Chronic Wasting Disease: State of the Science.

Chronic wasting disease (CWD) is a prion disease affecting cervid species, both free-ranging and captive populations. As the geographic range continues to expand and disease prevalence continues to increase, CWD will have an impact on cervid populations, local economies, and ecosystem health. Mitigation of this "wicked" disease will require input from many different stakeholders including hunters, landowners, research biologists, wildlife managers, and others, working together. The NC1209 (North American interdisciplinary chronic wasting disease research consortium) is composed of scientists from different disciplines involved with investigating and managing CWD. Leveraging this broad breadth of expertise, the Consortium has created a state-of-the-science review of five key aspects of CWD, including current diagnostic capabilities for detecting prions, requirements for validating these diagnostics, the role of environmental transmission in CWD dynamics, and potential zoonotic risks associated with CWD. The goal of this review is to increase stakeholders', managers', and decision-makers' understanding of this disease informed by current scientific knowledge.

Longitudinal detection of prion shedding in nasal secretions of CWD-infected white-tailed deer.

Chronic wasting disease (CWD) is an emergent prion disease spreading in cervid populations in North America, South Korea and Scandinavia. Rapid detection of CWD prions shed by live animals using minimally invasive methods remains an important need. Previous studies in deer, elk and hamsters have demonstrated prion replication in the nasal olfactory mucosa, yet the temporal profile of CWD prion shedding in nasal secretions has not been well characterized. Here we report nasal prion shedding in 18 deer orally exposed to low doses of CWD prions and monitored longitudinally by several parameters. Serially collected nasal swabs were assayed for CWD prion seeding activity using iron oxide magnetic extraction and real-time quaking-induced conversion (IOME RT-QuIC). These findings were correlated with the results from longitudinal tonsil biopsies, terminal tissues and PRNP genotype. We detected nasal prion shedding 3-16 months after the first positive tonsil biopsy in ten of the 18 deer; detectable shedding persisted thereafter in nine of the ten animals. Surprisingly, nasal swabs were negative in eight deer, even though all were CWD-infected as determined by tonsil biopsies and terminal tissue assays. Nasal shedding was detected more often in deer that were homozygous for glycine at codon 96, and those that were near or demonstrating symptoms of clinical disease shed earlier and more frequently, irrespective of prion exposure dose. The results of this study demonstrate nasal shedding of CWD prions that can be detected using minimally invasive nasal swab sampling and RT-QuIC analysis.

Detection of Chronic Wasting Disease Prions in Fetal Tissues of Free-Ranging White-Tailed Deer.

The transmission of chronic wasting disease (CWD) has largely been attributed to contact with infectious prions shed in excretions (saliva, urine, feces, blood) by direct animal-to-animal exposure or indirect contact with the environment. Less-well studied has been the role that mother-to-offspring transmission may play in the facile transmission of CWD, and whether mother-to-offspring transmission before birth may contribute to the extensive spread of CWD. We thereby focused on a population of free-ranging white-tailed deer from West Virginia, USA, in which CWD has been detected. Fetal tissues, ranging from 113 to 158 days of gestation, were harvested from the uteri of CWD+ dams in the asymptomatic phase of infection. Using serial protein misfolding amplification (sPMCA), we detected evidence of prion seeds in 7 of 14 fetuses (50%) from 7 of 9 pregnancies (78%), with the earliest detection at 113 gestational days. This is the first report of CWD detection in free ranging white-tailed deer fetal tissues. Further investigation within cervid populations across North America will help define the role and impact of mother-to-offspring vertical transmission of CWD.

Detection of chronic wasting disease in mule and white-tailed deer by RT-QuIC analysis of outer ear.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Involvement of N- and C-terminal region of recombinant cervid prion protein in its reactivity to CWD and atypical BSE prions in real-time quaking-induced conversion reaction in the presence of high concentrations of tissue homogenates.

The real-time quaking-induced conversion (RT-QuIC) reaction is a sensitive and specific method for detecting prions. However, inhibitory factors present in tissue homogenates can easily interfere with this reaction. To identify the RT-QuIC condition under which low levels of chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) prions can be detected in the presence of high concentrations of brain tissue homogenates, reactivities of various recombinant prion proteins (rPrPs) were tested. Among the tested rPrPs, recombinant cervid PrP (rCerPrP) showed a unique reactivity: the reactivity of rCerPrP to CWD and atypical BSE prions was not highly affected by high concentrations of normal brain homogenates. The unique reactivity of rCerPrP disappeared when the N-terminal region (aa 25-93) was truncated. Replacement of aa 23-149 of mouse (Mo) PrP with the corresponding region of CerPrP partially restored the unique reactivity of rCerPrP in RT-QuIC. Replacement of the extreme C-terminal region of MoPrP aa 219-231 to the corresponding region of CerPrP partially conferred the unique reactivity of rCerPrP to rMoPrP, suggesting the involvement of both N- and C-terminal regions. Additionally, rCerN-Mo-CerCPrP, a chimeric PrP comprising CerPrP aa 25-153, MoPrP aa 150-218, and CerPrP aa 223-233, showed an additive effect of the N- and C-terminal regions. These results provide a mechanistic implication for detecting CWD and atypical BSE prions using rCerPrP and are useful for further improvements of RT-QuIC.

