RT‑QuIC detection of CWD prion seeding activity in white‑tailed deer muscle tissues
Manci Li1,2, Marc D. Schwabenlander1,2, Gage R. Rowden1,2, Jeremy M. Schefers2,3, Christopher S. Jennelle4 , Michelle Carstensen4 , Davis Seelig2,5 & Peter A. Larsen1,2*
Chronic wasting disease (CWD) is a prion disease circulating in wild and farmed cervid populations throughout North America (United States and Canada), Europe (Finland, Norway, Sweden), and South Korea. CWD is a long-term threat to all cervid populations and to cervid hunting heritage, with the potential to cause substantial economic losses across multiple sectors. In North America, hunting and farming industries focused on the processing and consumption of white-tailed deer (WTD) venison are particularly vulnerable to CWD prion contamination, as millions of WTD are consumed annually. Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay amplifying misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues and excreta. To date, no study has comprehensively examined CWD prion content across bulk skeletal muscle tissues harvested from individual CWD infected WTD. Here, we use RT-QuIC to characterize prion-seeding activity in a variety of skeletal muscles from both wild and farmed CWD-positive WTD. We successfully detected CWD prions in muscles commonly used for consumption (e.g., backstrap, tenderloin, etc.) as well as within tongue and neck samples of WTD. Our results suggest that CWD prions are distributed across the skeletal muscles of infected WTD. We posit that RT-QuIC will be a useful tool for monitoring CWD prions in venison and that the method (with additional protocol optimization and high-throughput functionality) could be used to reduce and/or prevent CWD prions from entering animal and human food chains.
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Discussion
CWD is an emerging infectious prion disease currently affecting cervid populations across three continents and negatively influencing all cervid-related industries within impacted regions. Infected animals can remain asymptomatic for months while shedding CWD prions through excreta22,25, thus making the identification of early-stage CWD-infected animals based on external diseased phenotypes impossible. Antibody-based ELISA and IHC tests are the current diagnostic standards for CWD. Despite their reliability, such immunodetection methods have limited sensitivity and application across various tissues and body excreta in comparison to in vitro amplification methods for prion detection, such as PMCA and RT-QuIC9 . Of the available in vitro amplification methods, RT-QuIC is well-suited as a CWD screening tool because it can be easily scaled up as required by industrial applications. Given the continued spread of CWD, and uncertainty surrounding potential health risks to both animals and humans due to the consumption of CWD-positive venison1 , it is clear that a highly sensitive and reliable diagnostic method to detect CWD prions in skeletal muscles of cervids is needed.
In this study, we tested methods aimed at extracting and enriching PrPCWD from WTD skeletal muscles for prion detection by RT-QuIC. We first found that CWD prions were present in bulk sampled neck muscles (bra chiocephalicus/sternocephalicus) of CWD positive animals (Fig. 1a)32. This result prompted us to investigate the general distribution of prions in skeletal muscles from the tongue, forelimb, mid-truck, and hindlimb of CWD positive WTD tissue-sets available in our biorepository. We found that in addition to the neck, PrPCWD is also present in a variety of skeletal muscle tissues (described above; Table 2; Fig. 3). Our results, based on a sampling of various muscle groups, suggest that CWD prions are distributed across CWD infected WTD skeletal muscles. Additional research is needed to determine the full extent to which CWD prions occur within particular muscle tissue types of infected animals, including intra- and inter-individual variation of CWD prion accumulation in WTD muscles.
