the total recovery of CWD JB R296 from the foam swabs dried for 24 hours was higher than the other samples (p<0.05). Sc Recovery of CWD t1821 was improved by sonication extraction. From undried foam swabs, total CWD t1821 recovery was similar between gentle shaking and sonication (p>0.05) (Figures 1E and 1F and Figures 3A and 3B). Sonication extraction from dried foam swabs yielded higher (p<0.05) CWD PrP recovery compared to shaking extraction. Sc This resulted in a recovery rate (Figures 1E and 1F and Figures 3A and 3B).

[0051] Prion recovery from glass and stainless steel is higher than from wood Prions from three environmentally associated surfaces contaminated with the three CWD isolates were sampled using foam swabs. Sc was extracted from the swabs using a brief sonication extraction (15 s) (Figure 4). Immediate swabbing after surface contamination with the highest levels of CWD prions (1000 μg brain equivalent (BE) for CWD t1821, 500 μg BE for CWD JB R296 and CWD JB B188) and immediate swab extraction (control) resulted in the extraction of CWD PrPSc The recoveries of PrP ranged from 25% to 99% on glass and stainless steel surfaces, with the majority being around 30% (Table 2 and Figures 4A-4I). In contrast, contamination of wood with CWD resulted in significantly lower (p<0.05, except Figures 4G vs. 4J and 4I vs. 4L) PrP from unseasoned surfaces (controls) for all CWD isolates tested. Sc At the highest contamination levels, all CWD isolates had comparable (p>0.05) recoveries after 24 h drying from glass and stainless steel compared to their controls (Table 2 and Figures 4A-4I). However, at the highest contamination levels, CWD PrP extracted from wood after 24 h drying was significantly higher than that of wood (Table 2 and Figures 4A-4I). Sc was significantly decreased (p<0.05) compared to their controls (Table 2 and Figures 4A-4C and 4J-4L). At all other lower contamination levels, regardless of surface type, CWD PrP Sc Swab extracts from surfaces without prion contamination (negative controls) showed undetectable CWD PrP for all surface types. Sc was produced (Table 3).

[0052] [Table 2]

[0053] [Table 3]

[0054] In the swab-free experimental system, the PrP Sc 10-50% of the samples were not recovered (Figures 5B and 5D).

[0055] However, this contribution may be limited and / or may vary by prion strain / isolate, as the HY and one CWD isolate were not significantly reduced (p>0.05) (Figures 5A and 5C).

[0056] Taken together, these results demonstrate that misfolded polypeptides (e.g., CWD prions) can be recovered from stainless steel, glass and wood using swabs. The methods and materials for recovering misfolded polypeptides can be used for prion detection and potentially monitoring in the natural environment.

[0057] [Example 2] RT-QuIC detection sensitivity of CWD wiped off stainless steel surfaces Materials and Methods RT-QuIC reaction RT-QuIC was performed and analyzed as described elsewhere (Schwabenlander et al., J. Wildl. Dis., 58(1):50-62(2022); and Wilham et al., PloS Pathog., 6(12):e1001217(2010)). Briefly, the control serial dilutions and swab extracts from Example 1 were diluted 10-fold in 0.1% SDS / 1X PBS / 1x N2. 2 μL of control and swab extract dilutions were added to 98 μL of RT-QuIC reaction buffer. The reaction buffer was made at the following concentrations: 1X PBS, 170 mM NaCl, 1 mM EDTA, 10 μM Thioflavin T (ThT), and 0.1 mg / mL recombinant hamster prion protein (recHaPrP). Reactions were read for ThT fluorescence emitting at approximately 450 / 480 maximum excitation / emission every 45 minutes for 48 hours. Shaking was performed in a dual orbit at 700 rpm for 1 minute with 1 minute of rest. Maximum point ratios (MRPs) were calculated by taking the maximum fluorescence of each well and dividing it by the initial fluorescence (i.e., background fluorescence) as described elsewhere (Vendramelli et al., J. Clin. Microbiol., 56:e00423-18 (2018)). Amyloid formation rates (RAFs) were also calculated per well as the reciprocal of the time required for fluorescence to reach twice the background fluorescence.

