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.

A field-deployable diagnostic assay for the visual detection of misfolded prions.

Diagnostic tools for the detection of protein-misfolding diseases (i.e., proteopathies) are limited. Gold nanoparticles (AuNPs) facilitate sensitive diagnostic techniques via visual color change for the identification of a variety of targets. In parallel, recently developed quaking-induced conversion (QuIC) assays leverage protein-amplification and fluorescent signaling for the accurate detection of misfolded proteins. Here, we combine AuNP and QuIC technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with chronic wasting disease (CWD), a prion disease of cervids. Our newly developed assay, MN-QuIC, enables both naked-eye and light-absorbance measurements for detection of misfolded prions. MN-QuIC leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. In addition to laboratory-based tests, we deployed to a rural field-station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not-detected deer at the field site in 24 h, thus documenting the portability of the assay. White-tailed deer tissues used to validate MN-QuIC included medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsils. Importantly, all of the white-tailed deer (n = 63) were independently tested using ELISA, IHC, and/or RT-QuIC technologies and results secured with MN-QuIC were 95.7% and 100% consistent with these tests for positive and non-detected animals, respectively. We hypothesize that electrostatic forces help govern the AuNP/prion interactions and conclude that MN-QuIC has great potential for sensitive, field-deployable diagnostics for CWD, with future potential diagnostic applications for a variety of proteopathies.

Sensitive detection of chronic wasting disease prions recovered from environmentally relevant surfaces.

Chronic wasting disease (CWD) has been identified in 30 states in the United States, four provinces in Canada, and recently emerged in Scandinavia. The association of CWD prions with environmental materials such as soil, plants, and surfaces may enhance the persistence of CWD prion infectivity in the environment exacerbating disease transmission. Identifying and quantifying CWD prions in the environment is significant for prion monitoring and disease transmission control. A systematic method for CWD prion quantification from associated environmental materials, however, does not exist. In this study, we developed an innovative method for extracting prions from swabs and recovering CWD prions swabbed from different types of surfaces including glass, stainless steel, and wood. We found that samples dried on swabs were unfavorable for prion extraction, with the greatest prion recovery from wet swabs. Using this swabbing technique, the recovery of CWD prions dried to glass or stainless steel was approximately 30% in most cases, whereas that from wood was undetectable by conventional prion immunodetection techniques. Real-time quake-induced conversion (RT-QuIC) analysis of these same samples resulted in an increase of the detection limit of CWD prions from stainless steel by 4 orders of magnitude. More importantly, the RT-QuIC detection of CWD prions recovered from stainless steel surfaces using this method was similar to the original CWD prion load applied to the surface. This combined surface swabbing and RT-QuIC detection method provides an ultrasensitive means for prion detection across many settings and applications.

A Protocol for Prion Discovery in Plants.

Recently a likely prion was found in the proteome of Arabidopsis thaliana based on inclusive compositional similarity to known yeast prion-like domains (PrLDs) and gene ontology analysis. A total of 474 proteins in the Arabidopsis thaliana proteome showed significant compositional similarity to known PrLDs in yeast warranting further analysis. In this chapter, we describe the use and limitations of the PLAAC (Prion-Like Amino Acid Composition) software for the identification of prions, specifically as it has recently been applied to identifying the first prion in plants. Our interest in this method, though presented from a plant-based perspective here, is broad and is primarily in using the method for comparative assessment with novel prion identification algorithms currently under development in our lab. This chapter is not meant to serve as a replete description of the architecture and use of HMM in prion prediction in general but is intended to serve as a reference for implementation and interpretation of output from PLAAC and its application to plant proteomes.

POSCAbilities: The Application of the Prion Organotypic Slice Culture Assay to Neurodegenerative Disease Research.

Prion diseases are fatal, transmissible neurodegenerative disorders whose pathogenesis is driven by the misfolding, self-templating and cell-to-cell spread of the prion protein. Other neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease, share some of these prion-like features, with different aggregation-prone proteins. Consequently, researchers have begun to apply prion-specific techniques, like the prion organotypic slice culture assay (POSCA), to these disorders. In this review we explore the ways in which the prion phenomenon has been used in organotypic cultures to study neurodegenerative diseases from the perspective of protein aggregation and spreading, strain propagation, the role of glia in pathogenesis, and efficacy of drug treatments. We also present an overview of the advantages and disadvantages of this culture system compared to in vivo and in vitro models and provide suggestions for new directions.