It remains to be determined whether CWD prions are detectable in skeletal muscles that were not sampled herein or in similar studies using amplification-based methods, such as PMCA and RT-QuIC. Although we were unable to detect PrPCWD across all muscle types within a given CWD positive animal, this result is expected because the successful detection of PrPCWD within an infected individual, and particular tissue type, can be impacted by multiple factors. With respect to the neck samples screened here, we only had access to unilaterally sampled muscles harvested from individual WTD heads and it has been shown recently that PrPCWD may not be bilaterally present in select tissues32,35. The 10 positive animals (originating from wild WTD herds) selected for testing of neck muscles were strongly positive across multiple tissues and likely were in relatively advanced, yet pre-clinical stages of the disease (i.e., no clinical signs were observed at the time of euthanasia)32. The stability of prions may vary depending on strains36 and, to date, no RT-QuIC method is available to detect particular CWD strain differences. Further, the progression of CWD affects the deposition of prions in peripheral tissues and it is unclear at what time in the disease progression that prions accumulate in WTD muscles. We note the neck muscles used herein were frozen less than 12 h after collection; however, the other muscle tissues were at various stages of decomposition and underwent multiple freeze–thaw cycles prior to our possession. This difference in tissue preservation and quality potentially accounts for the reduced sensitivity of RT-QuIC upon application, an observation that suggests an altered balance of RT-QuIC inhibitors and active prion seeds and/or degradation of particular CWD prion strains in decaying tissue. Tus, we recommend muscles for RT-QuIC-based analyses of CWD prions be frozen (at either –20 °C or –80 °C) as soon as possible after collection, ideally less than 24 h.
Based on the results from well-preserved neck muscles, we posit that the freeze–thaw method has the most potential for large-scale diagnostic screening of venison, as it is cheaper and easier to perform. For samples with heavy prion loads, such as tongue, all methods used in this study agreed on the positivity of prion-seeding activity. For poorly preserved sample types, collagenase A outperformed the freeze–thaw method and trypsin digestion in terms of identifying more RT-QuIC positive muscle samples from CWD positive animals. Surprisingly, trypsin digestion yielded a high RAF and did not require additional dilutions of the final resuspension as needed by other methods. This could be due to the digestion of protein inhibitors by trypsin and/or superior ability of trypsin to free prions from examined tissues. Additional optimization of the methods presented here is needed for protocols focused on suboptimal sample types. It is possible that the prion seeding activity we detected in the collected muscle tissues is from non-muscle cell types as reported by Daus et al.28. However, the cellular origin of PrPCWD in skeletal muscle, whether in myocytes, erythrocytes, neurons, epithelial cells, or any other cell type, is inconsequential to the recommendations of not consuming venison from CWD-positive animals or the potential for RT-QuIC-based venison screening as venison products are a matrix of multiple tissues and cell types.
Our findings suggest that CWD prions occur throughout an array of WTD muscles and further investigation, from an anatomical perspective, is needed to understand the extent of this distribution. Future studies focusing on larger sample sizes with systematic, bilateral samplings of well-preserved muscle samples throughout the body are needed to assess, validate, and improve the presented method for its application, as well as quantify the load of CWD prions present. Longitudinal characterization of prion deposition (i.e., using cervid challenge experiments) in a variety of high-quality muscle samples, such as those conducted for saliva, lymphoid tissues, and feces is needed to better understand the pathophysiology of CWD in deer and other cervids. Our study provides the foundation for the development of RT-QuIC-based screening of venison and venison-related products associated with food processing pipelines for CWD-prions.
Prion Infectivity in Fat of Deer with Chronic Wasting Disease▿
Brent Race#, Kimberly Meade-White#, Richard Race and Bruce Chesebro* + Author Affiliations
In mice, prion infectivity was recently detected in fat. Since ruminant fat is consumed by humans and fed to animals, we determined infectivity titers in fat from two CWD-infected deer. Deer fat devoid of muscle contained low levels of CWD infectivity and might be a risk factor for prion infection of other species.
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.
Subject: Prions in Skeletal Muscles of Deer with Chronic Wasting Disease [SCIENCE FULL TEXT]
Date: January 26, 2006 at 12:23 pm PST
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Rachel C. Angers,1* Shawn R. Browning,1*† Tanya S. Seward,2 Christina J. Sigurdson,4‡ Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3§
1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders Brown Center on Aging, 3Department of Neurology, University of Kentucky, Lexington, KY 40536, USA. 4Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526, USA.
*These authors contributed equally to this work.
†Present address: Department of Infectology, Scripps Research Institute, 5353 Parkside Drive, RF-2, Jupiter, Florida, 33458, USA.