[0058] result RT-QuIC detection of CWD prions from swabs and stainless steel surfaces To apply the optimized prion swab extraction method to the detection of prions swabbed from surfaces, the following experimental method was utilized. First, serial 10-fold dilutions of CWD-infected brain (isolate 307CL) were extracted from 10 -2 (10μg brain equivalent / ml)~10 -6 (0.001 μg brain equivalent / ml) (Figure 8A). These dilutions were directly analyzed by RT-QuIC. -5 The same serial 10-fold dilutions of CWD-infected brains were then applied to foam-tipped swabs, extracted using the optimized protocol, and the extracts analyzed using RT-QuIC, resulting in a detection limit of 10 in all three experimental replicates. -5 Finally, the same serial 10-fold dilutions of CWD-infected brains were applied to a stainless steel surface, allowed to dry for 24 hours at 22°C, and then the surface was swabbed and the swabs were extracted using the optimized protocol, and analysis of the extracts using RT-QuIC resulted in a detection limit of 10 in all three experimental replicates. -5 A detection limit of 1 log was obtained (Figures 8A and 8C). The detection limit of RT-QuIC CWD was similar between direct analysis of serial 10-fold dilutions of CWD-infected brain, analysis of CWD applied directly to a swab (pool in Figure 8B), and CWD applied to a stainless steel surface, allowed to dry for 24 hours, and then wiped off (pool in Figure 8C). CWD was extracted a second time from the same swab to determine whether additional RT-QuIC seeding activity could be recovered. RT-QuIC seeding activity from the second extraction of CWD prions applied directly to a swab (Figure 8B) or recovered from a surface (Figure 8C) was reduced by 1 log compared to the first extraction (Figure 9). In the RAF analysis, consistent results were observed for both the first (Figure 10) and second (Figure 11) extracts. Extracts of the negative control, which was a DPBS-loaded swabs and a non-contaminated surface, did not initiate the RT-QuIC reaction (Figure 12). Overall, RT-QuIC detection of CWD prions recovered from stainless steel surfaces by swab sampling was comparable to detection of CWD prions when added directly to the RT-QuIC reaction.

[0059] Taken together, these results demonstrate that a prion sampling and recovery method combined with an ultrasensitive detection method allows for sensitive prion detection from contaminated environmental surfaces.

[0060] [Example 3] RT-QuIC detection of CWD and TSE wiped off stainless steel surfaces This example demonstrates that other prions, and other prion-like polypeptides in addition to CWD, can be detected by RT-QuIC. This example also demonstrates that RT-QuIC can successfully detect misfolded polypeptides under various sonication and concentration conditions.

[0061] Materials and Methods To investigate whether different sonication and concentration conditions could be used in RT-QuIC, the following experiment was performed.

[0062] Brain homogenates (10% w / v) affected by synthetic alpha-synuclein and four prion strains including sheep scrapie, transmissible mink encephalopathy (TME), bovine-adapted TME or squirrel monkey-adapted CWD prions were diluted to the desired levels in PBS to contaminate stainless steel surfaces (Table 4). CWD 307 CL and PBS were tested as positive and negative controls for extraction and detection. 50 μL samples from each dilution were added to the stainless steel surface in triplicate and allowed to dry at room temperature for 24 hours. Foam-tipped swabs were moistened with ultrapure water and wiped on the surface. Swabs were placed into 15 mL conical tubes preloaded with 500 μL PBS (one swab per tube) or the handle was cut to fit into 1.7 mL microcentrifuge tubes preloaded with 300 μL PBS (one swab per tube), and samples were then extracted and concentrated under the test conditions (Table 5). Each swab was extracted once. Extracts from 15 mL tubes were transferred to 1.7 mL microcentrifuge tubes for concentration. For vacuum concentrated samples, 50 μL of 0.1% SDS in PBS supplemented with N-2 (1x) was added for resuspension. For centrifugation concentrated samples, the supernatant was collected by pipette and the invisible pellet was resuspended in 50 μL (307CL) or 20 μL (synthetic alpha-synuclein) 0.1% SDS in PBS. After resuspension, samples were either loaded directly onto the RT-QuIC plate or further diluted for detection.

[0063] [Table 4]

[0064] result All samples under the conditions tested were successfully detected, ranging from about 0.72 to about 8.84 watts / cm 2These results show that ultrasonic intensities in the range of 1000 x g can extract various misfolded polypeptides swabbed from stainless steel surfaces using both vacuum concentration and centrifugation at 16,000 x g or higher. Both microcentrifuge and centrifuge tubes can be used for swab extraction. RT-QuIC detection of misfolded polypeptides swabbed from stainless steel surfaces as maximum point ratios (MPRs) is shown in Figure 13.

[0065] [Table 5] TIFF2024521944000006.tif253156

[0066] [Example 4] Herd-level monitoring of CWD in the environment A feeding apparatus having at least one sample collection surface (e.g., a stainless steel sample collection surface) is placed in an area (e.g., a particular geographic area and / or farm) known to contain a herd of deer. An exemplary feeding apparatus is shown in Figures 6 and 7.