Shedding and stability of CWD prion seeding activity in cervid feces.

CWD is an emergent prion disease that now affects cervid species on three continents. CWD is efficiently spread in wild and captive populations, likely through both direct animal contact and environmental contamination. Here, by longitudinally assaying in feces of CWD-exposed white-tailed deer by RT-QuIC, we demonstrate fecal shedding of prion seeding activity months before onset of clinical symptoms and continuing throughout the disease course. We also examine the impact of simulated environmental conditions such as repeated freeze-thaw cycles and desiccation on fecal prion seeding activity. We found that while multiple (n = 7) freeze-thaw cycles substantially decreased fecal seeding activity, desiccation had little to no effect on seeding activity. Finally, we examined whether RT-QuIC testing of landscape fecal deposits could distinguish two premises with substantial known CWD prevalence from one in which no CWD-infected animals had been detected. In the above pilot study, this distinction was possible. We conclude that fecal shedding of CWD prions occurs over much of the disease course, that environmental factors influence prion seeding activity, and that it is feasible to detect fecal prion contamination using RT-QuIC.

Preclinical biomarkers of prion infection and neurodegeneration.

Therapeutic strategies and study designs for neurodegenerative diseases have started to explore the potential of preventive treatment in healthy people, emphasising characterisation of biomarkers capable of indicating proximity to clinical onset. This need is even more pressing for individuals at risk of prion disease given its rarity which virtually precludes the probability of recruiting enough numbers for well powered preventive trials based on clinical endpoints. Experimental mouse inoculation studies have revealed a rapid exponential rise in infectious titres followed by a relative plateau of considerable duration before clinical onset. This clinically silent incubation period represents a potential window of opportunity for the adaptation of ultrasensitive prion seeding assays to define the onset of prion infection, and for neurodegenerative biomarker discovery through similarly sensitive digital immunoassay platforms.

What is strain in neurodegenerative diseases?

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by the aggregation of misfolded proteins, including Aß, tau and α-synuclein. It is well recognized that these misfolded proteins are able to self-propagate and spread throughout the nervous system and cause neuronal injury in a way that resembles prion disease. These disease-specific misfolded proteins demonstrate unique features, including the seeding barrier, the conformational memory effect, strain selection and strain evolution, based on the presence of various strains. However, the accurate definition of the term strain remains to be clarified. Here, a clear interpretation is proposed by a retrospective of its history in prion research and the recent progress in neurodegeneration research. Furthermore, the causes contributing to the genesis of various strains are also summarized. Deeper insight into strains helps us to understand the phenomena we observe in this field and it also enlightens us on the elusive mechanisms and management of neurodegeneration.

A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering.

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Clinicopathological findings of an MM2-cortical-type sporadic Creutzfeldt-Jakob disease patient with cortical blindness during a course of glaucoma and age-related macular degeneration.

Here, we report an autopsy-verified patient with MM2-coritical-type sporadic Creutzfeldt-Jakob disease (MM2C-type sCJD) presenting cortical blindness during a course of glaucoma and age-related macular degeneration, and focus on the difficulties involved in early clinical diagnosis. An 83-year-old man was admitted to our hospital 15 months after the onset of cortical blindness, and 9 months after the onset of progressive dementia. Neurological examination revealed dementia, frontal signs, visual disturbance, dysphagia, myoclonus and exaggerated tendon reflexes in the four extremities. Diffusion-weighted MRI (DW-MRI) showed cortical hyperintensities predominantly in the bilateral occipital lobes. PRNP gene analysis showed no mutations with methionine homozygosity at codon 129. Cerebrospinal fluid (CSF) examination revealed elevation of 14-3-3 and total tau protein. The symptoms progressed gradually, and the patient died of aspiration pneumonia, 30 months after the onset. Neuropathological examination revealed extensive large confluent vacuole-type spongiform changes in the cerebral cortices. Prion protein (PrP) immunostaining showed perivascular and plaque-type PrP deposits. We diagnosed our patient as MM2C-type sCJD. There are two difficulties in the early clinical diagnosis of MM2C-type sCJD with ocular disease in the elderly; delayed utilization of DW-MRI, and accompaniment of ocular disease. For early diagnosis of MM2C-type sCJD, we conclude that clinician should perform DW-MRI for patients with isolated dementia or cortical visual disturbance.

Comparison of conventional, amplification and bio-assay detection methods for a chronic wasting disease inoculum pool.