RT-QuIC detection of tauopathies using full-length tau substrates.

Early detection and diagnosis of neurodegenerative diseases has been hampered by the lack of sensitive testing. Real-time quaking induced conversion (RT-QuIC) has been used for the early and sensitive detection of prion-induced neurologic disease, and has more recently been adapted to detect misfolded alpha-synuclein and tau as biomarkers for neurodegenerative disease. Here we use full-length recombinant tau substrates to detect tau seeding activity in Alzheimer's disease and other human tauopathies.

Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease.

The minimum infectious dose required to induce CWD infection in cervids remains unknown, as does whether peripherally shed prions and/or multiple low dose exposures are important factors in CWD transmission. With the goal of better understand CWD infection in nature, we studied oral exposures of deer to very low doses of CWD prions and also examined whether the frequency of exposure or prion source may influence infection and pathogenesis. We orally inoculated white-tailed deer with either single or multiple divided doses of prions of brain or saliva origin and monitored infection by serial longitudinal tissue biopsies spanning over two years. We report that oral exposure to as little as 300 nanograms (ng) of CWD-positive brain or to saliva containing seeding activity equivalent to 300 ng of CWD-positive brain, were sufficient to transmit CWD disease. This was true whether the inoculum was administered as a single bolus or divided as three weekly 100 ng exposures. However, when the 300 ng total dose was apportioned as 10, 30 ng doses delivered over 12 weeks, no infection occurred. While low-dose exposures to prions of brain or saliva origin prolonged the time from inoculation to first detection of infection, once infection was established, we observed no differences in disease pathogenesis. These studies suggest that the CWD minimum infectious dose approximates 100 to 300 ng CWD-positive brain (or saliva equivalent), and that CWD infection appears to conform more with a threshold than a cumulative dose dynamic.

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.

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry.

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.

In vitro detection of haematogenous prions in white-tailed deer orally dosed with low concentrations of chronic wasting disease.

Infectivity associated with prion disease has been demonstrated in blood throughout the course of disease, yet the ability to detect blood-borne prions by in vitro methods remains challenging. We capitalized on longitudinal pathogenesis studies of chronic wasting disease (CWD) conducted in the native host to examine haematogenous prion load by real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification. Our study demonstrated in vitro detection of amyloid seeding activity (prions) in buffy-coat cells harvested from deer orally dosed with low concentrations of CWD positive (+) brain (1 gr and 300 ng) or saliva (300 ng RT-QuIC equivalent). These findings make possible the longitudinal assessment of prion disease and deeper investigation of the role haematogenous prions play in prion pathogenesis.

Detection of CWD in cervids by RT-QuIC assay of third eyelids.

The diagnosis of chronic wasting disease (CWD) relies on demonstration of the disease-associated misfolded CWD prion protein (PrPCWD) in brain or retropharyngeal lymph node tissue by immunodetection methods, e.g. ELISA and immunohistochemistry (IHC). The success of these methods relies on a quality sample of tissues, which requires both anatomical knowledge and considerable dissection to collect. As the prevalence of CWD continues to increase globally, the development of fast and cost-effective methods to detect the disease is vital to facilitate CWD detection and surveillance. To address these issues, we have evaluated third eyelids from CWD-infected deer and elk using real-time quaking induced conversion (RT-QuIC). We identified prion seeding activity in third eyelids in 24 of 25 (96%) CWD-infected white-tailed deer (Odocoileus virginianus). We detected RT-QuIC positivity in the third eyelid as early as 1 month after experimental CWD exposure. In addition, we identified prion seeding activity in third eyelids of 18 of 25 (72%) naturally exposed asymptomatic CWD-positive rocky mountain elk (Cervus canadensis nelson). We compared CWD detection by RT-QuIC and IHC in third eyelid, retropharyngeal lymph node, and brain in 10 deer in early symptomatic stage of disease. IHC detected PrPCWD deposition in third eyelid lymphoid follicles in 5 of 10 deer (50%) whereas third eyelids of all 10 animals were positive by RT-QuIC. This difference reflected in part a lower requirement for lymphoid follicle presence for seeding activity detection by RT-QuIC. In conclusion, RT-QuIC analysis of the third eyelid, an easily accessed tissue, has potential to advance CWD detection and testing compliance.

