Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

PMCA for ultrasensitive detection of prions and to study disease biology.

The emergence of a novel class of infectious agent composed exclusively of a misfolded protein (termed prions) has been a challenge in modern biomedicine. Despite similarities on the behavior of prions with respect to conventional pathogens, the many uncertainties regarding the biology and virulence of prions make them a worrisome paradigm. Since prions do not contain nucleic acids and rely on a very different way of replication and spreading, it was necessary to invent a novel technology to study them. In this article, we provide an overview of such a technology, termed protein misfolding cyclic amplification (PMCA), and summarize its many applications to detect prions and understand prion biology.

Preventive and therapeutic reduction of amyloid deposition and behavioral impairments in a model of Alzheimer's disease by whole blood exchange.

Alzheimer's disease (AD) is the major form of dementia in the elderly population. The main neuropathological changes in AD patients are neuronal death, synaptic alterations, brain inflammation, and the presence of cerebral protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Compelling evidence suggests that the misfolding, aggregation, and cerebral deposition of amyloid-beta (Aß) plays a central role in the disease. Thus, prevention and removal of misfolded protein aggregates is considered a promising strategy to treat AD. In the present study, we describe that the development of cerebral amyloid plaques in a transgenic mice model of AD (Tg2576) was significantly reduced by 40-80% through exchanging whole blood with normal blood from wild type mice having the same genetic background. Importantly, such reduction resulted in improvement in spatial memory performance in aged Tg2576 mice. The exact mechanism by which blood exchange reduces amyloid pathology and improves memory is presently unknown, but measurements of Aß in plasma soon after blood exchange suggest that mobilization of Aß from the brain to blood may be implicated. Our results suggest that a target for AD therapy may exist in the peripheral circulation, which could open a novel disease-modifying intervention for AD.

Prion Dissemination through the Environment and Medical Practices: Facts and Risks for Human Health.

Prion diseases are a group of fatal, infectious neurodegenerative disorders affecting various species of mammals, including humans. The infectious agent in these diseases, termed prion, is composed exclusively of a misfolded protein that can spread and multiply in the absence of genetic materials. In this article, we provide an overview of the mechanisms of prion replication, interindividual transmission, and dissemination in communities. In particular, we review the potential role of the natural environment in prion transmission, including the mechanisms and pathways for prion entry and accumulation in the environment as well as its roles in prion mutation, adaptation, evolution, and transmission. We also discuss the transmission of prion diseases through medical practices, scientific research, and use of biological products. Detailed knowledge of these aspects is crucial to limit the spreading of existing prion diseases as well as to prevent the emergence of new diseases with possible catastrophic consequences for public health.

North American and Norwegian Chronic Wasting Disease Prions Exhibit Different Potential for Interspecies Transmission and Zoonotic Risk.

BACKGROUND: Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting various species of wild and captive cervids. The risk that CWD poses to cohabiting animals or more importantly to humans is largely unknown. METHODS: In this study, we investigated differences in the capacity of CWD isolates obtained from 6 different cervid species to induce prion conversion in vitro by protein misfolding cyclic amplification. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at expenses of normal prion proteins from various mammals and human, respectively. RESULTS: Our data suggest that reindeer and red deer from Norway could be the most transmissible CWD prions to other mammals, whereas North American CWD prions were more prone to generate human prions in vitro. CONCLUSIONS: Our results suggest that Norway and North American CWD prions correspond to different strains with distinct spillover and zoonotic potentials.

Uptake, Retention, and Excretion of Infectious Prions by Experimentally Exposed Earthworms.

Prions are proteinaceous infectious agents that can be transmitted through various components of the environment, including soil particles. We found that earthworms exposed to prion-contaminated soil can bind, retain, and excrete prions, which remain highly infectious. Our results suggest that earthworms potentially contribute to prion disease spread in the environment.

Efficient prion disease transmission through common environmental materials.

Prion diseases are a group of fatal neurodegenerative diseases associated with a protein-based infectious agent, termed prion. Compelling evidence suggests that natural transmission of prion diseases is mediated by environmental contamination with infectious prions. We hypothesized that several natural and man-made materials, commonly found in the environments of wild and captive animals, can bind prions and may act as vectors for disease transmission. To test our hypothesis, we exposed surfaces composed of various common environmental materials (i.e. wood, rocks, plastic, glass, cement, stainless steel, aluminum, and brass) to hamster-adapted 263K scrapie prions and studied their attachment and retention of infectivity in vitro and in vivo Our results indicated that these surfaces, with the sole exception of brass, efficiently bind, retain, and release prions. Prion replication was studied in vitro using the protein misfolding cyclic amplification technology, and infectivity of surface-bound prions was analyzed by intracerebrally challenging hamsters with contaminated implants. Our results revealed that virtually all prion-contaminated materials transmitted the disease at high rates. To investigate a more natural form of exposure to environmental contamination, we simply housed animals with large contaminated spheres made of the different materials under study. Strikingly, most of the hamsters developed classical clinical signs of prion disease and typical disease-associated brain changes. Our findings suggest that prion contamination of surfaces commonly present in the environment can be a source of disease transmission, thus expanding our understanding of the mechanisms for prion spreading in nature.

Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid.

Misfolded alpha-synuclein (αSyn) aggregates are a hallmark event in Parkinson's disease (PD) and other synucleinopathies. Recently, αSyn seed amplification assays (αSyn-SAAs) have shown promise as a test for biochemical diagnosis of synucleinopathies. αSyn-SAAs use the intrinsic self-replicative nature of misfolded αSyn aggregates (seeds) to multiply them in vitro. In these assays, αSyn seeds circulating in biological fluids are amplified by a cyclical process that includes aggregate fragmentation into smaller self-propagating seeds, followed by elongation at the expense of recombinant αSyn (rec-αSyn). Amplification of the seeds allows detection by fluorescent dyes specific for amyloids, such as thioflavin T. Several αSyn-SAA reports have been published in the past under the names 'protein misfolding cyclic amplification' (αSyn-PMCA) and 'real-time quaking-induced conversion'. Here, we describe a protocol for αSyn-SAA, originally reported as αSyn-PMCA, which allows detection of αSyn aggregates in cerebrospinal fluid samples from patients affected by PD, dementia with Lewy bodies or multiple-system atrophy (MSA). Moreover, this αSyn-SAA can differentiate αSyn aggregates from patients with PD versus those from patients with MSA, even in retrospective samples from patients with pure autonomic failure who later developed PD or MSA. We also describe modifications to the original protocol introduced to develop an optimized version of the assay. The optimized version shortens the assay length, decreases the amount of rec-αSyn required and reduces the number of inconclusive results. The protocol has a hands-on time of ~2 h per 96-well plate and can be performed by personnel trained to perform basic experiments with specimens of human origin.

Development of a methodology for large-scale production of prions for biological and structural studies.

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies.

Detection of prions in the urine of patients affected by sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: Currently, it is unknown whether infectious prions are present in peripheral tissues and biological fluids of patients affected by sporadic Creutzfeldt-Jakob disease (sCJD), the most common prion disorder in humans. This represents a potential risk for inter-individual prion infection. The main goal of this study was to evaluate the presence of prions in urine of patients suffering from the major subtypes of sCJD. METHODS: Urine samples from sCJD patients spanning the six major subtypes were tested. As controls, we used urine samples from people affected by other neurological or neurodegenerative diseases as well as healthy controls. These samples were analyzed blinded. The presence of prions was detected by a modified version of the PMCA technology, specifically optimized for high sensitive detection of sCJD prions. RESULTS: The PMCA assay was first optimized to detect low quantities of prions in diluted brain homogenates from patients affected by all subtypes of sCJD spiked into healthy urine. Twenty-nine of the 81 patients affected by sCJD analyzed in this study were positive by PMCA testing, whereas none of the 160 controls showed any signal. These results indicate a 36% sensitivity and 100% specificity. The subtypes with the highest positivity rate were VV1 and VV2, which combined account for about 15-20% of all sCJD cases, and no detection was observed in MV1 and MM2. INTERPRETATION: Our findings indicate that potentially infectious prions are secreted in urine of some sCJD patients, suggesting a possible risk for inter-individual transmission. Prion detection in urine might be used as a noninvasive preliminary screening test to detect sCJD.

Central and peripheral α-synuclein in Parkinson disease detected by seed amplification assay.

OBJECTIVES: Detection of α-synuclein aggregates by seed amplification is a promising Parkinson disease biomarker assay. Understanding intraindividual relationships of α-synuclein measures could inform optimal biomarker development. The objectives were to test accuracy of α-synuclein seed amplification assay in central (cerebrospinal fluid) and peripheral (submandibular gland) sources, compare to total α-synuclein measures, and investigate within-subject relationships. METHODS: The Systemic Synuclein Sampling Study aimed to characterize α-synuclein in multiple tissues and biofluids within Parkinson disease subjects (n = 59) and compared to healthy controls (n = 21). Motor and non-motor measures and dopamine transporter scans were obtained. Four measures of α-synuclein were compared: seed amplification assay in cerebrospinal fluid and formalin-fixed paraffin-embedded submandibular gland, total α-synuclein quantified in biofluids using enzyme-linked immunoassay, and aggregated α-synuclein in submandibular gland detected by immunohistochemistry. Accuracy of seed amplification assay for Parkinson disease diagnosis was examined and within-subject α-synuclein measures were compared. RESULTS: Sensitivity and specificity of α-synuclein seed amplification assay for Parkinson disease diagnosis was 92.6% and 90.5% in cerebrospinal fluid, and 73.2% and 78.6% in submandibular gland, respectively. 25/38 (65.8%) Parkinson disease participants were positive for both cerebrospinal fluid and submandibular gland seed amplification assay. Comparing accuracy for Parkinson disease diagnosis of different α-synuclein measures, cerebrospinal fluid seed amplification assay was the highest (Youden Index = 83.1%). 98.3% of all Parkinson disease cases had ≥1 measure of α-synuclein positive. INTERPRETATION: α-synuclein seed amplification assay (cerebrospinal fluid>submandibular gland) had higher sensitivity and specificity compared to total α-synuclein measures, and within-subject relationships of central and peripheral α-synuclein measures emerged.