[0067] When feeding from a feeding device having at least one sample collection surface (e.g., a stainless steel sample collection surface), cervids leave a deposit of saliva and / or mucus on the sample collection surface. The sample collection surface (e.g., a stainless steel sample collection surface) of the feeding device is used to detect misfolded polypeptides (e.g., PrP Sc For example, the sample collection surface of the feeding apparatus (e.g., a stainless steel sample collection surface) is wiped with a foam swab and / or a cotton swab. Optionally, the swab is kept moist (e.g., in a buffer solution, e.g., a PBS solution). Polypeptides are extracted from the swabs as described in Example 1. Misfolded polypeptides (e.g., PrP Sc ), if present, is detected and quantified as described in Example 1.

[0068] Other embodiments Although the present invention has been described in conjunction with a detailed description thereof, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for evaluating a population of non-human mammals for prion disease, comprising: Obtaining a swab containing a sample by wiping a sample collection surface of a feeding device, wherein the feeding device includes a reservoir and a sample collection surface, the reservoir contains food, and the food can pass from the reservoir to the sample collection surface; Extracting a polypeptide from the sample to obtain an extract; Concentrating the extract to obtain a concentrated extract; Detecting the presence of misfolded polypeptide in the concentrated extract; When the presence of the misfolded polypeptide is detected, identifying the population of non-human mammals as having the prion disease; and When the presence of the misfolded polypeptide is not detected, identifying the population of non-human mammals as not having the prion disease. A method comprising the above steps.

2. The method according to claim 1, wherein the sample collection surface is a material selected from the group consisting of stainless steel, mica, slate, aluminum, ceramic, and glass.

3. The method according to claim 1, wherein the sample is selected from the group consisting of saliva, mucus, and tongue epithelial cells.

4. The method according to claim 1, wherein the feeding device is installed in a pristine natural environment conservation area, an urban environment, a suburban environment, or a rural environment.

5. The method according to claim 4, wherein the feeding device is unattended for about 1 to about 14 days.

6. The method according to claim 1, wherein the sample is obtained daily or weekly.

7. The method according to claim 1, wherein the swab is a cotton swab or a foam swab.

8. The method according to claim 7, wherein the swab is stored in a solution before extracting the polypeptide from the sample to obtain the extract. **Claim 9** The method according to claim 8, wherein the swab is stored at a temperature of about -80 °C to about 4 °C. **Claim 10** The method according to claim 1, wherein the extraction includes sonication. **Claim 11** The misfolded polypeptide is a misfolded polypeptide associated with scrapie (PrP Sc ), a misfolded polypeptide associated with chronic wasting disease (PrP CWD ), a misfolded polypeptide associated with bovine spongiform encephalopathy (PrP BSE ), a misfolded polypeptide associated with Creutzfeldt-Jakob disease (PrP CJD ), a misfolded polypeptide associated with feline spongiform encephalopathy (PrP FSE ), a misfolded polypeptide associated with transmissible mink encephalopathy (PrP TME ), and a misfolded polypeptide associated with ovine spongiform encephalopathy (PrP CSE ), and is selected from the group consisting of: the method according to claim 1. **Claim 12** The prion disease is selected from the group consisting of chronic wasting disease (CWD), transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE), scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, and ovine spongiform encephalopathy, the method according to claim 1. **Claim 13** A method for detecting a misfolded polypeptide, comprising: obtaining a swab containing a sample by wiping a sample collection surface; extracting a polypeptide from the sample to obtain an extract; concentrating the extract to obtain a concentrated extract; and detecting the presence of the misfolded polypeptide in the concentrated extract. The method comprising. **Claim 14** The method according to claim 13, wherein the sample collection surface is a material selected from the group consisting of stainless steel, mica, slate, aluminum, ceramic, and glass.

15. The method according to claim 13, wherein the collection surface is in a food processing facility, a water treatment facility, or a hospital.

16. The method according to claim 13, wherein the sample is selected from the group consisting of blood, urine, feces, saliva, and mucus.

17. The method according to claim 13, wherein the sample is obtained daily or weekly.

18. The method according to claim 13, wherein the swab is a cotton swab or a foam swab.

19. The method according to claim 18, wherein the swab is stored in a solution before extracting the polypeptide from the sample to obtain the extract.

20. The method according to claim 18, wherein the swab is stored at a temperature of about -80°C to about 4°C.

21. The method according to claim 13, wherein the extraction includes sonication.

22. The method according to claim 13, wherein the misfolded polypeptide is selected from the group consisting of misfolded tau polypeptide, misfolded alpha-synuclein polypeptide, and misfolded amyloid-beta polypeptide.

23. The method according to claim 13, wherein the misfolded polypeptide is associated with a prion disease.

24. The method according to claim 23, wherein the prion disease is selected from the group consisting of Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), Alzheimer's disease, and Parkinson's disease.

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