urine, feces, and chronic wasting disease cwd tse prion risk factors, loading up the environment
Keystone Journal of Undergraduate Research 2(1): 1-7. 2014 1Corresponding
author: aaron.haines@millersville.edu
Detection of Urine-Based Deer Lures to Mitigate CWD Transmission in
Pennsylvania
Kenneth Strauser Faculty Mentor: 1Dr. Aaron Haines Department of Biology
Millersville University
ABSTRACT
The Pennsylvania Game Commission contacted Millersville University to
perform tests to help combat the transmission and spread of Chronic Wasting
Disease (CWD) via better detection of urine-based deer lures. The objective of
this study was to use multiple methods and kits designed for detection of urine
and blood to determine the best method of detection for urine-based deer lures.
Methods included use of kits such as Uritrace®, Nite-SiteTM luminol, Hemascein®
and Ultra-violet (UV) light. We found that no one technique yielded a positive
for all urine-based deer lures. The UV light and Uritrace methods were the most
effective and the UV light was the best technique in field-testing. The spread
of CWD throughout the Commonwealth has been confirmed and these findings provide
a foundation to further develop methods to detect urine-based deer lures to help
mitigate the spread of CWD.
snip...
An executive order given by the Pennsylvania Game Commission established
several restrictions within the DMA, one of which was the prohibition of the
possession and use of deer urine (Pennsylvania Game Commission 2013).
Urine-based deer lures are used by white-tailed deer hunters to attract deer
into designated areas for harvest. It is estimated that between 10 and 40% of
Pennsylvania hunters have used urine-based deer lures to hunt deer (Pennsylvania
Game Commission 2013). At the same time, commercial urine-based deer lures to
attract white–tailed deer have become readily available to consumers at common
retail sporting goods stores such as Wal-Mart, Bass Pro Shops, and Cabela’s.
However, urineStrauser - Urine-Based Deer Lures and CWD 2 based deer lures have
been known to be collected from domestic white-tailed deer herds, with the
potential of an animal infected with CWD residing within the herd. This can lead
to the risk of CWD infecting wild populations of white-tailed deer via hunter
use of deer lures. Thus, the use of urine-based deer lures is prohibited within
the DMA (Pennsylvania Game Commission 2013).
Due to this risk, the Pennsylvania Game Commission contacted Millersville
University to investigate if urine-based deer lures can be identified in the
field using forensic tests. Based on a review of scientific literature and a
lack of published reports, we know of no other study that has conducted these
types of tests. The objective of this study was to determine if there were
reliable field based tests to detect urine-based deer lures. The Pennsylvania
Game Commission could then use these tests as part of a CWD prevention strategy
in Pennsylvania. Our null hypothesis was that none of our forensics tests would
be able to detect the presence of urine-based deer lures.
snip...
DISCUSSION
We rejected our null hypothesis that none of the tests would yield a
positive result for detecting urine-based deer lures. However, our tentative
results suggest that each test has limitations in regards to detecting
urine-based lures. The most promise in results was provided by the UV tests.
However, we found Strauser - Urine-Based Deer Lures and CWD that urine-based
lures using UV could not be detected in certain fabrics like leather and treated
cotton shirts, but could be seen under UV light on solid ground surfaces (i.e.,
leaves, sticks) and on untreated cotton and shoe foam. A combination of methods
(sight, smell, UV, identification of key wicks, and Uritrace® for those which
yielded a positive) could be helpful to detect presence of most of these lures,
whether on scrapes or on the different fabric material which tested positive. We
are hopeful that the knowledge gained from these tests provides insight into
better techniques to detect urine-based deer lures to help mitigate the spread
of CWD throughout Pennsylvania.
Some commercial lures were collected from individual deer (Code Blue®
Scrape Mate, Code Blue® Buck, Code Blue® Doe) and stated on their bottles that
the urine will smell like a “real, single deer” and give a “true-tolife hunting
experience”. On the back of the three Wildlife Research Center® products, a
guarantee stated that the urine types were sourced from deer tested for (and
free of) CWD. However, the only way to ensure that a deer is free of CWD is to
test its brain tissue, which would require the deer to be deceased (Williams et
al. 2002). In addition, the maximum disease course for CWD (time from exposure
to end-stage clinical disease) is not known, but can exceed 25 months in
experimentally infected deer (Williams et al. 2002).
***Therefore, we recommend that the Pennsylvania Game Commission should
continue to ban urine-based deer lures on Pennsylvania DMAs.
Keywords: Chronic Wasting Disease; detection; urine-based lure;
white-tailed deer
Prion Amplification and Hierarchical Bayesian Modeling Refine Detection of
Prion Infection
A. Christy Wyckoff1, 2 n1 , Nathan Galloway3 n1 , Crystal Meyerett-Reid1 ,
Jenny Powers4 , Terry Spraker1 , Ryan J. Monello4 , Bruce Pulford1 , Margaret
Wild4 , Michael Antolin3 , Kurt VerCauteren2 […] & Mark Zabel1 - Show fewer
authors Scientific Reports 5, Article number: 8358 (2015) doi:10.1038/srep08358
Download Citation
Molecular ecology | Proteins | Statistics Received: 27 June 2014 Accepted:
19 January 2015 Published online: 10 February 2015
Prions are unique infectious agents that replicate without a genome and
cause neurodegenerative diseases that include chronic wasting disease (CWD) of
cervids. Immunohistochemistry (IHC) is currently considered the gold standard
for diagnosis of a prion infection but may be insensitive to early or
sub-clinical CWD that are important to understanding CWD transmission and
ecology. We assessed the potential of serial protein misfolding cyclic
amplification (sPMCA) to improve detection of CWD prior to the onset of clinical
signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus
elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the
specificity and sensitivity of IHC and sPMCA conditional on simultaneously
estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%,
credible interval (CI) 97.15–100%) than IHC of obex (brain stem, 76.56%, CI
57.00–91.46%) or retropharyngeal lymph node (90.06%, CI 74.13–98.70%) tissues,
or both (98.99%, CI 90.01–100%). Our hierarchical Bayesian model predicts the
prevalence of prion infection in this elk population to be 18.90% (CI
15.50–32.72%), compared to previous estimates of 12.90%. Our data reveal a
previously unidentified sub-clinical prion-positive portion of the elk
population that could represent silent carriers capable of significantly
impacting CWD ecology.
snip...
Infected animals shed prions into the environment through saliva, feces,
urine and even antler velvet15,20,21,22,23,24. Studies have successfully
transmitted PrPCWD through a single dose of urine or feces from animals
displaying signs of CWD, indicating that at the time of clinical disease
sufficient prions are shed to result in an infectious dose24,25. However, at
what stage(s) of disease animals shed prions into the environment remains
unclear. If shedding occurs early in disease, a sub-clinical animal may not only
shed prions into the environment, increasing the infectious reservoir, but may
also transmit CWD horizontally to their associates. Unfortunately little is
known about the prevalence of early or sub-clinical infection, and what role
they may play in CWD transmission ecology. This ignorance raises a critical
question that must be answered: how long do free-ranging animals live once
infected? Answering it requires detection of prion infections as early as
possible in the course of infection.
snip...
Our results suggest that prevalence of prion infection in this free-ranging
RMNP elk herd is much higher than previously reported. Prior to 2013, the CWD
prevalence in elk surrounding RMNP was estimated at <2 12.9="" 18.90="" 2008="" 2013="" 28="" 4="" adult="" al.="" all="" an="" and="" animals="" based="" biopsy="" by="" can="" cases="" conclude="" conservatively="" consistent="" course="" cwd="" detect="" div="" during="" early="" elk="" estimated="" et="" female="" finding="" found="" from="" here="" higher="" ihc="" in="" infected="" infection="" known="" model="" monello="" of="" on="" our="" over="" overall="" population.="" positive="" prevalence="" previous="" prpcwd="" ramalt19="" ramalt="" removed="" report="" reported="" reports.="" rmnp="" sample="" since="" spmca="" study.="" tested="" than="" that="" the="" their="" three-year="" times="" we="" were="" which="" who="" with="">
The higher overall prevalence estimate in this herd suggests previous
measurements have been missing a large portion of PrPCWD -positive animals and
that a long history of exposure to prions and decades of relatively high
densities on the winter range may have led to increased prevalence19,42,43.
Further study is required to identify possible ecological differences in this
herd compared to neighboring ones.
As an amplifying assay, sPMCA has previously been shown to be extremely
specific and sensitive in prion detection studies19,22,40,44,45 but had not been
directly compared to IHC in elk or in samples from free-ranging animals. This
study has shown that sPMCA on the obex alone is more sensitive than IHC on obex
or RPLN. sPMCA also detected several positive obex samples, which were
IHC-negative from 2011. We argue that this increased detection represents early
stage infections or sub-clinical animals, which may or may not shed PrPCWD or
develop clinical disease at a later time point.
Similar to Monello et al.19, our sensitivity analysis of IHC by tissue
indicates that in this study population, that IHC in the RPLN was actually more
effective in detecting positives animals than the obex. These results indicate
that IHC on the obex might not be the best method to detect nascent PrPCWD in
elk, and perhaps the premise that the infection course is different between deer
and elk is not absolute. Determining whether sPMCA in the RPLN would show a
similar improvement on sensitivity compared to obex requires further
study.
Our data demonstrate that previous IHC-based studies are possibly missing
early stage or sub-clinical cases in sampled populations. It is widely accepted
that IHC is sensitive enough to detect pre-clinical cases, but we propose that
sPMCA can detect additional cases even earlier, possibly soon after infection.
In previous work we found sPMCA had a detection limit of 10−9 35,46 which is
much more sensitive than the sensitivity of a mouse bioassay at 10−4. This
suggests that animals found positive by sPMCA have much lower levels of PrPCWD
than animals with clinical disease, but are indeed infected. The detection of
very early sub-clinical cases raises the question of biological relevance at the
population level. We propose that this sub-clinical subset of the population may
be ecologically important to the disease transmission cycle because of potential
preclinical vertical transmission from mother to offspring47, horizontal
transmission through direct contact, or indirect transmission through
environmental deposits of prions.
It remains unclear when animals begin shedding prions into the environment.
Through the use of a mouse bioassay Tamguney et al.22,24,44,45 showed
asymptomatic deer were capable of shedding infectious levels of CWD as early as
10 months prior to clinical disease. Bioassays, both in mice and deer, have
limited sensitivity so shedding could be occurring much earlier than 10 months
post-infection but at levels insufficient to cause clinical disease in the
infected host. It is also unclear if genotype plays a role in prion shedding, as
well as disease course. Our data suggest that having at least one L allele at
codon 132 does not alter the disease prevalence within the ML genotype,
supporting data reported by Perucchini et al.27. The slow disease course and the
potential existence of a carrier state facilitate a high prevalence and frequent
opportunity for transmission between animals with the MM and ML genotypes.
It is commonly stated in the literature that CWD is an invariably fatal
disease, but it may be more accurate to state that once animals begin to show
clinical signs they are certain to succumb to CWD or other associated causes of
death such as predation4,24,48. Perhaps other carrier states exist within the
population, which may or may not contribute to the transmission and deposition
of prions in the population and the environment. Further research is required to
address the role of a carrier state in the ecology of CWD transmission.