Longitudinal studies of chronic wasting disease (CWD) in the native host have provided considerable understanding of how this prion disease continues to efficiently spread among cervid species. These studies entail great cost in animal, time and financial support. A variety of methods have emerged including transgenic mouse bioassay, western blot, enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), serial protein misfolding cyclic amplification (sPMCA) and real time quaking-induced conversion (RT-QuIC), that deepen our understanding of this and other protein misfolding disorders. To further characterize an inoculum source used for ongoing CWD studies and to determine how the readouts from each of these assays compare, we assayed a CWD-positive brain pool homogenate (CBP6) and a mouse dilutional bioassay of this homogenate using the above detection methods. We demonstrate that: (i) amplification assays enhanced detection of amyloid seeding activity in the CWD+ cervid brain pool to levels beyond mouse LD50, (ii) conventional detection methods (IHC and western blot) performed well in identifying the presence of PrPSc in terminal brain tissue yet lack sufficient detection sensitivity to identify all CWD-infected mice, and (iii) the incorporation of amplification assays enhanced detection of CWD-infected mice near the LD50. This cross-platform analysis provides a basis to calibrate the relative sensitivities of CWD detection assays.

Use of faecal volatile organic compound analysis for ante-mortem discrimination between CWD-positive, -negative exposed, and -known negative white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a naturally occurring infectious, fatal, transmissible spongiform encephalopathy of cervids. Currently, disease confirmation relies on post-mortem detection of infectious prions in the medial retropharyngeal lymph nodes or obex in the brain via immunohistochemistry (IHC). Detection of CWD in living animals using this method is impractical, and IHC and other experimental assays are not reliable in detecting low concentrations of prion present in biofluids or faeces. Here, we evaluate the capability of faecal volatile organic compound analysis to discriminate between CWD-positive and -exposed white-tailed deer located at two positive cervid farms, and two groups of CWD-negative deer from two separate disease-free farms.

Modified Protein Misfolding Cyclic Amplification Overcomes Real-Time Quaking-Induced Conversion Assay Inhibitors in Deer Saliva To Detect Chronic Wasting Disease Prions.

Chronic wasting disease (CWD), a fatal neurodegenerative prion disease of cervids, has spread across North America and has been detected in The Republic of Korea, Finland, and Norway. CWD appears to spread by horizontal transmission, and prions shed in saliva, feces, and urine are thought to contribute. However, studies investigating the rapid spread of CWD have been hampered by assay inhibitors and a lack of consistent and sensitive means to detect the relatively low levels of prions in these samples. Here we show that saliva frequently contains an inhibitor of the real-time quaking-induced conversion assay (RT-QuIC) and that the inhibitor is a member of the mucin family. To circumvent the inhibitor, we developed a modified protein misfolding cyclic amplification (PMCA) method to amplify CWD prions in saliva that were undetectable or ambiguous by RT-QuIC. Our results reinforce the impact of saliva in horizontal CWD transmission and highlight the importance of detection optimization.

Food Forensics: Using Mass Spectrometry To Detect Foodborne Protein Contaminants, as Exemplified by Shiga Toxin Variants and Prion Strains.

Food forensicists need a variety of tools to detect the many possible food contaminants. As a result of its analytical flexibility, mass spectrometry is one of those tools. Use of the multiple reaction monitoring (MRM) method expands its use to quantitation as well as detection of infectious proteins (prions) and protein toxins, such as Shiga toxins. The sample processing steps inactivate prions and Shiga toxins; the proteins are digested with proteases to yield peptides suitable for MRM-based analysis. Prions are detected by their distinct physicochemical properties and differential covalent modification. Shiga toxin analysis is based on detecting peptides derived from the five identical binding B subunits comprising the toxin. 15N-labeled internal standards are prepared from cloned proteins. These examples illustrate the power of MRM, in that the same instrument can be used to safely detect and quantitate protein toxins, prions, and small molecules that might contaminate our food.

Detection of amyloid fibrils in Parkinson's disease using plasmonic chirality.