Transmission, Strain Diversity, and Zoonotic Potential of Chronic Wasting Disease.

Chronic wasting disease (CWD) is a prion disease affecting several species of captive and free-ranging cervids. In the past few decades, CWD has been spreading uncontrollably, mostly in North America, resulting in a high increase of CWD incidence but also a substantially higher number of geographical regions affected. The massive increase in CWD poses risks at several levels, including contamination of the environment, transmission to animals cohabiting with cervids, and more importantly, a putative transmission to humans. In this review, I will describe the mechanisms and routes responsible for the efficient transmission of CWD, the strain diversity of natural CWD, its spillover and zoonotic potential and strategies to minimize the CWD threat.

Bacterial DNA promotes Tau aggregation.

A hallmark feature of Alzheimer's disease (AD) and other tauopathies is the misfolding, aggregation and cerebral accumulation of tau deposits. Compelling evidence indicates that misfolded tau aggregates are neurotoxic, producing synaptic loss and neuronal damage. Misfolded tau aggregates are able to spread the pathology from cell-to-cell by a prion like seeding mechanism. The factors implicated in the initiation and progression of tau misfolding and aggregation are largely unclear. In this study, we evaluated the effect of DNA extracted from diverse prokaryotic and eukaryotic cells in tau misfolding and aggregation. Our results show that DNA from various, unrelated gram-positive and gram-negative bacteria results in a more pronounced tau misfolding compared to eukaryotic DNA. Interestingly, a higher effect in promoting tau aggregation was observed for DNA extracted from certain bacterial species previously detected in the brain, CSF or oral cavity of patients with AD. Our findings indicate that microbial DNA may play a previously overlooked role in the propagation of tau protein misfolding and AD pathogenesis, providing a new conceptual framework that positions the compromised blood-brain and intestinal barriers as important sources of microbial DNA in the CNS, opening novel opportunities for therapeutic interventions.

The ecology of chronic wasting disease in wildlife.

Prions are misfolded infectious proteins responsible for a group of fatal neurodegenerative diseases termed transmissible spongiform encephalopathy or prion diseases. Chronic Wasting Disease (CWD) is the prion disease with the highest spillover potential, affecting at least seven Cervidae (deer) species. The zoonotic potential of CWD is inconclusive and cannot be ruled out. A risk of infection for other domestic and wildlife species is also plausible. Here, we review the current status of the knowledge with respect to CWD ecology in wildlife. Our current understanding of the geographic distribution of CWD lacks spatial and temporal detail, does not consider the biogeography of infectious diseases, and is largely biased by sampling based on hunters' cooperation and funding available for each region. Limitations of the methods used for data collection suggest that the extent and prevalence of CWD in wildlife is underestimated. If the zoonotic potential of CWD is confirmed in the short term, as suggested by recent results obtained in experimental animal models, there will be limited accurate epidemiological data to inform public health. Research gaps in CWD prion ecology include the need to identify specific biological characteristics of potential CWD reservoir species that better explain susceptibility to spillover, landscape and climate configurations that are suitable for CWD transmission, and the magnitude of sampling bias in our current understanding of CWD distribution and risk. Addressing these research gaps will help anticipate novel areas and species where CWD spillover is expected, which will inform control strategies. From an ecological perspective, control strategies could include assessing restoration of natural predators of CWD reservoirs, ultrasensitive CWD detection in biotic and abiotic reservoirs, and deer density and landscape modification to reduce CWD spread and prevalence.

PrPTSE in muscle-associated lymphatic tissue during the preclinical stage of mice infected orally with bovine spongiform encephalopathy.

The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.

Application of PMCA to screen for prion infection in a human cell line used to produce biological therapeutics.