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.

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.

Comparative analysis of prions in nervous and lymphoid tissues of chronic wasting disease-infected cervids.

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.

Design, implementation, and interpretation of amplification studies for prion detection.

Amplification assays for transmissible spongiform encephalopathies have been in development for close to 15 years, with critical implications for the postmortem and antemortem diagnosis of human and animal prion diseases. Little has been published regarding the structured development, implementation and interpretation of experiments making use of protein misfolding cyclic amplification (PMCA) and real time quaking-induced conversion (RT-QuIC), and our goal with this Perspectives manuscript is to offer a framework which might allow for more efficient expansion of pilot studies into diagnostic trials in both human and animal subjects. This framework is made up of approaches common to diagnostic medicine, including a thorough understanding of analytical and diagnostic sensitivity and specificity, an a priori development of amplification strategy, and an effective experimental design. It is our hope that a structured framework for prion amplification assays will benefit not only experiments seeking to sensitively detect naturally-occurring cases of prion diseases and describe the pathogenesis of TSEs, but ultimately assist with future endeavors seeking to use these methods more broadly for other protein misfolding disorders, including Alzheimer's and Parkinson's disease.

Chronic wasting disease management in ranched elk using rectal biopsy testing.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting members of the cervid species, and is one of the few TSEs with an expanding geographic range. Diagnostic limitations, efficient transmission, and the movement of infected animals are important contributing factors in the ongoing spread of disease. Managing CWD in affected populations has proven difficult, relying on population reduction in the case of wild deer and elk, or quarantine and depopulation in farmed cervids. In the present study, we evaluated the effectiveness of managing endemic CWD in a closed elk herd using antemortem sampling combined with both conventional and experimental diagnostic testing, and selective, targeted culling of infected animals. We hypothesized that the real-time quaking-induced conversion (RT-QuIC) assay, a developing amplification assay, would offer greater detection capabilities over immunohistochemistry (IHC) in the identification of infected animals using recto-anal mucosa associated lymphoid tissue (RAMALT). We further sought to develop a better understanding of CWD epidemiology in elk with various PRNP alleles, and predicted that CWD prevalence would decrease with targeted culling. We found that RT-QuIC identified significantly more CWD-positive animals than IHC using RAMALT tissues (121 vs. 86, respectively, out of 553 unique animals), and that longstanding disease presence was associated with an increasing frequency of less susceptible PRNP alleles. Prevalence of CWD increased significantly over the first two years of the study, implying that refinements in our management strategy are necessary to reduce the prevalence of CWD in this herd.

A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing.

Feline leukemia virus (FeLV) was the first feline retrovirus discovered, and is associated with multiple fatal disease syndromes in cats, including lymphoma. The original research conducted on FeLV employed classical virological techniques. As methods have evolved to allow FeLV genetic characterization, investigators have continued to unravel the molecular pathology associated with this fascinating agent. In this review, we discuss how FeLV classification, transmission, and disease-inducing potential have been defined sequentially by viral interference assays, Sanger sequencing, PCR, and next-generation sequencing. In particular, we highlight the influences of endogenous FeLV and host genetics that represent FeLV research opportunities on the near horizon.

Assessment of Chronic Wasting Disease Prion Shedding in Deer Saliva with Occupancy Modeling.

The detection of prions is difficult due to the peculiarity of the pathogen, which is a misfolded form of a normal protein. The specificity and sensitivity of detection methods are imperfect in complex samples, including in excreta. Here, we combined optimized prion amplification procedures with a statistical method that accounts for false-positive and false-negative errors to test deer saliva for chronic wasting disease (CWD) prions. This approach enabled us to discriminate the shedding of prions in saliva and the detection of prions in saliva-a distinction crucial to understanding the role of prion shedding in disease transmission and for diagnosis. We found that assay sensitivity and specificity were indeed imperfect, and we were able to draw several conclusions pertinent to CWD biology from our analyses: (i) the shedding of prions in saliva increases with time postinoculation, but is common throughout the preclinical phase of disease; (ii) the shedding propensity is influenced neither by sex nor by prion protein genotype at codon 96; and (iii) the source of prion-containing inoculum used to infect deer affects the likelihood of prion shedding in saliva; oral inoculation of deer with CWD-positive saliva resulted in 2.77 times the likelihood of prion shedding in saliva compared to that from inoculation with CWD-positive brain. These results are pertinent to horizontal CWD transmission in wild cervids. Moreover, the approach described is applicable to other diagnostic assays with imperfect detection.

PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer.

The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs.

Chronic wasting disease prion infection of differentiated neurospheres.

A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.