The application of sPMCA will be important both to research and for
diagnostic investigation, and may improve state and federal surveillance
programs for CWD in both naïve and endemic host populations. Increased
sensitivity, and the need for only obex tissue, may lead to detection of new
focal points prior to clinical disease emerging in otherwise CWD-free
populations. Additionally, in the economically and politically difficult
scenario of culling captive herds that tested positive for CWD, extremely
sensitive assays such as sPMCA of prions from tissue and excreta are essential
to verify that more animals besides the index case were infected, and if any
sub-clinical carriers may have been shedding into the environment.
Overall, our data contribute to the increasing evidence that a portion of a
herd may be infected, but die from other causes while infected with PrPCWD
because of age, genetic susceptibility or other unknown factors. However, the
contribution of prions shed into the environment from this sub-clinical
population may be important and requires further investigation. The existence of
an infectious PrPCWD carrier state aligns with disease ecology theory, which
proposes balance between transmissibility and pathogenesis of a pathogen. As
such, through selection pressures from the host and external environment the
pathogen will tend towards the greatest transmissibility strategy. CWD
transmission may be more complicated than disease ecology might predict, since
prolonged persistence and indirect transmission of prions in the environment may
potentiate spread without affecting pathogenesis.
Despite the fact that prions are only protein, studies continue to point at
evolutionary behavior and selection pressures of prions which indicate that like
other pathogens, prions are capable of evolving and adapting to their
environment4,27,48,49. With increasing prevalence at the population level, as is
reported in this study, sPMCA will continue to be an important tool to
investigate CWD in wildlife.
Quantitative assessment of prion infectivity in tissues and body fluids by
real-time quaking-induced conversion
Authors: Davin M. Henderson1, Kristen A. Davenport1, Nicholas J. Haley2,
Nathaniel D. Denkers1, Candace K. Mathiason1, Edward A. Hoover1
VIEW AFFILIATIONS Affiliations: 1 1Prion Research Center, College of
Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort
Collins, CO 80523, USA 2 2Department of Diagnostic Medicine and Pathobiology,
Kansas State University, College of Veterinary Medicine, Manhattan, KS 66506,
USA Correspondence Edward A. Hoover edward.hoover@colostate.edu J. Gen. Virol.,
January 2015 96: 210-219, doi: 10.1099/vir.0.069906-0 Subject: TSE Agents
Received: 08/07/2014 Accepted: 06/10/2014 Published Online: 01/01/2015
Prions are amyloid-forming proteins that cause transmissible spongiform
encephalopathies through a process involving the templated conversion of the
normal cellular prion protein (PrPC) to a pathogenic misfolded conformation.
Templated conversion has been modelled in several in vitro assays, including
serial protein misfolding amplification, amyloid seeding and real-time
quaking-induced conversion (RT-QuIC). As RT-QuIC measures formation of amyloid
fibrils in real-time, it can be used to estimate the rate of seeded conversion.
Here, we used samples from deer infected with chronic wasting disease (CWD) in
RT-QuIC to show that serial dilution of prion seed was linearly related to the
rate of amyloid formation over a range of 10−3 to 10−8 µg. We then used an
amyloid formation rate standard curve derived from a bioassayed reference sample
(CWD+ brain homogenate) to estimate the prion seed concentration and infectivity
in tissues, body fluids and excreta. Using these methods, we estimated that
urine and saliva from CWD-infected deer both contained 1–5 LD50 per 10 ml. Thus,
over the 1–2 year course of an infection, a substantial environmental reservoir
of CWD prion contamination accumulates.
Research Article
Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse
Bioassay
Nicholas J. Haley, Davis M. Seelig, Mark D. Zabel, Glenn C. Telling, Edward
A. Hoover
Published: March 18, 2009 DOI: 10.1371/journal.pone.0004848
Abstract Chronic wasting disease (CWD) is a prion disease affecting
captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of
CWD transmission are poorly understood, though bodily fluids are thought to play
an important role. Here we report the presence of infectious prions in the urine
and saliva of deer with chronic wasting disease (CWD). Prion infectivity was
detected by bioassay of concentrated, dialyzed urine and saliva in transgenic
mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrPCWD was
detected in pooled and concentrated urine by protein misfolding cyclic
amplification (PMCA). The concentration of abnormal prion protein in bodily
fluids was very low, as indicated by: undetectable PrPCWD levels by traditional
assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE
attack rates in inoculated Tg(CerPrP) mice (373±3days in 2 of 9 urine-inoculated
mice and 342±109 days in 8 of 9 saliva-inoculated mice). These findings help
extend our understanding of CWD prion shedding and transmission and portend the
detection of infectious prions in body fluids in other prion infections.
snip...
While our findings point to urine as an additional vehicle for CWD
transmission, only 2 of 9 inoculated tg1536 mice were confirmed WB/IHC-positive
for prion infection, with a third PrPCWD+ animal later identified by PMCA. This
contrasts with 8 of 9 positive mice receiving saliva and infers a much lower
concentration of prion infectivity in urine. The wide range of survival times in
inoculated mice suggests relatively low levels of infectious prions and/or
uneven distribution of infectious PrP moieties in the inocula [21]. Differing
[CerPrP] zygosity in tg1536 mice (homozygous vs. hemizygous) may also have
played a role in this variation.
Using sPMCA, PrPCWD was repeatedly identified in test urine and spiked
urine and saliva used as positive control, but was not detected in test saliva
after three rounds of amplification. The reasons for our inability to identify
PrPCWD in saliva – given the definitive bioassay findings – remain unknown, and
we propose the presence of as-yet unidentified inhibitors such as mucin or
salivary proteases which are thought to negatively affect other in vitro assays
[22], [23].
The finding of PrPCWD in urine and saliva calls for the identification of
the pathological processes and cellular associations of the prion protein
involved in shedding. Previous studies have related renal pathology to prionuria
[24], [25], a finding which corresponds to our identification of mild to
moderate nephritis in those deer providing samples for the current study. It is
plausible that renal pathology contributed to prionuria in each of these
animals; as samples were pooled, however, we cannot identify specific animals in
which it may have been occurring, nor can we accurately estimate the relative
level of prionuria occurring in each donor as ultrastructural studies were not
performed [26]. While we have not yet identified pathologic prions in renal
source tissues [Unpublished data], protease-resistant PrPCWD has been identified
by immunostaining in renal tissue of prion-infected deer [27], sheep [28],
hamsters and most intriguingly humans [29], foreshadowing the potential for
prionuria in other transmissible spongiform encephalopathies. We continue to
examine tissues from CWD+ deer in an effort to determine the pathogenesis and
kinetics of CWD prion excretion and shedding.
Evidence for excretion and shedding of infectious prions is also
accumulating in the scrapie system. PrPC-converting activity has been identified
by sPMCA in the urine of scrapie-infected sheep, hamsters and mice [21], [30],
[31], [32]. Prion infectivity has also been demonstrated in the feces of
hamsters orally infected with scrapie [33]. Other studies point to infectious
prions in the milk of scrapie-infected ewes [34], [35]. As noted above, it
remains unknown whether other prion diseases (e.g. Kuru, BSE, CJD, TME) may be
transmitted by bodily fluids or excreta other than blood. Additional studies
examining feces, milk, and other body fluids are therefore necessary in CWD and
other prion diseases, studies currently underway in our laboratory.
As CWD transmission may model communicability of other TSE's, the
transmissible nature of prion diseases may serve as a model for other
protein-misfolding diseases. For example, feces, but not urine, from both mice
and cheetahs affected with systemic amyloidosis A (SAA) was recently shown to
induce SAA in a mouse model, although negative controls were not available in
those studies [36]. In light of the prionuria detected in CWD and in models of
scrapie, further investigations of infectivity in body fluids in other protein
folding diseases may be warranted in the event that prion diseases are not the
only infectious proteinopathies.
In summary, we confirm prionsialia in CWD-affected deer by bioassay in
cervidized mice and demonstrate for the first time infectious prions in the
urine of these cervids by both bioassay and sPMCA. We are currently evaluating
urine and saliva from individual animals in hopes of identifying predisposing
factors, such as genotypic background and underlying pathology, which may
contribute to prionuria and prionsialia. Concurrently, we have begun to explore
the tissue origins and protease sensitivity of the infectious prions as well as
the onset and duration of shedding in these bodily fluids.
P179
Detection of CWD prion in fecal samples by RT-QuIC
Yo Ching Cheng1, Theodore Ralph John2, Sampson Law3, Stephanie Czub4,
Sabine Gilch1
1Department of Ecosystem and Public Health, Faculty of Veterinary Medicine,
University of Calgary, Calgary, Alberta, Canada, 2Department of Molecular
Biology, University of Wyoming, Laramie, Wyoming, USA, 3Department of
Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine,
University of Calgary, Calgary, Alberta, Canada, 4Canadian Food Inspection
Agency (CFIA) Lethbridge Laboratory, Lethbridge, Alberta, Canada
Chronic wasting disease (CWD) is a prion disease which mainly affects
captive and wild cervids in North America. To date, cases in elk, mule deer,
white-tailed deer and moose h a v e b e e n r e p o r t e d . I n C W D ,
infectious prions are transported from the CNS into a wide range of peripheral
tissues, body fluids, excreta and eventually shed into the environment.
In order to detect the disease, the use of easily accessible specimens such
as feces would be a practical way for prion detection. However, those excretions
and secretions harbor relatively low concentrations of prions which challenge
current diagnosis methods. In an earlier study, we demonstrated that CWD prions
are detectable in urine collected from pre-symptomatic deer and in fecal
extracts by real-time quaking-induced conversion assay (RT-QuIC). RT-QuIC
employs minute amounts of PrPSc as a seed to initiate conformational transition
of recombinant PrP (rPrP) by vigorous intermittent shaking.
In this study, we aim to improve the detection of CWD pions in fecal
extracts by RT-QuIC, and to determine the shedding pattern in feces of elk which
were challenged orally with CWD prions in an experimental study. We u s e d m e
t h a n o l p r e c i p i t a t i o n , u l t r a c e n t r i f u g a t i o n a
n d s o d i u m phosphotungstic acid (NaPTA) precipitation to purify and to
concentrate CWD prions in feces. We found that NaPTA precipitation of fecal
extracts dose not interfere with seeding activity, but increases the sensitivity
of detection. Our data demonstrate that concentration and purification of PrPSc
enhances detection of CWD prions in feces, which will eventually enable the use
of RTQuIC for CWD surveillance.
Prion2015 Program Guide 27
P196
Prion peripheralization is a host-driven trait of prion infection,
independent of strain
Nicholas Haley1, Chris Siepker1, Sarena Olsen2, Glenn Telling2, Edward
Hoover2, Justin Greenlee3, Juergen Richt1 1Department of Diagnostic Medicine and
Pathobiology, Kansas State University, Manhattan, KS, USA, 2Department of MIP,
Colorado State University, Fort Collins, CO, USA, 3Virus and Prion Research
Unit, National Animal Disease Center, ARS, USDA, Ames, IA, USA
Chronic wasting disease (CWD), like scrapie of sheep, is a horizontally
transmissible spongiform encephalopathy. Proposed natural routes of transmission
for both agents include saliva, urine, and feces, and are likely related to an
accumulation of misfolded prion proteins in excretory tissues, including
salivary glands and both gastrointestinal and urogenital tracts. In recent
years, prion research has focused on the prion strain (e.g. bovine spongiform
encephalopathy – BSE, scrapie, or CWD) as the source code for pathogenic
information – encoded by tertiary conformation for example – with strain
commonly used as the predominant criteria for classifying prions. In the present
study, we compared the peripheral distribution of CWD prions in two experimental
host species: transgenic cervidized mice and cattle. Following inoculation and
demonstrable infection with CWD, a variety of central and peripheral tissues
were collected at necropsy, including brain and both peripheral lymphoid and
excretory tissues, and analyzed for prion seeding activity by real time
quaking-induced conversion (RT-QuIC). While transgenic mice displayed broad and
elevated levels of CWD prions in the periphery, as has been demonstrated with
scrapie in mice and CWD in deer, the peripheralization of CWD prions in cattle
was limited and similar to that reported in BSE. Our findings imply that prion
peripheralization is likely a trait imparted by the host, independent of the
prion strain, though additional research investigating the peripheralization of
BSE prions in cervids, for example, is required. It also remains to be seen if
other prion phenotypes, e.g. zoonotic potential, are hostderived in nature.