‡Present address: Institute of Neuropathology, University of Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
§To whom correspondence should be addressed: E-mail: gtell2@uky.edu
Prions are transmissible proteinaceous agents of mammals that cause fatal neurodegenerative diseases of the central nervous system (CNS). The presence of infectivity in skeletal muscle of experimentally infected mice raised the possibility that dietary exposure to prions might occur through meat consumption (1). Chronic wasting disease (CWD), an enigmatic and contagious prion disease of North American cervids, is of particular concern. The emergence of CWD in an increasingly wide geographic area and the interspecies transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt Jakob disease (vCJD) have raised concerns about zoonotic transmission of CWD.
To test whether skeletal muscle of diseased cervids contained prion infectivity, Tg(CerPrP)1536 mice (2) expressing cervid prion protein (CerPrP), were inoculated intracerebrally with extracts prepared from the semitendinosus/semimembranosus muscle group of CWD-affected mule deer or from CWD-negative deer. The availability of CNS materials also afforded direct comparisons of prion infectivity in skeletal muscle and brain. All skeletal muscle extracts from CWD-affected deer induced progressive neurological dysfunction in Tg(CerPrP)1536 mice with mean incubation times ranging between 360 and ~490 d, whereas the incubation times of prions from the CNS ranged from ~230 to 280 d (Table 1). For each inoculation group, the diagnosis of prion disease was confirmed by the presence of PrPSc in the brains of multiple infected Tg(CerPrP)1536 mice (see supporting online material for examples). In contrast, skeletal muscle and brain material from CWD-negative deer failed to induce disease in Tg(CerPrP)1536 mice (Table 1) and PrPSc was not detected in the brains of sacrificed asymptomatic mice as late as 523 d after inoculation (supporting online material).
Our results show that skeletal muscle as well as CNS tissue of deer with CWD contains infectious prions. Similar analyses of skeletal muscle BSE-affected cattle did not reveal high levels of prion infectivity (3). It will be important to assess the cellular location of PrPSc in muscle. Notably, while PrPSc has been detected in muscles of scrapie-affected sheep (4), previous studies failed to detect PrPSc by immunohistochemical analysis of skeletal muscle from deer with natural or experimental CWD (5, 6). Since the time of disease onset is inversely proportional to prion dose (7), the longer incubation times of prions from skeletal muscle extracts compared to matched brain samples indicated that prion titers were lower in muscle than in CNS where infectivity titers are known to reach high levels. Although possible effects of CWD strains or strain mixtures on these incubation times cannot be excluded, the variable 360 to ~490 d incubation times suggested a range of prion titers in skeletal muscles of CWD-affected deer. Muscle prion titers at the high end of the range produced the fastest incubation times that were ~30% longer than the incubation times of prions from the CNS of the same animal. Since all mice in each inoculation group developed disease, prion titers in muscle samples producing the longest incubation times were higher than the end point of the bioassay, defined as the infectious dose at which half the inoculated mice develop disease. Studies are in progress to accurately assess prion titers.
While the risk of exposure to CWD infectivity following consumption of prions in muscle is mitigated by relatively inefficient prion transmission via the oral route (8), these results show that semitendinosus/semimembranosus muscle, which is likely to be consumed by humans, is a significant source of prion infectivity. Humans consuming or handling meat from CWD-infected deer are therefore at risk to prion exposure.
References and Notes
1. P. J. Bosque et al., Proc. Natl. Acad. Sci. U.S.A. 99, 3812 (2002).
2. S. R. Browning et al., J. Virol. 78, 13345 (2004).
3. A. Buschmann, M. H. Groschup, J. Infect. Dis. 192, 934 (2005).
4. O. Andreoletti et al., Nat. Med. 10, 591 (2004).
5. T. R. Spraker et al., Vet. Pathol. 39, 110 (2002).
6. A. N. Hamir, J. M. Miller, R. C. Cutlip, Vet. Pathol. 41, 78 (2004).
7. S. B. Prusiner et al., Biochemistry 21, 4883 (1980).
8. M. Prinz et al., Am. J. Pathol. 162, 1103 (2003).
9. This work was supported by grants from the U.S. Public Health Service 2RO1 NS040334-04 from the National Institute of Neurological Disorders and Stroke and N01-AI-25491 from the National Institute of Allergy and Infectious Diseases.
Supporting Online Material
www.sciencemag.org/
Materials and Methods
Fig. S1
21 November 2005; accepted 13 January 2006 Published online 26 January 2006; 10.1126/science.1122864 Include this information when citing this paper.