Advances in biotechnology have led to the development of a number of biological therapies for the treatment of diverse human diseases. Since these products may contain or are made using human or animal (e.g. cattle) derived materials, it is crucial to test their safety by ensuring the absence of infectious agents; specifically prions, which are highly resilient to elimination and produce fatal diseases in humans. Many cases of iatrogenic Creutzfeldt-Jakob disease have been caused by the use of biological materials (e.g. human growth hormone) contaminated with prions. For this reason, it is important to screen cells and biological materials for the presence of prions. Here we show the utility of the Protein Misfolding Cyclic Amplification (PMCA) technology as a screening tool for the presence of human (vCJD) and bovine (BSE) prions in a human cell therapy product candidate. First, we demonstrated the sensitivity of PMCA to detect a single cell infected with prions. For these experiments, we used RKM7 cells chronically infected with murine RML prions. Serial dilutions of an infected cell culture showed that PMCA enabled prion amplification from a sample comprised of only one cell. Next, we determined that PMCA performance was robust and uncompromised by the spiking of large quantities of uninfected cells into the reaction. Finally, to demonstrate the practical application of this technology, we analyzed a human cell line being developed for therapeutic use and found it to be PMCA-negative for vCJD and BSE prions. Our findings demonstrate that the PMCA technology has unparalleled sensitivity and specificity for the detection of prions, making it an ideal quality control procedure in the production of biological therapeutics.

Identification of circulating microRNA signatures as potential biomarkers in the serum of elk infected with chronic wasting disease.

Chronic wasting disease (CWD) is an emerging infectious prion disorder that is spreading rapidly in wild populations of cervids in North America. The risk of zoonotic transmission of CWD is as yet unclear but a high priority must be to minimize further spread of the disease. No simple diagnostic tests are available to detect CWD quickly or in live animals; therefore, easily accessible biomarkers may be useful in identifying infected animals. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that circulate in blood and are promising biomarkers for several infectious diseases. In this study we used next-generation sequencing to characterize the serum miRNA profiles of 35 naturally infected elk that tested positive for CWD in addition to 35 elk that tested negative for CWD. A total of 21 miRNAs that are highly conserved amongst mammals were altered in abundance in sera, irrespective of hemolysis in the samples. A number of these miRNAs have previously been associated with prion diseases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminative potential of these miRNAs as biomarkers for the diagnosis of CWD. We also determined that a subgroup of 6 of these miRNAs were consistently altered in abundance in serum from hamsters experimentally infected with scrapie. This suggests that common miRNA candidate biomarkers could be selected for prion diseases in multiple species. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pointed to a strong correlation for 3 of these miRNAs, miR-148a-3p, miR-186-5p, miR-30e-3p, with prion disease.

Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases.

A hallmark event in neurodegenerative diseases (NDs) is the misfolding, aggregation, and accumulation of proteins, leading to cellular dysfunction, loss of synaptic connections, and brain damage. Despite the involvement of distinct proteins in different NDs, the process of protein misfolding and aggregation is remarkably similar. A recent breakthrough in the field was the discovery that misfolded protein aggregates can self-propagate through seeding and spread the pathological abnormalities between cells and tissues in a manner akin to the behavior of infectious prions in prion diseases. This discovery has vast implications for understanding the mechanisms involved in the initiation and progression of NDs, as well as for the design of novel strategies for treatment and diagnosis. In this Review, we provide a critical discussion of the role of protein misfolding and aggregation in NDs. Commonalities and differences between distinct protein aggregates will be highlighted, in addition to evidence supporting the hypothesis that misfolded aggregates can be transmissible by the prion principle. We will also describe the molecular basis and implications for prion-like conformational strains, cross-interaction between different misfolded proteins in the brain, and how these concepts can be applied to the development of novel strategies for therapy and diagnosis.

Detection of Prions in Blood of Cervids at the Asymptomatic Stage of Chronic Wasting Disease.

Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting captive and free-ranging cervids. The zoonotic potential of CWD is unknown, as well as the mechanism for its highly efficient transmission. A top priority to minimize further spreading of this disease and its potential impact on environmental prion contamination is the development of a non-invasive, sensitive, and specific test for ante-mortem detection of infected animals. Here, we optimized the protein misfolding cyclic amplification (PMCA) assay for highly efficient detection of CWD prions in blood samples. Studies were done using a blind panel of 98 field-collected samples of whole blood from codon 96 glycine/glycine, captive white-tailed deer that were analyzed for prion infection post-mortem by immunohistochemistry (IHC). The results showed a sensitivity of 100% in animals with very poor body condition that were IHC-positive in both brain and lymph nodes, 96% in asymptomatic deer IHC-positive in brain and lymph nodes and 53% in animals at early stages of infection that were IHC-positive only in lymph nodes. The overall mean diagnostic sensitivity was 79.3% with 100% specificity. These findings show that PMCA might be useful as a blood test for routine, live animal diagnosis of CWD.