Longitudinal Detection of Prion Shedding in Saliva and Urine by
CWD-Infected Deer by RT-QuIC
Davin M. Henderson1, Nathaniel D. Denkers1, Clare E. Hoover1, Nina
Garbino1, Candace K. Mathiason1 and Edward A. Hoover1# + Author
Affiliations
1Prion Research Center, Department of Microbiology, Immunology, and
Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado
State University, Fort Collins, CO 80523 ABSTRACT Chronic Wasting Disease (CWD)
is an emergent, rapidly spreading prion disease of cervids. Shedding of
infectious prions in saliva and urine is thought to be an important factor in
CWD transmission. To help elucidate this issue, we applied an in vitro
amplification assay to determine the onset, duration, and magnitude of prion
shedding in longitudinally collected saliva and urine samples from CWD-exposed
white-tailed deer. We detected prion shedding as early as 3 months after CWD
exposure and sustained shedding throughout the disease course. We estimated that
a 50% lethal dose (LD50) for cervidized transgenic mice would be contained in 1
ml of infected deer saliva or 10 ml or urine. Given the average course of
infection and daily production of these body fluids, an infected deer would shed
thousands of prion infectious dosesover the course of CWD infection. The direct
and indirect environmental impact of this magnitude of prion shedding for cervid
and non-cervid species is surely significant.
Importance: Chronic wasting disease (CWD) is an emerging and uniformly
fatal prion disease affecting free ranging deer and elk and now recognized in 22
United States and 2 C anadian Provinces. It is unique among prion diseases in
that it is transmitted naturally though wild populations. A major hypothesis for
CWD's florid spread is that prions are shed in excreta and transmitted via
direct or indirect environmental contact. Here we use a rapid in vitro assay to
show that infectious doses of CWD prions are in fact shed throughout the
multi-year disease course in deer. This finding is an important advance in
assessing the risks posed by shed CWD prions to animals as well as humans.
FOOTNOTES
↵#To whom correspondence should be addressed: Edward A. Hoover, Prion
Research Center, Department of Microbiology, Immunology and Pathology, Colorado
State University, Fort Collins, Colorado, US Email: edward.hoover@colostate.edu
Longitudinal Detection of Prion Shedding in Saliva and Urine by Chronic
Wasting Disease-Infected Deer by Real-Time Quaking-Induced Conversion
Davin M. Henderson, Nathaniel D. Denkers, Clare E. Hoover, Nina Garbino,
Candace K. Mathiason and Edward A. Hoover
K. L. Beemon, Editor
+ Author Affiliations
ABSTRACT
Chronic wasting disease (CWD) is an emergent, rapidly spreading prion
disease of cervids. Shedding of infectious prions in saliva and urine is thought
to be an important factor in CWD transmission. To help to elucidate this issue,
we applied an in vitro amplification assay to determine the onset, duration, and
magnitude of prion shedding in longitudinally collected saliva and urine samples
from CWD-exposed white-tailed deer. We detected prion shedding as early as 3
months after CWD exposure and sustained shedding throughout the disease course.
We estimated that the 50% lethal dose (LD50) for cervidized transgenic mice
would be contained in 1 ml of infected deer saliva or 10 ml of urine. Given the
average course of infection and daily production of these body fluids, an
infected deer would shed thousands of prion infectious doses over the course of
CWD infection. The direct and indirect environmental impacts of this magnitude
of prion shedding on cervid and noncervid species are surely significant.
IMPORTANCE Chronic wasting disease (CWD) is an emerging and uniformly fatal
prion disease affecting free-ranging deer and elk and is now recognized in 22
U.S. states and 2 Canadian provinces. It is unique among prion diseases in that
it is transmitted naturally through wild populations. A major hypothesis to
explain CWD's florid spread is that prions are shed in excreta and transmitted
via direct or indirect environmental contact. Here we use a rapid in vitro assay
to show that infectious doses of CWD prions are in fact shed throughout the
multiyear disease course in deer. This finding is an important advance in
assessing the risks posed by shed CWD prions to animals as well as humans.
FOOTNOTES Received 14 May 2015. Accepted 23 June 2015. Accepted manuscript
posted online 1 July 2015. Address correspondence to Edward A. Hoover,
edward.hoover@colostate.edu.
D.M.H. and N.D.D. contributed equally to this article.
Citation Henderson DM, Denkers ND, Hoover CE, Garbino N, Mathiason CK,
Hoover EA. 2015. Longitudinal detection of prion shedding in saliva and urine by
chronic wasting disease-infected deer by real-time quaking-induced conversion. J
Virol 89:9338–9347. doi:10.1128/JVI.01118-15.
Insights into Chronic Wasting Disease and Bovine Spongiform Encephalopathy
Species Barriers by Use of Real-Time Conversion
Kristen A. Davenport, Davin M. Henderson, Jifeng Bian, Glenn C. Telling,
Candace K. Mathiason and Edward A. Hoover Prion Research Center, Department of
Microbiology, Immunology and Pathology, College of Veterinary Medicine and
Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA
K. L. Beemon, Editor
+ Author Affiliations
ABSTRACT
The propensity for transspecies prion transmission is related to the
structural characteristics of the enciphering and new host PrP, although the
exact mechanism remains incompletely understood. The effects of variability in
prion protein on cross-species prion transmission have been studied with animal
bioassays, but the influence of prion protein structure versus that of host
cofactors (e.g., cellular constituents, trafficking, and innate immune
interactions) remains difficult to dissect. To isolate the effects of
protein-protein interactions on transspecies conversion, we used recombinant
PrPC and real-time quaking-induced conversion (RT-QuIC) and compared chronic
wasting disease (CWD) and classical bovine spongiform encephalopathy (cBSE)
prions. To assess the impact of transmission to a new species, we studied feline
CWD (fCWD) and feline BSE (i.e., feline spongiform encephalopathy [FSE]). We
cross-seeded fCWD and FSE into each species' full-length, recombinant PrPC and
measured the time required for conversion to the amyloid (PrPRes) form, which we
describe here as the rate of amyloid conversion. These studies revealed the
following: (i) CWD and BSE seeded their homologous species' PrP best; (ii) fCWD
was a more efficient seed for feline rPrP than for white-tailed deer rPrP; (iii)
conversely, FSE more efficiently converted bovine than feline rPrP; (iv) and
CWD, fCWD, BSE, and FSE all converted human rPrP, although not as efficiently as
homologous sCJD prions. These results suggest that (i) at the level of
protein-protein interactions, CWD adapts to a new species more readily than does
BSE and (ii) the barrier preventing transmission of CWD to humans may be less
robust than estimated.
IMPORTANCE We demonstrate that bovine spongiform encephalopathy prions
maintain their transspecies conversion characteristics upon passage to cats but
that chronic wasting disease prions adapt to the cat and are distinguishable
from the original prion. Additionally, we showed that chronic wasting disease
prions are effective at seeding the conversion of normal human prion protein to
an amyloid conformation, perhaps the first step in crossing the species barrier.
FOOTNOTES Received 3 June 2015. Accepted 1 July 2015. Accepted manuscript
posted online 8 July 2015. Address correspondence to Edward A. Hoover,
edward.hoover@colostate.edu.
Citation Davenport KA, Henderson DM, Bian J, Telling GC, Mathiason CK,
Hoover EA. 2015. Insights into chronic wasting disease and bovine spongiform
encephalopathy species barriers by use of real-time conversion. J Virol
89:9524–9531. doi:10.1128/JVI.01439-15.
PL1
Using in vitro prion replication for high sensitive detection of prions and
prionlike proteins and for understanding mechanisms of transmission.
Claudio Soto
Mitchell Center for Alzheimer's diseases and related Brain disorders,
Department of Neurology, University of Texas Medical School at Houston.
Prion and prion-like proteins are misfolded protein aggregates with the
ability to selfpropagate to spread disease between cells, organs and in some
cases across individuals. I n T r a n s m i s s i b l e s p o n g i f o r m
encephalopathies (TSEs), prions are mostly composed by a misfolded form of the
prion protein (PrPSc), which propagates by transmitting its misfolding to the
normal prion protein (PrPC). The availability of a procedure to replicate prions
in the laboratory may be important to study the mechanism of prion and
prion-like spreading and to develop high sensitive detection of small quantities
of misfolded proteins in biological fluids, tissues and environmental samples.
Protein Misfolding Cyclic Amplification (PMCA) is a simple, fast and efficient
methodology to mimic prion replication in the test tube. PMCA is a platform
technology that may enable amplification of any prion-like misfolded protein
aggregating through a seeding/nucleation process. In TSEs, PMCA is able to
detect the equivalent of one single molecule of infectious PrPSc and propagate
prions that maintain high infectivity, strain properties and species
specificity. Using PMCA we have been able to detect PrPSc in blood and urine of
experimentally infected animals and humans affected by vCJD with high
sensitivity and specificity. Recently, we have expanded the principles of PMCA
to amplify amyloid-beta (Aβ) and alphasynuclein (α-syn) aggregates implicated in
Alzheimer's and Parkinson's diseases, respectively. Experiments are ongoing to
study the utility of this technology to detect Aβ and α-syn aggregates in
samples of CSF and blood from patients affected by these diseases.
***Recently, we have been using PMCA to study the role of environmental
prion contamination on the horizontal spreading of TSEs. These experiments have
focused on the study of the interaction of prions with plants and
environmentally relevant surfaces. Our results show that plants (both leaves and
roots) bind tightly to prions present in brain extracts and excreta (urine and
feces) and retain even small quantities of PrPSc for long periods of time.
Strikingly, ingestion of prioncontaminated leaves and roots produced disease
with a 100% attack rate and an incubation period not substantially longer than
feeding animals directly with scrapie brain homogenate. Furthermore, plants can
uptake prions from contaminated soil and transport them to different parts of
the plant tissue (stem and leaves). Similarly, prions bind tightly to a variety
of environmentally relevant surfaces, including stones, wood, metals, plastic,
glass, cement, etc. Prion contaminated surfaces efficiently transmit prion
disease when these materials were directly injected into the brain of animals
and strikingly when the contaminated surfaces were just placed in the animal
cage. These findings demonstrate that environmental materials can efficiently
bind infectious prions and act as carriers of infectivity, suggesting that they
may play an important role in the horizontal transmission of the disease.