Table 1. Incubation times following inoculation of Tg(CerPrP)1536 mice with prions from skeletal muscle and brain samples of CWD-affected deer.
Inocula Incubation time, mean d ± SEM (n/n0)*
Skeletal muscle Brain
CWD-affected deer
H92 360 ± 2 d (6/6) 283 ± 7 d (6/6)
33968 367 ± 9 d (8/8) 278 ± 11 d (6/6)
5941 427 ± 18 d (7/7)
D10 483 ± 8 d (8/8) 231 ± 17 d (7/7)
D08 492 ± 4 d (7/7)
Averages 426 d 264 d
Non-diseased deer
FPS 6.98 >523 d (0/6)
FPS 9.98 >454 d (0/7) >454 d (0/6)
None >490 d (0/6)
PBS >589 d (0/5)
*The number of mice developing prion disease divided by the original number of inoculated mice is shown in parentheses. Mice dying of intercurrent illnesses were excluded.
http://www.sciencemag.org/
www.sciencemag.org/
Supporting Online Material for
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Rachel C. Angers, Shawn R. Browning, Tanya S. Seward, Christina J. Sigurdson,
Michael W. Miller, Edward A. Hoover, Glenn C. Telling§
§To whom correspondence should be addressed: E-mail: gtell2@uky.edu
Published 26 January 2006 on Science Express
DOI: 10.1126/science.1122864
This PDF file includes:
Materials and Methods
Fig. S1
Supporting Online Materials
Materials and Methods
Homogenates of semitendinosus/semimembranosus muscle (10% w/v in phosphate
buffered saline) were prepared from five emaciated and somnolent mule deer, naturally
infected with CWD at the Colorado Division of Wildlife, Wildlife Research Center.
These deer were identified as D10, D08, 33968, H92, and 5941. CWD infection was
confirmed in all cases by the presence of histologic lesions in the brain including
spongiform degeneration of the perikaryon, the immunohistochemical detection of
disease-associated PrP in brain and tonsil, or by immunoblotting of protease-resistant,
disease associated PrP (CerPrPSc). Semitendinosus/semimembranosus muscle was also
obtained from two asymptomatic, mock inoculated deer, referred to as FPS 6.68 and 9.98,
that originated from a CWD non-endemic area and which were held indoors at Colorado
State University from ten days of age. These control deer were confirmed negative for
CWD by histopathological and immunohistochemical analysis of brain tissue at autopsy.
The utmost care was taken to avoid inclusion of obvious nervous tissue when muscle
biopsies were prepared and to ensure that contamination of skeletal muscle samples with
CNS tissue did not occur. Fresh, single-use instruments were used to collect each sample
biopsy and a central piece from each sample was prepared with fresh, disposable
instruments to further isolate muscle tissue for inoculum preparation. Brain samples for
transmission were prepared separately from muscle as additional insurance against cross
contamination.
1
Groups of anesthetized Tg(CerPrP)1536 mice were inoculated intracerebrally with 30 µl
of 1 % skeletal muscle or brain extracts prepared in phosphate buffered saline (PBS).
Inoculated Tg(CerPrP) mice were diagnosed with prion disease following the progressive
development of at least three neurologic symptoms including truncal ataxia, ‘plastic’ tail,
loss of extensor reflex, difficultly righting, and slowed movement. The time from
inoculation to the onset of clinical signs is referred to as the incubation time.
For PrP analysis in brain extracts of Tg(CerPrP)1536 mice, 10 % homogenates prepared
in PBS were either untreated (-) or treated (+) with 40 µg/ml proteinase K (PK) for one
hour at 37oC in the presence of 2% sarkosyl. Proteins were separated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis, analyzed by immunoblotting using anti PrP
monoclonal antibody 6H4 (Prionics AG, Switzerland), incubated with appropriate
secondary antibody, developed using ECL-plus detection (Amersham), and analyzed
using a FLA-5000 scanner (Fuji).