Amyloid fibrils, which are closely associated with various neurodegenerative diseases, are the final products in many protein aggregation pathways. The identification of fibrils at low concentration is, therefore, pivotal in disease diagnosis and development of therapeutic strategies. We report a methodology for the specific identification of amyloid fibrils using chiroptical effects in plasmonic nanoparticles. The formation of amyloid fibrils based on α-synuclein was probed using gold nanorods, which showed no apparent interaction with monomeric proteins but effective adsorption onto fibril structures via noncovalent interactions. The amyloid structure drives a helical nanorod arrangement, resulting in intense optical activity at the surface plasmon resonance wavelengths. This sensing technique was successfully applied to human brain homogenates of patients affected by Parkinson's disease, wherein protein fibrils related to the disease were identified through chiral signals from Au nanorods in the visible and near IR, whereas healthy brain samples did not exhibit any meaningful optical activity. The technique was additionally extended to the specific detection of infectious amyloids formed by prion proteins, thereby confirming the wide potential of the technique. The intense chiral response driven by strong dipolar coupling in helical Au nanorod arrangements allowed us to detect amyloid fibrils down to nanomolar concentrations.

Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl.

In this report we describe the use of a novel anti-prion monoclonal antibody (DRM2-118) for the direct detection of infectious prions by ELISA. Epitope mapping using overlapping hamster (SHa) prion peptides indicates DRM2-118 binding occurs between residues 93-100 and at the 310-helix (residues 163-170) between alpha helix-A and -B. This antibody shows broad species binding to endogenous prions from brain homogenates and corresponding recombinant prion proteins. To evaluate the performance of this MAb for the detection of prion proteins we performed an animal time course and evaluated prion detection from both crude brain homogenates and lipid raft fractions (DRM) by direct ELISA. Prion detection was significantly enhanced by the addition of the chaotropic guanidine-HCl (Gdn-HCl) during protein immobilization with detection of PK-resistant prion from asymptomatic animal brains at (45-DPI) and from lipid rafts at (24-DPI). Our data demonstrates enhanced prion detection from brain lipid rafts of asymptomatic animals by a simple direct ELISA using the DRM2-118 MAb combined with Gdn-HCl.

Cerebrospinal fluid in Creutzfeldt-Jakob disease.

Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which reflects physiologic or pathologic states of the central nervous system. Changes in CSF proteome have been described in various neurodegenerative disorders. Earliest publications came from the field of prion disease. Two major approaches have been followed aiming to detect the pathologic form of prion protein (PrPSc) in various peripheral tissues on one hand, but also looking for surrogate parameters as a consequence of the underlying neurodegenerative process. First observations were made using two-dimensional gel electrophoresis for proteins named p130/131, identified as belonging to the 14-3-3 protein family group. This protein became known as the first "wet" biomarker part of clinical diagnostic criteria. Other proteins were identified; most of the work in addition to 14-3-3 has been done on tau/p-tau. The development of PrPSc-based biomarkers was hampered by technical problems and detection limits. A novel technique which uses an amplification procedure followed by an aggregation step (real-time quaking-induced conversion: RT-QuIC) emerged and allows the detection of abnormally folded PrPSc in the CSF. This chapter summarizes the current knowledge of biomarker development in prion disease and discusses perspectives for new approaches.

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with ᵦ-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in cattle. During passaging of H-BSE in transgenic bovinized (TgBoPrP) mice, a novel phenotype of BSE, termed BSE-SW emerged and was characterized by a short incubation time and host weight loss. To investigate the biological and biochemical properties of the BSE-SW prion, a transmission study was conducted in cattle, which were inoculated intracerebrally with brain homogenate from BSE-SW-infected TgBoPrP mice. The disease incubation period was approximately 15 months. The animals showed characteristic neurological signs of dullness, and severe spongiform changes and a widespread, uniform distribution of disease-associated prion protein (PrPSc) were observed throughout the brain of infected cattle. Immunohistochemical PrPSc staining of the brain revealed the presence of intraglial accumulations and plaque-like deposits. No remarkable differences were identified in vacuolar lesion scores, topographical distribution patterns, and staining types of PrPSc in the brains of BSE-SW- vs H-BSE-infected cattle. PrPSc deposition was detected in the ganglia, vagus nerve, spinal nerve, cauda equina, adrenal medulla, and ocular muscle. Western blot analysis revealed that the specific biochemical properties of the BSE-SW prion, with an additional 10- to 12-kDa fragment, were well maintained after transmission. These findings indicated that the BSE-SW prion has biochemical properties distinct from those of H-BSE in cattle, although clinical and pathologic features of BSW-SW in cattle are indistinguishable from those of H-BSE. The results suggest that the 2 infectious agents, BSE-SW and H-BSE, are closely related strains.