Since its invention 13 years ago, PMCA has helped to answer fundamental
questions of prion propagation and has broad applications in research areas
including the food industry, blood bank safety and human and veterinary disease
diagnosis.
see soil ;
I7 Early Trafficking and Dissemination of CWD Prions in Deer
Edward A. Hoover1*, Clare E. Hoover1, Davin M. Henderson1, Nathaniel D.
Denkers1, Kristin A. Davenport1, Shannon Bartelt-Hunt2, Alan M. Elder1, Anthony
E. Kincaid3, 4, Jason C. Bartz3, Mark D. Zabel1, Candace K. Mathiason1
1Prion Research Center, Department of Microbiology, Immunology, and
Pathology, Colorado State University, Fort Collins, Colorado, USA 2Department of
Civil Engineering, University of Nebraska-Lincoln, Prion2015 Program Guide
10 invited speakers PRION 2015 Omaha, Nebraska, USA, 3Department of Medical
Microbiology and immunology Creighton University, Omaha, Nebraska, USA
4Department of Pharmacy Science, Creighton University, Omaha, Nebraska,
USA
* P r e s e n t i n g a u t h o r ’ s e - m a i l :
edward.hoover@colostate.edu
Efficient horizontal infection is a hallmark of chronic wasting disease
(CWD) in freeranging cervids. The mechanisms and pathways that enable this
remarkable process, however, remain incompletely understood--in particular the
facile transmucosal entry, exit, and environmental persistence of CWD prions. We
have focused on trans-mucosal CWD infection in white-tailed deer, specifically
on early prion tissue tropism and later stage prion shedding and association
with environmental constituents using modifications of real-time quaking-induced
conversion combined with amplified immunohistochemistry. We have documented very
early trans-mucosal prion passage (within hours), followed by uptake and
amplification in upper digestive tract lymphoid t i s s u e s ( 4 w e e k s ) ,
and dissemination to more distant lymphoid and non-lymphoid tissue sites (8-12
weeks). We have used quantitative approaches to realtime conversion to estimate
the relatively low (i.e. vs. tissues) prion concentrations in body fluids and
excreta; i.e. >100 (cervidized mouse) LD50 are shed daily in the urine of one
CWD infected deer. Using similar methods, we have also demonstrated and
quantified the impressive
===========================
P183
Novel Detection of PrPCWD on Plants Collected from Rocky Mountain National
Park
Aimee Ortega1, Jeffrey Seligman1, Jan Leach2, Mark Zabel1 1Colorado State
University, Prion Research Center, Department of Microbiology, Immunology and
Pathology, Fort Collins, CO, USA, 2Colorado State University, Department of
Bioagricultural Sciences and Pest Management, Fort Collins, CO, USA
Chronic wasting disease (CWD) affects animals such as elk, deer, and moose
and has become endemic over the last decade. The disease is one of many
transmissible spongiform encephalopathies which occur due to the accumulation of
an abnormally folded, proteinase K resistant, form of the normal cellular prion
protein PrPC. This abnormally folded form, PrPCWD, seeds conversion of PrPC into
PrPCWD and eventually forms amyloid fibrils. The exact mechanisms behind
transmission and spread of CWD are unknown but research has shown that it can be
spread through h o r i z o n t a l , v e r t i c a l , and i n d i r e c t /
environmental routes. PrPCWD has been found in both soil and water.
Additionally, PrPCWD is very resistant to degradation which makes it stable in
the environment for long periods of time. A study has shown that the
Prion2015 Program Guide 29
abnormal prion protein can remain viable in the environment for as long as
16 years. We wanted to further explore the latter and determine whether prions
could be detected in grasses and other plants in Rocky Mountain National Park
(RMNP) by use of the protein misfolding cyclic amplification (PMCA) assay.
This past summer we surveyed three sites within RMNP and collected a total
of 32 plants. Plants were collected from both outside and inside enclosures that
serve to keep wildlife out and allow for restoration and regrowth of the flora.
Plant samples were assayed by PMCA and we are now reporting for the first time
the novel detection of PrPCWD from the surface on a number of plants assayed.
P195 Chronic wasting disease prions detected during early stages of
infection by mbPMCA in tissues from white-tailed deer o r a l l y inoculated
with f r e e and microparticle-bound prions
Alexandra Chesney1, Chad Johnson1, Tracy Nichols2, Hannah Kornely1, Dania
Shoukfeh1, Joel Pedersen1
1University of Wisconsin, Madison, WI, USA, 2United States Department of
Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Wildlife
Services (WS), National Wildlife Research Center (NWRC), Fort Collins, CO,
USA
Enhanced oral transmission of rodent a d a p t e d p r i o n d i s e a s e
has been demonstrated with the disease agent bound to several types of mineral
microparticles; however, the generalizability of this finding to ruminants has
not been established. Contaminated soil is believed to represent a reservoir for
environmental prions and may contribute to horizontal transmission of chronic
wasting disease (CWD) in captive and wild cervid populations. Here, we examined
the impact of CWD agent association with microparticles o f montmorillonite, an
aluminosilicate clay mineral that showed the largest disease transmission
enhancement in rodent bioassays, on early disease in orally inoculated
white-tailed deer. Amplification of prions by PMCA has been achieved from
various contaminated organs and excretions at late stages in disease. Using
microplatebased PMCA (mbPMCA), we detected different accumulation patterns in
white-tailed deer tissues 42 days after oral inoculation with CWD prions bound
to montmorillonite. We expected mbPMCA to be more sensitive than
immunohistochemistry (IHC) to determine prion accumulations in tissues. Through
evaluation of mb-PMCA positive tissues, we found that mbPMCA is more sensitive
than IHC by at least a factor of 106.3, and detected CWD prions in multiple
tissue types that were negative by IHC. These findings suggest that
microparticles can enhance the transmission of CWD in white-tailed deer and also
demonstrates the consistency and high-throughput utility of the mbPMCA assay.
Furthermore, our results indicate that enhanced transmission of
microparticle-bound CWD agent warrants consideration in evaluating the relative
importance o f d i r e c t and i n d i r e c t (environmental) transmission of
CWD in natural populations and in disease management.
Prion2015 Program Guide 35
Saturday, September 12, 2015
*** In utero transmission and tissue distribution of chronic wasting
disease-associated prions in free-ranging Rocky Mountain elk
>>>Interestingly, five of fifteen sPMCA positive dams showed no
evidence of PrPCWD in either CNS or LRS, sites typically assessed in diagnosing
CWD. Analysis of fetal tissues harvested from the fifteen sPMCA positive dams
revealed PrPCWD in 80% of fetuses (12/15), regardless of gestational stage.
These findings demonstrate that PrPCWD is more abundant in peripheral tissues of
CWD exposed elk than current diagnostic methods suggest, and that transmission
of prions from mother to offspring may contribute to the efficient transmission
of the CWD in naturally exposed cervid populations.<<<
P.141: Abundant prion shedding in CWD-infected deer revealed by Realtime
conversion
Edward A Hoover,1 Davin M Henderson,1 Nathaniel D Denkers,1 Candace K
Mathiason,1 Matteo Manca,2,3 and Byron Caughey2 1Prion Research Center, Colorado
State University; Fort Collins, CO USA; 2Laboratory of Persistent Viral
Diseases, NI AID; Hamilton, MT USA; 3Department of Biomedical Sciences,
University of Cagliari; Monserrato, Italy
Background/Introduction. Chronic wasting disease (CWD) is unique among
prion diseases in its efficient lateral transmission in nature. While the
presence of infectious prions in body fluids and excreta of infected cervids has
been demonstrated by bioassay, the dynamics, magnitude, and consequences of
prion shedding remain unknown. The present studies were undertaken to determine
the kinetics, duration, and magnitude of prion shedding in infected white-tailed
deer.
Materials and Methods. Longitudinal samples were collected from
white-tailed deer over a 2-year span after either oral (n=11)] aerosol (n = 6)
CWD exposure. The assay protocol employed phosphotungstic acid precipitation of
either whole saliva or the pelleted fraction of urine to seed recombinant Syrian
hamster prion PrP substrate in RT-QuIC reactions. Prion seeding activity was
assayed in 8 replicates of each sample employing thioflavin T detection in a
96-well plate-based fluorometer. Prion seeding reaction rate was determined by
taking the inverse of the time at which samples exceeded a threshold of 5
standard deviations above the mean fluorescence of negative controls (1/time to
threshold). Seeding activity was quantitated by comparing the realtime
conversion reaction rate to a standard curve derived from a reference bioassayed
brain pool homogenate from deer with terminal CWD.
Results. We analyzed >200 longitudinally collected, blinded, then
randomized saliva and urine samples from 17 CWDinfected and 3 uninfected
white-tailed deer. We detected prion shedding as early as 3 months post exposure
and sustained thereafter throughout the disease course in both aerosol and
orally exposed deer. The incidence of non-specific false positive results from
> 500 saliva and urine samples from negative control deer was 0.8%. By
comparing real-time reaction rates for these body fluids to a bioassayed
serially diluted brain control, we estimated that ≤1 ml of saliva or urine from
pre-symptomatic infected deer constitutes a lethal infectious prion dose.
Conclusion. CWD prions are shed in saliva and urine of infected deer as
early as 3 months post infection and throughout the subsequent >1.5 year
course of infection. In current work we are examining the relationship of
prionemia to excretion and the impact of excreted prion binding to surfaces and
particulates in the environment.
Acknowledgments. Support: NIH-RO1-NS-061902; Morris Animal Foundation
D12ZO-045
P.154: Urinary shedding of prions in Chronic Wasting Disease infected
white-tailed deer
Nathaniel D Denkers,1 Davin M Henderson, 1 Candace K Mathiason,1 and Edward
A Hoover1 1Prion Research Center, Department of Microbiology, Immunology, and
Pathology, Colorado State University; Fort Collins, CO USA
Background/Introduction. Chronic wasting disease (CWD) is unique among
prion diseases in its efficient lateral transmission in nature, yet the dynamics
and magnitude of shedding and its immediate and long term consequences remain
unknown. The present study was designed to determine the frequency and time span
in which CWD prions are shed in urine from infected white-tailed deer using
adapted real-time quaking-induced conversion (RT-QuIC) methodology.
Materials and Methods. Longitudinal urine samples were collected by free
catch or catheterization over a 2-year period from oral-route infected [CWD+ (n
= 11)] and aerosol-route-infected [CWD+ (n = 6); CWD- (n = 3)] white-tailed
deer. High speed centrifugation pelleted material from 500 µl of urine was
treated with sodium phosphotungstic acid (Na-PTA), resuspended in 0.05% SDS
buffer, and used as seed in RT-QuIC assays employing recombinant Syrian hamster
prion PrP substrate. Eight (8) replicates of each sample were run and prion
seeding activity was recorded as thioflavin T binding fluorescence (480 nm
emission) using a fluorimeter-shaker. Samples were considered positive if they
crossed an established threshold (5 standard deviations above the negative mean
fluorescence).
Results. In our oral-route inoculation studies, prion seeding activity has
been demonstrated in urine collected at 6 months post-inoculation in 6 of 10
deer (11 of 80 replicates; 14%), and intermittently at later time points in all
11 CWD+ exposed deer. Our aerosol-route inoculation studies also showed prion
seeding activity in urine collected at 6 months post-inoculation in 1 of 2 deer
(3 of 16 replicates; 19%), and intermittently at later time points in 4 of 6
CWD+ exposed deer. Urine from sham-inoculated control deer and all baseline
samples yielded 3 false-positive prion seeding activities (3 of 352 replicates;
0.8%).
Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC)
are shed in urine of infected deer as early as 6 months post inoculation and
throughout the subsequent disease course. Further studies are in progress
refining the real-time urinary prion assay sensitivity and we are examining more
closely the excretion time frame, magnitude, and sample variables in
relationship to inoculation route and prionemia in naturally and experimentally
CWD-infected cervids.
Acknowledgments. Support: NIH: RO1-NS-061902 and Morris Animal Foundation:
D12ZO-045 P.178: Longitudinal quantitative analysis of CWD prions shed in saliva
of deer
Davin M Henderson, Nina Garbino, Nathaniel D Denkers, Amy V Nalls, Candace
K Mathiason, and Edward A Hoover Prion Research Center, College of Veterinary
Medicine and Biomedical Sciences, Colorado State University; Fort Collins, CO
USA
Background/Introduction. Chronic Wasting Disease (CWD) is an emergent
rapidly spreading fatal prion disease of cervids (deer, elk and moose). CWD has
now been identified in 22 States (including two new states within the last
year), 2 Canadian provinces, and South Korea. Shedding of infectious prions in
excreta (saliva, urine, feces) may be an important factor in CWD transmission.
Here we apply an adapted version of a rapid in vitro assay [real-time
quaking-induced conversion (RT-QuIC)] to determine the time of onset, length,
pattern, and magnitude of prion shedding in saliva of infected deer.
Materials and Methods. The RT-QuIC assay was performed as previously
described in Henderson et al. PLoS-One (2013). Saliva samples were quantitated
by comparison to a RT-QuIC reaction rate standard curve of a bioassayed obex
sample from a terminally ill cervid.
Results. To better understand the onset and length of CWD prion shedding we
analyzed >150 longitudinally collected, blinded, then randomized saliva
samples from 17 CWD-infected and 3 uninfected white-tailed deer. We observed
prion shedding, as detected by the RT-QuIC assay, as early as 3 months from
inoculation and sustained shedding throughout the disease course in both aerosol
and orally exposed deer. We estimated the infectious lethal dose of prions shed
in saliva from infected deer by comparing real-time reaction rates of saliva
samples to a bioassayed serially diluted brain control. Our results indicate
that as little as 1 ml of saliva from pre-symptomatic infected deer constitutes
a lethal CWD prion dose.
Conclusions. During the pre-symptomatic stage of CWD infection and
throughout the course of disease deer may be shedding multiple LD50 doses per
day in their saliva. CWD prion shedding through saliva and excreta may account
for the unprecedented spread of this prion disease in nature.
Acknowledgments. Supported by NIH grant RO1-NS-061902 and grant D12ZO-045
from the Morris Animal Foundation.
Friday, August 14, 2015
Susceptibility of cattle to the agent of chronic wasting disease from elk
after intracranial inoculation
Saturday, January 31, 2015
European red deer (Cervus elaphus elaphus) are susceptible to Bovine
Spongiform Encephalopathy BSE by Oral Alimentary route
I strenuously once again urge the FDA and its industry constituents, to
make it MANDATORY that all ruminant feed be banned to all ruminants, and this
should include all cervids as soon as possible for the following
reasons...
======
In the USA, under the Food and Drug Administrations BSE Feed Regulation (21
CFR 589.2000) most material (exceptions include milk, tallow, and gelatin) from
deer and elk is prohibited for use in feed for ruminant animals. With regards to
feed for non-ruminant animals, under FDA law, CWD positive deer may not be used
for any animal feed or feed ingredients. For elk and deer considered at high
risk for CWD, the FDA recommends that these animals do not enter the animal feed
system.
***However, this recommendation is guidance and not a requirement by law.
======
31 Jan 2015 at 20:14 GMT
*** Ruminant feed ban for cervids in the United States? ***
31 Jan 2015 at 20:14 GMT
P.126: Successful transmission of chronic wasting disease (CWD) into mice
over-expressing bovine prion protein (TgSB3985)
Larisa Cervenakova,1 Christina J Sigurdson,2 Pedro Piccardo,3 Oksana
Yakovleva,1 Irina Vasilyeva,1 Jorge de Castro,1 Paula Saá,1 and Anton Cervenak1
1American Red Cross, Holland Laboratory; Rockville, MD USA; 2University of
California; San Diego, CA USA; 3Lab TSE/OBRR /CBER/FDA; Rockville, MD USA
Keywords: chronic wasting disease, transmission, transgenic mouse, bovine
prion protein
Background. CWD is a disease affecting wild and farmraised cervids in North
America. Epidemiological studies provide no evidence of CWD transmission to
humans. Multiple attempts have failed to infect transgenic mice expressing human
PRNP gene with CWD. The extremely low efficiency of PrPCWD to convert normal
human PrPC in vitro provides additional evidence that transmission of CWD to
humans cannot be easily achieved. However, a concern about the risk of CWD
transmission to humans still exists. This study aimed to establish and
characterize an experimental model of CWD in TgSB3985 mice with the following
attempt of transmission to TgHu mice.
Materials and Methods. TgSB3985 mice and wild-type FVB/ NCrl mice were
intracranially injected with 1% brain homogenate from a CWD-infected Tga20 mouse
(CWD/Tga20). TgSB3985 and TgRM (over-expressing human PrP) were similarly
injected with 5% brain homogenates from CWD-infected white-tailed deer (CWD/WTD)
or elk (CWD/Elk). Animals were observed for clinical signs of neurological
disease and were euthanized when moribund. Brains and spleens were removed from
all mice for PrPCWD detection by Western blotting (WB). A histological analysis
of brains from selected animals was performed: brains were scored for the
severity of spongiform change, astrogliosis, and PrPCWD deposition in ten brain
regions.
Results. Clinical presentation was consistent with TSE. More than 90% of
TgSB3985 and wild-type mice infected with CWD/Tga20, tested positive for PrPres
in the brain but only mice in the latter group carried PrPCWD in their spleens.
We found evidence for co-existence or divergence of two CWD/ Tga20 strains based
on biochemical and histological profiles. In TgSB3985 mice infected with CWD-elk
or CWD-WTD, no animals tested positive for PrPCWD in the brain or in the spleen
by WB. However, on neuropathological examination we found presence of amyloid
plaques that stained positive for PrPCWD in three CWD/WTD- and two
CWD/Elk-infected TgSB3985 mice. The neuropathologic profiles in CWD/WTD- and
CWD/Elkinfected mice were similar but unique as compared to profiles of BSE,
BSE-H or CWD/Tg20 agents propagated in TgSB3985 mice. None of CWD-infected TgRM
mice tested positive for PrPCWD by WB or by immunohistochemical detection.
Conclusions. To our knowledge, this is the first established experimental
model of CWD in TgSB3985. We found evidence for co-existence or divergence of
two CWD strains adapted to Tga20 mice and their replication in TgSB3985 mice.
Finally, we observed phenotypic differences between cervid-derived CWD and
CWD/Tg20 strains upon propagation in TgSB3985 mice. Further studies are underway
to characterize these strains.
P.89: Prions survive long-term burial in soil with some groundwater
dissemination
Allister JA Smith,1 Karen Fernie,1 Ben Maddison,2 Keith Bishop,2 Kevin
Gough,3 and Robert A Somerville1 1The Roslin Institute; University of Edinburgh;
Edinburgh, UK; 2ADAS Biotechnology Group, University of Nottingham; Nottingham,
UK; 3University of Nottingham; Nottingham, UK
An intrinsic property of prions is their extreme resistance to degradation.
When they are deposited within the environment, whether from inappropriate
disposal by man or from fallen diseased livestock, there is the potential to
further propagate cases of disease for many years. It is evidenced that the
spread of scrapie in sheep and chronic wasting disease in deer have occurred in
this manner.
We mimicked such scenarios under large-scale field conditions to determine
the extent to which TSE infectivity survives or disseminates in soil and soil
water over five years. The mouse passaged BSE strain, 301V, was used to spike
buried bovine heads, or was buried as an uncontained bolus in large soil-filled
lysimeters. Two soils were examined, a free-draining sandy loam and a
water-retentive clay loam.
Infectivity, determined by bioassay in mice, was recovered from all heads
exhumed annually for 5 years from both soil types, with little reduction in the
amount of infectivity over time. Small amounts of infectivity were found in soil
samples immediately surrounding the heads but not in samples remote from them.
Commensurate with this there was no evidence of significant lateral movement of
infectivity from the bolus buried in a large soil mass. However large amounts of
infectivity were recovered at the original bolus burial site in both soils.
There was limited vertical upward movement of infectivity from the bolus buried
in clay and downward movement from the bolus buried in sand perhaps reflecting
the clay soils propensity to flood.
Throughout the course of the experiment rainwater particulate from several
lysimeters was trapped on glass-fibre filters. Extracts from these filters were
subject to serial PMCA (protein misfolding cyclic amplification) which was
optimised using 301V-spiked samples and blinded controls. All positive and
negative control samples were correctly determined. We have tested 44 samples
from rainwater passed through the clay lysimeter filters, and found 9 positive
samples, mainly from the initial 8 months of the experiment.
We conclude that TSE infectivity is likely to survive burial for long time
periods with minimal loss of infectivity and limited movement from the original
burial site. However PMCA results have shown that there is the potential for
rainwater to elute TSErelated material from soil which could lead to the
contamination of a wider area. These experiments reinforce the importance of
risk assessment when disposing of TSE risk materials.
P.121: Efficient transmission of prion disease through environmental
contamination
Sandra Pritzkow, Rodrigo Morales, and Claudio Soto Mitchell Center for
Alzheimer’s disease and related Brain disorders; University of Texas Medical
School at Houston; Hourston, TX USA
Chronic wasting disease (CWD) is a prion disorder effecting captive and
free-ranging deer and elk. The efficient propagation suggests that horizontal
transmission through contaminated environment may play an important role. It has
been shown that infectious prions enter the environment through saliva, feces,
urine, blood or placenta tissue from infected animals, as well as by carcasses
from diseased animals and can stay infectious inside soil over several
years.
82 Prion Volume 8 Supplement
We hypothesize that environmental components getting in contact with
infectious prions can also play a role for the horizontal transmission of prion
diseases. To study this issue, surfaces composed of various environmentally
relevant materials were exposed to infectious prions and the attachment and
retention of infectious material was studied in vitro and in vivo. We analyzed
polypropylene, glass, stainless steel, wood, stone, aluminum, concrete and brass
surfaces exposed to 263K-infected brain homogenate. For in vitro analyses, the
material was incubated in serial dilutions of 263K-brain homogenate, washed
thoroughly and analyzed for the presence of PrPSc by PMCA. The results show that
even highly diluted PrPSc can bind efficiently to polypropylene, stainless
steel, glass, wood and stone and propagate the conversion of normal prion
protein. For in vivo experiments, hamsters were ic injected with implants
incubated in 1% 263K-infected brain homogenate. Hamsters, inoculated with
263K-contaminated implants of all groups, developed typical signs of prion
disease, whereas control animals inoculated with non-contaminated materials did
not.
In addition, in order to study the transmission in a more natural setting,
we exposed a group of hamster to habit in the presence of spheres composed of
various materials that were pretreated with 263K prions. Many of the hamsters
exposed to these contaminated materials developed typical signs of the disease
that were confirmed by immunohistological and biochemical analyses.