2
Fig. S1
PrP in brain extracts from representative Tg(CerPrP)1536 mice receiving muscle or CNS
tissue inocula from CWD-affected or CWD-negative deer. Extracts were either treated
(+) or untreated (-) with proteinase K (PK) as indicated. The positions of protein
molecular weight markers at 21.3, 28.7, 33.5 kDa (from bottom to top) are shown to the
left of the immunoblot.
3
http://www.sciencemag.org/
ARS RESEARCH Generation of human chronic wasting disease in transgenic mice
Publication Acceptance Date: 9/8/2021
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: Generation of human chronic wasting disease in transgenic mice
Author item WANG, ZERUI - Case Western Reserve University (CWRU) item QIN, KEFENG - University Of Chicago item CAMACHO, MANUEL - Case Western Reserve University (CWRU) item SHEN, PINGPING - Case Western Reserve University (CWRU) item YUAN, JUE - Case Western Reserve University (CWRU) item Greenlee, Justin item CUI, LI - Jilin University item KONG, QINGZHONG - Case Western Reserve University (CWRU) item MASTRIANNI, JAMES - University Of Chicago item ZOU, WEN-QUAN - Case Western Reserve University (CWRU)
Submitted to: Acta Neuropathologica Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/8/2021 Publication Date: N/A Citation: N/A
Interpretive Summary: Prion diseases are invariably fatal neurologic diseases for which there is no known prevention or cure. Chronic wasting disease (CWD) is the prion disease of deer and elk and is present in farmed and free ranging herds throughout North America. To date there is no clear evidence that the CWD agent could be transmitted to humans. This manuscript describes the use of an in vitro technique, cell-free serial protein misfolding cyclic amplification (sPMCA), to generate a CWD prion that is infectious to transgenic mice expressing the human prion protein. This study provides the first evidence that CWD prions may be able to cause misfolding in the human prion protein. This information will impact medical experts and those involved in making policy for farmed cervids and wildlife.
Technical Abstract: Chronic wasting disease (CWD) is a cervid spongiform encephalopathy or prion disease caused by the infectious prion or PrPSc, a misfolded conformer of cellular prion protein (PrPC). It has rapidly spread in North America and also has been found in Asia and Europe. In contrast to the zoonotic mad cow disease that is the first animal prion disease found transmissible to humans, the transmissibility of CWD to humans remains uncertain although most previous studies have suggested that humans may not be susceptible to CWD. Here we report the generation of an infectious human PrPSc by seeding CWD PrPSc in normal human brain PrPC through the in vitro cell-free serial protein misfolding cyclic amplification (sPMCA). Western blotting confirms that the sPMCA-induced proteinase K-resistant PrPSc is a human form, evidenced by a PrP-specific antibody that recognizes human but not cervid PrP. Remarkably, two lines of humanized transgenic (Tg) mice expressing human PrP-129Val/Val (VV) or -129Met/Met (MM) polymorphism develop prion disease at 233 ± 6 (mean ± SE) days post-inoculation (dpi) and 552 ± 27 dpi, respectively, upon intracerebral inoculation with the sPMCA-generated PrPSc. The brain of diseased Tg mice reveals the electrophoretic profile of PrPSc similar to sporadic Creutzfeldt-Jakob disease (sCJD) MM1 or VV2 subtype but different neuropathological patterns. We believe that our study provides the first evidence that CWD PrPSc is able to convert human PrPC into PrPSc in vitro and the CWD-derived human PrPSc mimics atypical sCJD subtypes in humanized Tg mice.
''The brain of diseased Tg mice reveals the electrophoretic profile of PrPSc similar to sporadic Creutzfeldt-Jakob disease (sCJD) MM1 or VV2 subtype but different neuropathological patterns.''
''We believe that our study provides the first evidence that CWD PrPSc is able to convert human PrPC into PrPSc in vitro and the CWD-derived human PrPSc mimics atypical sCJD subtypes in humanized Tg mice.''
