Tracy A. Nichols

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10-7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.

Immunization with a Synthetic Peptide Vaccine Fails to Protect Mule Deer (Odocoileus hemionus) from Chronic Wasting Disease

Chronic wasting disease (CWD) adversely affects both wild and captive cervid populations. A vaccine to prevent CWD would be a highly desirable tool to aid in disease management. To this end, we tested in mule deer a combination of CWD vaccines consisting of cervid prion peptide sequences 168-VDQYNNQNTFVHDC-182 and 145-NDYEDRYYRENMYRYPNQ-164 that had previously been shown to delay onset of clinical disease and increase survival in a mouse-adapted scrapie model. Thirteen captive mule deer (Odocoileus hemionus) were divided into vaccine (n=7) and control groups (n=6), and given prime and boost vaccinations intramuscularly 5 wk apart. Eight weeks postprime (3 wk postboost), all animals were challenged via natural exposure to an environment contaminated with infective CWD prions. Deer were monitored intermittently for prion infection by rectal and tonsil biopsies beginning 275 days postchallenge. All vaccinates responded to both peptide conjugates present in the combination vaccine as measured by enzyme-linked immunosorbent assay. However, all deer eventually became infected regardless of vaccine status.

Detection of prion protein in the cerebrospinal fluid of elk (Cervus canadensis nelsoni) with chronic wasting disease using protein misfolding cyclic amplification

Cerebrospinal fluid (CSF) has been examined as a possible source for preclinical diagnosis of prion diseases in hamsters and sheep. The present report describes the detection of chronic wasting disease (CWD) in the CSF of elk and evaluates its usefulness as an antemortem test for CWD. The CSF from 6 captive and 31 free-ranging adult elk was collected at necropsy and evaluated for the presence of the abnormal isoform of the prion protein that has been associated with CWD (PrPCWD) via protein misfolding cyclic amplification. Additionally, the obex from each animal was examined by immunohistochemistry (IHC). Four out of 6 captive animals were CWD-positive and euthanized due to signs of terminal CWD. The remaining 2 were CWD negative. None of the 31 free-range animals showed overt signs of CWD, but 12 out of 31 tested positive for CWD by IHC. Protein misfolding cyclic amplification detected PrPCWD from 3 of the 4 captive animals showing clinical signs of CWD and none of the nonclinical animals that were CWD positive by IHC. The data suggests that CWD prions can be detected in the CSF of elk, but only relatively late in the course of the disease.

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.

Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease

The purpose of our study was to describe the progressive accumulation of the abnormal conformer of the prion protein (PrPCWD) and spongiform degeneration in a single section of brain stem in Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease (CWD). A section of obex from 85 CWD-positive elk was scored using the presence and abundance of PrPCWD immunoreactivity and spongiform degeneration in 10 nuclear regions and the presence and abundance of PrPCWD in 10 axonal tracts, the subependymal area of the fourth ventricle, and the thin subpial astrocytic layer (glial limitans). Data was placed in a formula to generate an overall obex score. Data suggests that PrPCWD immunoreactivity and spongiform degeneration has a unique and relatively consistent pattern of progression throughout a section of obex. This scoring technique utilizing a single section of obex may prove useful in future work for estimating the presence and abundance of PrPCWD in peripheral tissues and the nervous system in elk with CWD.

Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

ABSTRACT Prion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52–99.8%) and 95% relative specificity (95% confidence limits: 85.13–99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.

Could avian scavengers translocate infectious prions to disease-free areas initiating new foci of chronic wasting disease?

Mechanisms for the spread of transmissible spongiform encephalopathy diseases, including chronic wasting disease (CWD) in North American cervids, are incompletely understood, but primary routes include horizontal and environmental transmission. Birds have been identified as potential vectors for a number of diseases, where they ingest or are exposed to infected material and later shed the disease agent in new areas after flying substantial distances. We recently identified American crows (Corvus brachyrhynchos) as having the potential to translocate infectious prions in their feces. Our results suggest that this common, migratory North American scavenger is capable of translocating infectious prions to disease-free areas, potentially seeding CWD infection where no other initial source of pathogen establishment is forthcoming. Here we speculate on the role avian scavengers, like American crows, might play in the spatial dissemination of CWD. We also consider the role mammalian scavengers may play in dispersing prions.

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrP(Res)) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases(1). Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal(2,3) as well as from environmental sources(4-6). Scavengers and carnivores have potential to translocate PrP(Res) material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrP(Res) material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger(7). We describe procedures used to document passage of PrP(Res) material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.