These findings suggest that various surfaces can efficiently bind
infectious prions and act as carriers of infectivity, suggesting that diverse
elements in the environment may play an important role in horizontal prion
transmission.
P.138: Phenotypic diversity in meadow vole (Microtus pennsylvanicus) prion
diseases following challenge with chronic wasting disease isolates
Christopher J Johnson,1 Christina M Carlson,1,2 Jay R Schneider,1 Jamie K
Wiepz,1 Crystal L Meyerett-Reid,3 Mark D Zabel,3 Joel A Pedersen,2 and Dennis M
Heisey1 1USGS National Wildlife Health Center; Madison, WI USA; 2University of
Wisconsin— Madison; Madison, WI USA; 3Colorado State University; Fort Collins,
CO USA
Chronic wasting disease (CWD), a prion disease of cervids (deer, elk and
moose), is spreading unchecked through large sections of North America.
Transmission of CWD among cervids is especially facile and can occur through
direct animal-toanimal contact and indirectly through contact with prions shed
from infected animals. The disease transmission threat posed by CWD to other
wildlife species remains unknown, but other species are inevitably exposed to
CWD by consumption of infectious materials and through contact with
environmental CWD contamination.
In this study, we investigated the transmission and adaptation of various
white-tailed deer CWD isolates in the meadow vole (Microtus pennsylvanicus), a
native North American rodent that is sympatric with current CWD epizootics that
we have previously established is susceptible to CWD. We found that serial
subpassage of CWD from white-tailed deer homozygous for glycine at position 96
(96GG) of the prion protein in meadow voles resulted in the selection of a
single prion strain that was characterized by homogeneity in incubation period,
abnormal prion protein (PrPTSE) glycoform ratio, lesion profile and PrPTSE
deposition pattern. In contrast, passage of CWD from heterozygous 96GS genotype
deer produced four unique disease phenotypes upon first passage. Subpassage of
these types ultimately resulted in selection of a single strain by third passage
that was distinct from the 96GG genotype CWD-derived strain.
We also establish that meadow voles are susceptible to CWD via peripheral
challenge, albeit with lower attack rates and longer incubation periods.
Interestingly, oral challenge of meadow voles with CWD resulted in subclinical
infection in primary passage animals, but manifested as clinical prion disease
upon subpassage.
Our data establish that meadow voles are permissive to CWD via peripheral
exposure route, suggesting they could serve as an environmental reservoir for
CWD. Additionally, our data are consistent with the hypothesis that at least two
strains of CWD circulate in naturally-infected cervid populations and provide
evidence that meadow voles are a useful tool for CWD strain typing.
Prion Disease Detection, PMCA Kinetics, and IgG in Urine from Sheep
Naturally/Experimentally Infected with Scrapie and Deer with
Preclinical/Clinical Chronic Wasting Disease
▿ Richard Rubenstein1,*, Binggong Chang1, Perry Gray2, Martin Piltch2,
Marie S. Bulgin3, Sharon Sorensen-Melson3 and Michael W. Miller4 + Author
Affiliations
1Departments of Neurology and Physiology/Pharmacology, SUNY Downstate
Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203 2Los Alamos
National Laboratory, Los Alamos, New Mexico 87545 3University of Idaho, Caine
Veterinary Teaching and Research Center, 1020 E. Homedale Road, Caldwell, Idaho
83607 4Colorado Division of Wildlife, Wildlife Research Center, 317 West
Prospect Road, Fort Collins, Colorado 80526-2097 ABSTRACT Prion diseases, also
known as transmissible spongiform encephalopathies, are fatal neurodegenerative
disorders. Low levels of infectious agent and limited, infrequent success of
disease transmissibility and PrPSc detection have been reported with urine from
experimentally infected clinical cervids and rodents. We report the detection of
prion disease-associated seeding activity (PASA) in urine from naturally and
orally infected sheep with clinical scrapie agent and orally infected
preclinical and infected white-tailed deer with clinical chronic wasting disease
(CWD). This is the first report on PASA detection of PrPSc from the urine of
naturally or preclinical prion-diseased ovine or cervids. Detection was achieved
by using the surround optical fiber immunoassay (SOFIA) to measure the products
of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of
PrPC to PrPSc was not influenced by the presence of poly(A) during sPMCA or by
the homogeneity of the PrP genotypes between the PrPC source and urine donor
animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an
exponential, course. Compared to uninfected animals, there was a 2- to 4-log
increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments
in scrapie-infected sheep but not in infected CWD-infected deer. The
higher-than-normal range of IgG levels found in the naturally and experimentally
infected clinical scrapie-infected sheep were independent of their genotypes.
Although analysis of urine samples throughout the course of infection would be
necessary to determine the usefulness of altered IgG levels as a disease
biomarker, detection of PrPSc from PASA in urine points to its potential value
for antemortem diagnosis of prion diseases.
FOOTNOTES Received 13 May 2011. Accepted 14 June 2011. ↵*Corresponding
author. Mailing address: Downstate Medical Center, Departments of Neurology and
Physiology/Pharmacology, Box 1213, 450 Clarkson Avenue, Brooklyn, NY 11203.
Phone: (718) 270-2019. Fax: (718) 270-2459. E-mail:
richard.rubenstein@downstate.edu. ↵▿ Published ahead of print on 29 June 2011.
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Thursday, August 13, 2015
Iatrogenic CJD due to pituitary-derived growth hormone with genetically
determined incubation times of up to 40 years
SHORT REPORT
Creutzfeldt-Jakob disease 38 years after diagnostic use of human growth
hormone
the warning shots fired over the bow of the boat that were never heard ;
PITUITARY EXTRACT
This was used to help cows super ovulate. This tissue was considered to be
of greatest risk of containing BSE and consequently transmitting the
disease...
Early detection of chronic wasting disease prions in urine of
pre-symptomatic deer by real-time quaking-induced conversion assay
Theodore R. John,1 Hermann M. Schätzl1,2,3 and Sabine Gilch1,4,*
1Department of Veterinary Sciences; University of Wyoming; Laramie, WY USA;
2Department of Molecular Biology; University of Wyoming; Laramie, WY USA;
3Faculty of Veterinary Medicine; Department of Comparative Biology and
Experimental Medicine; University of Calgary; Calgary, AB Canada; 4Faculty of
Veterinary Medicine; Department of Ecosystem and Public Health; University of
Calgary; Calgary, AB Canada Keywords: prion, chronic wasting disease, diagnosis,
surveillance, RT-QuIC, urine, feces been demonstrated that prions can persist in
soil21 and that water in endemic areas can contain CWD-associated PrPSc.22
Prion 7:3, 253–258; May/June 2013; © 2013 Landes Bioscience
Short Communication Short Communication
*Correspondence to: Sabine Gilch; Email: sgilch@ucalgary.ca Submitted:
02/07/13; Revised: 03/17/13; Accepted: 03/24/13 http://dx.doi.org/10.4161/pri.24430
Chronic wasting disease (CWD) is a prion disease of captive and
free-ranging deer (Odocoileus spp.), elk (Cervus elaphus nelsonii) and moose
(Alces alces shirasi). Unlike in most other prion diseases, in CWD prions are
shed in urine and feces, which most likely contributes to the horizontal
transmission within and between cervid species. To date, CWD ante mortem
diagnosis is only possible by immunohistochemical detection of protease
resistant prion protein (PrPSc) in tonsil or recto-anal mucosa-associated
lymphoid tissue (RA MALT) biopsies, which requires anesthesia of animals. We
report on detection of CWD prions in urine collected from pre-symptomatic deer
and in fecal extracts by using real time quakinginduced conversion (RT -QuIC).
This assay can be useful for non-invasive pre-symptomatic diagnosis and
surveillance of CWD.
Introduction
Chronic wasting disease (CWD) is to date the most contagious prion disease
and affects captive and free-ranging elk, deer and moose in North America.1,2
The disease is caused by the accumulation of an abnormally folded isoform of the
cellular prion protein PrPC, denominated PrPSc.3,4 CWD is the cervid equivalent
of bovine spongiform encephalopathy (BSE), scrapie in sheep and goat5 or
Creutzfeldt-Jakob disease (CJD) in humans.6 Although transmission studies of CWD
prions to humanized transgenic mice or non-human primates suggest a strong
species barrier,7-9 recent in vitro studies have demonstrated that human PrP can
be converted by CWD prions into PrPSc upon adaptation.10 Therefore, a potential
for zoonotic transmission, as exemplified by BSE,11 cannot be completely
excluded.
A huge body of evidence suggests that CWD can be efficiently transmitted
horizontally within and between cervid species,12 which may be the reason for
geographical spread and increase in case numbers. Horizontal transmission is
explained by the rather unusual peripheral distribution of prions in CWD
affected animals and the high susceptibility to the disease by oral infection.
13,14 Unlike in most other prion diseases, CWD prions can be found in a wide
variety of tissues, such as skeletal and cardiac muscle15,16 or kidney,17 in
addition to the lymphoreticular system and blood.18 Furthermore, they are shed
in significant amounts in saliva,18,19 urine19 or feces,20 which enables oral
infection of animals by foraging on contaminated pastures. In addition, it has
been demonstrated that prions can persist in soil21 and that water in endemic
areas can contain CWD-associated PrPSc.22
snip...see full text ;
Friday, December 14, 2012
DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced
into Great Britain? A Qualitative Risk Assessment October 2012
snip...
In the USA, under the Food and Drug Administration’s BSE Feed Regulation
(21 CFR 589.2000) most material (exceptions include milk, tallow, and gelatin)
from deer and elk is prohibited for use in feed for ruminant animals. With
regards to feed for non-ruminant animals, under FDA law, CWD positive deer may
not be used for any animal feed or feed ingredients. For elk and deer considered
at high risk for CWD, the FDA recommends that these animals do not enter the
animal feed system. However, this recommendation is guidance and not a
requirement by law.
Animals considered at high risk for CWD include:
1) animals from areas declared to be endemic for CWD and/or to be CWD
eradication zones and
2) deer and elk that at some time during the 60-month period prior to
slaughter were in a captive herd that contained a CWD-positive animal.
Therefore, in the USA, materials from cervids other than CWD positive
animals may be used in animal feed and feed ingredients for non-ruminants.
The amount of animal PAP that is of deer and/or elk origin imported from
the USA to GB can not be determined, however, as it is not specified in TRACES.
It may constitute a small percentage of the 8412 kilos of non-fish origin
processed animal proteins that were imported from US into GB in 2011.
Overall, therefore, it is considered there is a __greater than negligible
risk___ that (nonruminant) animal feed and pet food containing deer and/or elk
protein is imported into GB.
There is uncertainty associated with this estimate given the lack of data
on the amount of deer and/or elk protein possibly being imported in these
products.
snip...
36% in 2007 (Almberg et al., 2011). In such areas, population declines of
deer of up to 30 to 50% have been observed (Almberg et al., 2011). In areas of
Colorado, the prevalence can be as high as 30% (EFSA, 2011). The clinical signs
of CWD in affected adults are weight loss and behavioural changes that can span
weeks or months (Williams, 2005). In addition, signs might include excessive
salivation, behavioural alterations including a fixed stare and changes in
interaction with other animals in the herd, and an altered stance (Williams,
2005). These signs are indistinguishable from cervids experimentally infected
with bovine spongiform encephalopathy (BSE). Given this, if CWD was to be
introduced into countries with BSE such as GB, for example, infected deer
populations would need to be tested to differentiate if they were infected with
CWD or BSE to minimise the risk of BSE entering the human food-chain via
affected venison.
snip...