Published: 26 September 2021
Generation of human chronic wasting disease in transgenic mice
Zerui Wang, Kefeng Qin, Manuel V. Camacho, Ignazio Cali, Jue Yuan, Pingping Shen, Justin Greenlee, Qingzhong Kong, James A. Mastrianni & Wen-Quan Zou
Acta Neuropathologica Communications volume 9, Article number: 158 (2021)
Abstract
Chronic wasting disease (CWD) is a cervid prion disease caused by the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC). It has been spreading rapidly in North America and also found in Asia and Europe. Although bovine spongiform encephalopathy (i.e. mad cow disease) is the only animal prion disease known to be zoonotic, the transmissibility of CWD to humans remains uncertain. Here we report the generation of the first CWD-derived infectious human PrPSc by elk CWD PrPSc-seeded conversion of PrPC in normal human brain homogenates using in vitro protein misfolding cyclic amplification (PMCA). Western blotting with human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPSc was derived from the human PrPC substrate. Two lines of humanized transgenic mice expressing human PrP with either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPSc patterns and neuropathological changes in the brain. Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSc can cross the species barrier to convert human PrPC into infectious PrPSc that can produce bona fide prion disease when inoculated into humanized transgenic mice.
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It is worth noting that the annual number of sporadic CJD (sCJD) cases in the USA has increased, with the total number of suspected and confirmed sCJD cases rising from 284 in 2003 to 511 in 2017 (https://www.cdc.gov/prions/cjd/occurrence-transmission.html). The greatly enhanced CJD surveillance and an aging population in the USA certainly contributed to the observed increase in annual sCJD case numbers in recent years, but the possibility cannot be excluded that some of the increased sCJD prevalence is linked to CWD exposure.
In the present study, using serial protein misfolding cyclic amplification (sPMCA) assay we generate PrPSc by seeding CWD prions in normal human brain homogenates. Importantly, we reveal that two lines of humanized Tg mice expressing human PrP-129VV and 129MM develop prion diseases upon intracerebral inoculation of the abnormal PrP generated by sPMCA. We believe that our study provides the first opportunity to dissect the clinical, pathological and biochemical features of the CWD-derived human prion disease in two lines of humanized Tg mice expressing two major human PrP genotypes, respectively.
Cervid to human prion transmission
Kong, Qingzhong
Case Western Reserve University, Cleveland, OH, United States
Abstract
Prion disease is transmissible and invariably fatal. Chronic wasting disease (CWD) is the prion disease affecting deer, elk and moose, and it is a widespread and expanding epidemic affecting 22 US States and 2 Canadian provinces so far. CWD poses the most serious zoonotic prion transmission risks in North America because of huge venison consumption (>6 million deer/elk hunted and consumed annually in the USA alone), significant prion infectivity in muscles and other tissues/fluids from CWD-affected cervids, and usually high levels of individual exposure to CWD resulting from consumption of the affected animal among often just family and friends. However, we still do not know whether CWD prions can infect humans in the brain or peripheral tissues or whether clinical/asymptomatic CWD zoonosis has already occurred, and we have no essays to reliably detect CWD infection in humans. We hypothesize that:
(1) The classic CWD prion strain can infect humans at low levels in the brain and peripheral lymphoid tissues;
(2) The cervid-to-human transmission barrier is dependent on the cervid prion strain and influenced by the host (human) prion protein (PrP) primary sequence;
(3) Reliable essays can be established to detect CWD infection in humans; and
(4) CWD transmission to humans has already occurred. We will test these hypotheses in 4 Aims using transgenic (Tg) mouse models and complementary in vitro approaches.
Aim 1 will prove that the classical CWD strain may infect humans in brain or peripheral lymphoid tissues at low levels by conducting systemic bioassays in a set of "humanized" Tg mouse lines expressing common human PrP variants using a number of CWD isolates at varying doses and routes. Experimental "human CWD" samples will also be generated for Aim 3.
Aim 2 will test the hypothesis that the cervid-to-human prion transmission barrier is dependent on prion strain and influenced by the host (human) PrP sequence by examining and comparing the transmission efficiency and phenotypes of several atypical/unusual CWD isolates/strains as well as a few prion strains from other species that have adapted to cervid PrP sequence, utilizing the same panel of humanized Tg mouse lines as in Aim 1.
Aim 3 will establish reliable essays for detection and surveillance of CWD infection in humans by examining in details the clinical, pathological, biochemical and in vitro seeding properties of existing and future experimental "human CWD" samples generated from Aims 1-2 and compare them with those of common sporadic human Creutzfeldt-Jakob disease (sCJD) prions.