The rate of transmission of CWD has been reported to be as high as 30% and
can approach 100% among captive animals in endemic areas (Safar et al., 2008).
snip...
In summary, in endemic areas, there is a medium probability that the soil
and surrounding environment is contaminated with CWD prions and in a
bioavailable form. In rural areas where CWD has not been reported and deer are
present, there is a greater than negligible risk the soil is contaminated with
CWD prion.
snip...
In summary, given the volume of tourists, hunters and servicemen moving
between GB and North America, the probability of at least one person travelling
to/from a CWD affected area and, in doing so, contaminating their clothing,
footwear and/or equipment prior to arriving in GB is greater than negligible.
For deer hunters, specifically, the risk is likely to be greater given the
increased contact with deer and their environment. However, there is significant
uncertainty associated with these estimates.
snip...
Therefore, it is considered that farmed and park deer may have a higher
probability of exposure to CWD transferred to the environment than wild deer
given the restricted habitat range and higher frequency of contact with tourists
and returning GB residents.
snip...
*** In areas of North America where CWD has been reported, given that CWD
is excreted in faeces, saliva, urine and blood, and survives in the environment
for several years where it is able to bind to the soil, there is a medium
probability that the environment (including soil) contains CWD. ***
Urine
Pooled urine from five terminally CWD infected white-tailed deer was
inoculated into nine tg mice. Two of the nine mice developed disease consistent
with a TSE at 370 and 376 days post inoculation suggesting infectious prions are
present in the urine of infected cervids but at a lower infectivity than other
bodily fluids such as saliva.
Haley et al., (2009)
Friday, March 25, 2011
Detection of Prion Protein in Urine-Derived Injectable Fertility Products
by a Targeted Proteomic Approach
Friday, March 25, 2011
Detection of Prion Protein in Urine-Derived Injectable Fertility Products
by a Targeted Proteomic Approach
Thursday, June 09, 2011
Detection of CWD prions in salivary, urinary, and intestinal tissues of
deer: potential mechanisms of prion shedding and transmission
Wednesday, September 08, 2010
CWD PRION CONGRESS SEPTEMBER 8-11 2010
snip...
PPo3-19:
Detection of CWD Prions in Salivary and Urinary Tissues of Deer: Potential
Mechanisms of Pathogenesis and Prion Shedding
Nicholas J. Haley,1 Candace K. Mathiason,1 Glenn C. Telling2 and Edward A.
Hoover1 1Department of Microbiology, Immunology and Pathology; College of
Veterinary Medicine and Biomedical Sciences; Colorado State University; Fort
Collins, Colorado USA; 2Department of Molecular Biology and Genetics; University
of Kentucky; Lexington, Kentucky USA
Key words: chronic wasting disease, transmission, PMCA, pathogenesis,
excretion, urine, saliva, salivary gland, urinary bladder, kidney, blood
Saliva and urine are thought to play an important role in the transmission
and pathogenesis of chronic wasting disease (CWD) in captive and free-ranging
cervids. We have previously identified PrPCWD in a variety of excreta using
serial PMCA (sPMCA) and bioassay; however the source of infectious prions in
urine and saliva has yet to be identified. In the present study, we applied
sPMCA to tissues associated with saliva and urine production and excretion in an
effort to seek proximal sources of prion shedding. Oropharyngeal and urogenital
tissues, along with blood and obex from CWD-exposed cervids (comprising over 300
individual samples) were analyzed blindly in duplicate and scored based on
apparent CWD burden. PrPCWD was detected by three rounds of sPMCA in tissues
associated with saliva and urine production and excretion, notably salivary
gland and urinary bladder; whereas blood samples from the same animals and
concurrent negative controls (n = 116 of 117) remained negative. Route of
inoculation and CNS burden appeared to play an important role in terminal prion
distribution, in that IV-inoculated animals and those with increasing CNS levels
of PrPCWD had higher and more widely distributed accumulation in excretory
tissues. PMCA identification of PrPCWD in oropharyngeal and urogenital
tissues—in the absence of detection by conventional methods—may indicate the
presence of protease- sensitive infectious prions in excretory tissues not
revealed by assays employing PK digestion or other means to remove PrPC
reactivity. Thus, evaluation of peripheral tissues via sPMCA may allow
additional insights into prion transmission, trafficking and pathogenesis.
PPo3-26:
Identification of Renal Origin for CWD Urinary Prion Excretion in
Deer
Davis M. Seelig,1 Nicholas J. Haley,1 Jan P. Langeveld and Edward A.
Hoover1 1Colorado State University; Department of Microbiology, Immunology and
Pathology; Fort Collins, CO USA; 2Central Institute for Animal Disease Control
(CIDC-Lelystad); Lelystad, The Netherlands
Chronic wasting disease (CWD) is an efficiently transmitted prion disease
of cervids. Although bioassays have confirmed the presence of infectious prions
in urine and other body fluids of infected deer, origin and mechanisms of prion
transfer to and shedding in excreta remains unknown. To address these questions,
we have developed enhanced immunohistochemistry (IHC) methods employing tyramide
signal amplification (TSA) on formalin-fixed, paraffin-embedded (FFPE) tissues
of n = 20 CWD-infected white-tailed deer. Using these methods we have
demonstrated PrPCWD present granular to clumped aggregates both within the
cytoplasm of renal tubule cells and in the interstitium. Cytoplasmic PrPCWD
aggregates were detected most commonly in proximal convoluted tubule epithelial
cells. PrPCWD was not identified in the lower urinary tract (ureters or bladder)
of any CWD-infected animal. In summary, we present evidence for PrPCWD
accumulation within the renal tubule cells, which may identify a proximate
tissue source and explain the manner by which infectious prions are excreted in
the urine of infected deer, thereby leading to the high degree of direct and
indirect horizontal transmission of chronic wasting disease.
snip...see more ;
EFSA Journal 2010;8(10):1861
SCIENTIFIC OPINION
Scientific Opinion on the results of the EU survey for Chronic Wasting
Disease (CWD) in cervids1
EFSA Panel on Biological Hazards (BIOHAZ)2, 3 European Food Safety
Authority (EFSA), Parma, Italy
ABSTRACT
The BIOHAZ Panel was asked to provide a scientific opinion drawing
conclusions on the occurrence of CWD in the cervid population in the EU, based
on the results of a survey set up by the European Commission and aimed at
detecting the possible presence of CWD and other TSEs in wild and farmed cervids
in the EU during years 2006 to 2010. The survey was designed taking into account
recommendations from an earlier 2004 EFSA opinion and established the minimum
sample size to be collected from wild and farmed red deer (Cervus elaphus
elaphus) from a number of Member States and from wild white-tailed deer
(Odocoileus virginianus) from Finland. It also required all Member States to
collect additional samples from all cervid species. Overall, approximately
13,000 brain stem samples were collected from cervids of different species in 21
Member States and Norway. No TSE positive results were found. The opinion
presents, analyses and discusses the results of the survey, explains the
uncertainties involved and outlines the limitations of the survey and its
results. It is concluded that the lack of one positive TSE test in the farmed
and wild red deer and wild white-tailed deer which were sampled indicates that
there is not a cervid TSE epidemic in the EU. It is also concluded that,
considering the spreading of CWD within and from clusters in North America, the
limitations of the sampling performed in the EU CWD/TSEs survey and the known
susceptibility of certain cervid species to CWD, occurrence of cases of TSEs,
especially in remote and presently unsampled geographic areas, may not be
excluded in cervids in the EU. A few recommendations for further experimental
studies and possible future monitoring of CWD/TSEs in EU cervids are also
provided.
ASSESSMENT
1. Introduction
1.1. CWD pathogenesis and transmission
Chronic Wasting Disease (CWD) belongs to a group of fatal,
neurodegenerative disorders in humans and animals called Transmissible
Spongiform Encephalopathies or TSEs. Other TSEs include scrapie in sheep, Bovine
Spongiform Encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease in
humans. TSEs are thought to be caused by an abnormal form of proteinaceous
agents called prions that are devoid of nucleic acid. According to the prion
hypothesis, infection occurs by conversion of normal prion proteins (PrPc) into
the disease-associated, misfolded form (PrPres, or PrPCWD when referring to CWD)
that is highly resistant to degradation by proteolytic enzymes. Disease is
characterized by progressive accumulation of abnormal prions in lymphoid and
nervous tissue. During initial stages of infection, PrPCWD is present in tonsil,
gut-associated lymphoid tissues and in the enteric nervous system. Thereafter,
CWD prions extend to the central nervous system (Williams, 2005). At later
stages of infection, PrPCWD is widely distributed in the body and is present in:
tonsils, spleen, retina, skeletal muscle, fat, peripheral nervous system,
adrenal glands, blood, saliva, lymph nodes, brain and spinal cord and antler
velvet. The brainstem at obex, tonsils and retropharyngeal lymph node were
determined as the best tissue targets for PrPSc testing (Angers et al., 2009;
Race et al., 2009; Spraker et al., 2002; Williams, 2005).
Clinical signs of the disease typically appear after over 1.5 years. The
majority of CWD-affected animals are between 3-5 years of age. In experimentally
infected deer, minimum incubation was approximately 15 months and mean time from
oral infection to death was approximately 23 months (20-25 months). Animals in
the later stages of the disease exhibit behavioural changes and progressive loss
of body condition. The clinical signs of CWD are not specific and CWD can be
confused with other diseases. Most CWD positive tested animals do not show
clinical signs. There is no immune response produced in an affected host.
Currently there are no treatments or vaccines for prion diseases, and all
infections are believed fatal (Williams et al., 2001).
In CWD the horizontal route and the environment seem to play an important
role in transmission. Within endemic areas, transmission occurs
intra-specifically (among deer of the same species) and inter-specifically
(among cervids of different species). Transmission by direct contact occurs too,
but the mechanisms are not well known (Miller et al., 2000). Recently, it was
shown experimentally in cervid PrP-expressing transgenic mice that CWD can be
acquired by aerosols and by nasal exposure, but with long incubation periods
(Denkers et al., 2010). Until recently maternal transmission has not been
considered important for the maintenance of CWD and PrPCWD has not been detected
in the placenta of cervids as in sheep with scrapie (Williams, 2005). However, a
recent study demonstrated experimental maternal transmission of CWD in the Asian
muntjac deer model (Muntiacus reevesi) and presence of PrPCWD in maternal
placenta and mammary tissue of the dam (Mathiason et al., 2010). PrPCWD is shed
in the urine and saliva of terminal CWD-infected and pre-symptomatic deer (Haley
et al., 2009; Mathiason et al., 2006). CWD prions are also excreted in faeces.