Aim 4 will attempt to detect clinical CWD-affected human cases by examining a significant number of brain samples from prion-affected human subjects in the USA and Canada who have consumed venison from CWD-endemic areas utilizing the criteria and essays established in Aim 3. The findings from this proposal will greatly advance our understandings on the potential and characteristics of cervid prion transmission in humans, establish reliable essays for CWD zoonosis and potentially discover the first case(s) of CWD infection in humans.
Public Health Relevance There are significant and increasing human exposure to cervid prions because chronic wasting disease (CWD, a widespread and highly infectious prion disease among deer and elk in North America) continues spreading and consumption of venison remains popular, but our understanding on cervid-to-human prion transmission is still very limited, raising public health concerns. This proposal aims to define the zoonotic risks of cervid prions and set up and apply essays to detect CWD zoonosis using mouse models and in vitro methods. The findings will greatly expand our knowledge on the potentials and characteristics of cervid prion transmission in humans, establish reliable essays for such infections and may discover the first case(s) of CWD infection in humans.
Prion 2017 Conference Abstracts
First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress Stefanie Czub1, Walter Schulz-Schaeffer2, Christiane Stahl-Hennig3, Michael Beekes4, Hermann Schaetzl5 and Dirk Motzkus6 1
University of Calgary Faculty of Veterinary Medicine/Canadian Food Inspection Agency; 2Universitatsklinikum des Saarlandes und Medizinische Fakultat der Universitat des Saarlandes; 3 Deutsches Primaten Zentrum/Goettingen; 4 Robert-Koch-Institut Berlin; 5 University of Calgary Faculty of Veterinary Medicine; 6 presently: Boehringer Ingelheim Veterinary Research Center; previously: Deutsches Primaten Zentrum/Goettingen
This is a progress report of a project which started in 2009.
21 cynomolgus macaques were challenged with characterized CWD material from white-tailed deer (WTD) or elk by intracerebral (ic), oral, and skin exposure routes. Additional blood transfusion experiments are supposed to assess the CWD contamination risk of human blood product. Challenge materials originated from symptomatic cervids for ic, skin scarification and partially per oral routes (WTD brain). Challenge material for feeding of muscle derived from preclinical WTD and from preclinical macaques for blood transfusion experiments. We have confirmed that the CWD challenge material contained at least two different CWD agents (brain material) as well as CWD prions in muscle-associated nerves.
Here we present first data on a group of animals either challenged ic with steel wires or per orally and sacrificed with incubation times ranging from 4.5 to 6.9 years at postmortem. Three animals displayed signs of mild clinical disease, including anxiety, apathy, ataxia and/or tremor. In four animals wasting was observed, two of those had confirmed diabetes. All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals. Protein misfolding cyclic amplification (PMCA), real-time quaking-induced conversion (RT-QuiC) and PET-blot assays to further substantiate these findings are on the way, as well as bioassays in bank voles and transgenic mice.
At present, a total of 10 animals are sacrificed and read-outs are ongoing. Preclinical incubation of the remaining macaques covers a range from 6.4 to 7.10 years. Based on the species barrier and an incubation time of > 5 years for BSE in macaques and about 10 years for scrapie in macaques, we expected an onset of clinical disease beyond 6 years post inoculation.
PRION 2017 DECIPHERING NEURODEGENERATIVE DISORDERS ABSTRACTS REFERENCE
8. Even though human TSE‐exposure risk through consumption of game from European cervids can be assumed to be minor, if at all existing, no final conclusion can be drawn due to the overall lack of scientific data. In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids. It might be prudent considering appropriate measures to reduce such a risk, e.g. excluding tissues such as CNS and lymphoid tissues from the human food chain, which would greatly reduce any potential risk for consumers. However, it is stressed that currently, no data regarding a risk of TSE infections from cervid products are available.
TUESDAY, DECEMBER 14, 2021
Transmissible Spongiform Encephalopathy TSE Prion end of year report December 14, 2021
SUNDAY, DECEMBER 12, 2021
ARS RESEARCH Generation of human chronic wasting disease in transgenic mice
Terry S. Singeltary Sr.
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