This has been shown even during the incubation period; infected mule deer
(Odocoileus hemionus) shed CWD prions in faeces 7–11 months before the onset of
neurological signs (Tamguney et al., 2009). Environmental contamination by
carcass remains and excreta is considered a main source of CWD infectivity
(Miller and Williams, 2003; Miller et al., 2004). CWD-prions have been shown to
contaminate soil persisting for years. Clay components of soil bind CWD prion
proteins with high affinity (Johnson et al., 2006b). PrPCWD has been detected in
water too. Very low PrPCWD levels were detected in an environmental water sample
from an endemic area and in water processing sample by a very sensitive method,
sPMCA (Nichols et al., 2009). CWD can apparently be transmitted by fomites. In
an Results of the EU survey for CWD in cervids
EFSA Journal 2010;8(10):1861 6
experimental model, CWD naïve white tailed deer exposed to bedding material
and water from pens occupied by CWD-infected animals contracted infection after
15 months (Mathiason et al., 2009). Because CWD is readily transmitted among
captive deer and elk concentrated in pens, it is believed that transmission is
facilitated by the concentration of animals related to artificial feeding and
baiting. Relative susceptibility to transmission among cervids and for other
wildlife species has not been established. Some polymorphisms like the G96S
polymorphism in white tail deer (Johnson et al., 2006a) and S225F in mule deer
(Jewell et al., 2005) seem to provide a reduced susceptibility. The potential
for genetic influences on susceptibility relating to the CWD strains identified
in North America, as well as to other TSEs, remains under investigation.
The likelihood of interspecies transmission of prion diseases is influenced
by the degree of homology of the infective prion proteins (PrPres) with that of
the host prion protein (PrPc), giving rise to the concept of a “species barrier”
which must be overcome before an infective prion strain from one species causes
disease in another species. In addition, different strains of prions may occur
within one animal species. At present, research on biological strain typing
involves a variety of methods including biological models using laboratory
rodents, molecular, and immunohistochemistry (IHC) methods. In vitro conversion
experiments indicate that CWD prions can convert human as well as bovine and
sheep prion proteins into its abnormal conformer (PrPres), albeit at a very low
rate (Raymond et al., 2000). CWD has been experimentally transmitted after
intracerebral inoculation to a number of animals, including cattle; however,
cattle did not become infected when exposed orally to infective prion proteins
specific to CWD (Sigurdson, 2008; Williams, 2005). At present, it can be
concluded that the species barrier may not completely protect other cervid
species, including reindeer, from CWD (Li et al., 2007).
The former Scientific Steering Committee (SSC) of the European Commission
(SSC, 2003) concluded that a theoretical risk for prion transmission to humans
consuming products of CWD affected-cervids of all ages in countries where CWD
exists cannot be excluded and that transmission risk of prions to domestic
animals cannot be excluded either. Later on, an EFSA opinion (EFSA, 2004)
concluded that even though human TSE-exposure risk through consumption of game
from European cervids could be assumed to be minor, if at all existing, no final
conclusion could be drawn due to the overall lack of scientific data. The
opinion recognized a potential risk to consumers if a TSE would be present in
European cervids; however, it also stressed that at that time no data regarding
a risk of TSE infections from cervid products were available.
1.2. CWD occurrence in cervids...
snip...
© European Food Safety Authority, 2010
KEY WORDS
Chronic Wasting Disease, Transmissible Spongiform Encephalopathies,
monitoring, cervids, red deer, whitetailed deer.
1 On request from the European Commission, Question No EFSA-Q-2010-00145,
adopted on 22 September 2010.
2 Panel members: Olivier Andreoletti, Herbert Budka, Sava Buncic, John D
Collins, John Griffin, Tine Hald, Arie Havelaar, James Hope, Günter Klein, James
McLauchlin, Christine Müller-Graf, Christophe Nguyen-The, Birgit Noerrung, Luisa
Peixe, Miguel Prieto Maradona, Antonia Ricci, John Sofos, John Threlfall, Ivar
Vågsholm and Emmanuel Vanopdenbosch. Correspondence: biohaz@efsa.europa.eu
3 Acknowledgement: The Panel wishes to thank the members of the Working
Group on the Results of EU survey for CWD in cervids: Olivier Andreoletti, Dirk
Berkvens, Christian Ducrot, Dolores Gavier-Widen, John Griffin, James Hope and
Emmanuel Vanopdenbosch for the preparatory work on this scientific opinion and
the hearing expert Matthias Greiner for the support provided to this scientific
opinion.
Sunday, December 06, 2009
Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer
Long after Oral Exposure to Urine and Feces from CWD+ Deer
Abstract Background Chronic wasting disease (CWD) of cervids is a prion
disease distinguished by high levels of transmissibility, wherein bodily fluids
and excretions are thought to play an important role. Using cervid bioassay and
established CWD detection methods, we have previously identified infectious
prions in saliva and blood but not urine or feces of CWD+ donors. More recently,
we identified very low concentrations of CWD prions in urine of deer by cervid
PrP transgenic (Tg[CerPrP]) mouse bioassay and serial protein misfolding cyclic
amplification (sPMCA). This finding led us to examine further our initial cervid
bioassay experiments using sPMCA.
Objectives We sought to investigate whether conventional test-negative
deer, previously exposed orally to urine and feces from CWD+ sources, may be
harboring low level CWD infection not evident in the 19 month observation
period. We further attempted to determine the peripheral PrPCWD distribution in
these animals.
Methods Various neural and lymphoid tissues from conventional test-negative
deer were reanalyzed for CWD prions by sPMCA and cervid transgenic mouse
bioassay in parallel with appropriate tissue-matched positive and negative
controls.
Results PrPCWD was detected in the tissues of orally exposed deer by both
sPMCA and Tg[CerPrP] mouse bioassay; each assay revealed very low levels of CWD
prions previously undetectable by western blot, ELISA, or IHC. Serial PMCA
analysis of individual tissues identified that obex alone was positive in 4 of 5
urine/feces exposed deer. PrPCWD was amplified from both lymphoid and neural
tissues of positive control deer but not from identical tissues of negative
control deer.
Discussion Detection of subclinical infection in deer orally exposed to
urine and feces (1) suggests that a prolonged subclinical state can exist,
necessitating observation periods in excess of two years to detect CWD
infection, and (2) illustrates the sensitive and specific application of sPMCA
in the diagnosis of low-level prion infection. Based on these results, it is
possible that low doses of prions, e.g. following oral exposure to urine and
saliva of CWD-infected deer, bypass significant amplification in the LRS,
perhaps utilizing a neural conduit between the alimentary tract and CNS, as has
been demonstrated in some other prion diseases.
snip...
In summary, we provide evidence for the presence of infectious prions in
the brains of conventional prion-assay-negative deer orally exposed 19 months
earlier to urine and feces from CWD-infected donor deer. This apparent low level
of prion infection was amplified by sPMCA, confirmed by Tg[CerPrP] mouse
bioassay, and detected only in the obex region of the brain. These results
demonstrate the potential for CWD prion transmission via urine and/or feces, and
highlight the application of more sensitive assays such as sPMCA in
identification of CWD infection, pathogenesis, and prevalence.
Wednesday, March 18, 2009
Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse
Bioassay
Detection of infectious prions in urine
Dennisse Gonzalez-Romero, Marcelo A. Barria, Patricia Leon, Rodrigo
Morales, Claudio Soto* George and Cynthia Mitchell Center for Neurodegenerative
diseases, Departments of Neurology, Neuroscience and Cell Biology and
Biochemistry and Molecular Biology, University of Texas Medical Branch, 301
University Boulevard, Galveston, TX 77555-0646, USA Received 26 July 2008;
accepted 4 August 2008
Available online 13 August 2008
Edited by Aleksander Benjak
Abstract
Prions are the infectious agents responsible for prion diseases, which
appear to be composed exclusively by the misfolded prion protein (PrPSc). The
mechanism of prion transmission is unknown. In this study, we attempted to
detect prions in urine of experimentally infected animals. PrPSc was detected in
80% of the animals studied, whereas no false positives were observed among the
control animals. Semi-quantitative calculations suggest that PrPSc concentration
in urine is around 10-fold lower than in blood. Interestingly, PrPSc present in
urine maintains its infectious properties. Our data indicate that low quantities
of infectious prions are excreted in the urine. These findings suggest that
urine is a possible source of prion transmission.
2008 Federation of European Biochemical Societies. Published by Elsevier
B.V. All rights reserved. Keywords: Prion; Transmissible
see full text ;
pdf
Tuesday, September 02, 2008
Detection of infectious prions in urine (Soto et al Available online 13
August 2008.)
doi:10.1016/j.febslet.2008.08.003 Copyright © 2008 Published by Elsevier
B.V.
Detection of infectious prions in urine
Dennisse Gonzalez-Romeroa, Marcelo A. Barriaa, Patricia Leona, Rodrigo
Moralesa and Claudio Soto, a,
Sunday, August 02, 2015 HAVE YOU BEEN THUNDERSTRUCK ?
RAW, UNCUT, AND UNCENSORED
Sunday, August 23, 2015
TAHC Chronic Wasting Disease CWD TSE Prion and how to put lipstick on a pig
and take her to the dance in Texas
TEXAS DEER CZAR SENT TO WISCONSIN TO SOLVE CWD CRISIS, WHILE ROME (TEXAS)
BURNS
Tuesday, August 11, 2015
*** Wisconsin doing what it does best, procrastinating about CWD yet again
thanks to Governor Walker
Wednesday, March 04, 2015
*** Disease sampling results provide current snapshot of CWD in Wisconsin
finding 324 positive detections statewide in 2014
Friday, June 01, 2012
*** TEXAS DEER CZAR TO WISCONSIN ASK TO EXPLAIN COMMENTS
Wednesday, March 18, 2015
Chronic Wasting Disease CWD Confirmed Texas Trans Pecos March 18, 2015
Wednesday, March 25, 2015
Chronic Wasting Disease CWD Cases Confirmed In New Mexico 2013 and 2014
UPDATE 2015
Wednesday, July 01, 2015
TEXAS Chronic Wasting Disease Detected in Medina County Captive Deer
Tuesday, July 21, 2015
*** Texas CWD Medina County Herd Investigation Update July 16, 2015 ***
Thursday, August 06, 2015
WE HAVE LOST TEXAS TO CWD TASK FORCE CATERING TO INDUSTRY
Friday, August 07, 2015
Texas CWD Captive, and then there were 4 ?
Thursday, August 20, 2015
*** TEXAS TAHC DEER BREEDER CWD PERMIT RULES EMERGENCY ADOPTION PREAMBLE
***
Thursday, August 20, 2015
TEXAS CAPTIVE Deer Industry, Pens, Breeding, Big Business, Invites Crooks
and CWD
Wednesday, March 18, 2015
Chronic Wasting Disease CWD Confirmed Texas Trans Pecos March 18, 2015
Wednesday, March 25, 2015
Chronic Wasting Disease CWD Cases Confirmed In New Mexico 2013 and 2014
UPDATE 2015
*** Danger of Canned Hunting Indiana Wildlife ***
a review since the TEXAS 84th Legislature commencing this January, deer
breeders are expected to advocate for bills that will seek to further deregulate
their industry...
Sunday, December 14, 2014
TEXAS 84th Legislature commencing this January, deer breeders are expected
to advocate for bills that will seek to further deregulate their industry
Tuesday, December 16, 2014
Texas 84th Legislature 2015 H.R. No. 2597 Kuempel Deer Breeding Industry
TAHC TPWD CWD TSE PRION
Under Texas law, though, breeder deer belong to the state, not the
permittee. See, e.g., TEX. PARKS & WILD. CODE §§ 1.011 (“All wild
animals . . . inside the borders of this state are the property of the people of
this state.”); 43.364 (“All breeder deer . . . are under the full force of the
laws of [Texas] pertaining to deer . . . .”). While a permittee may have
possession of the breeder deer, the deer are only “held under a permit[.]” Id. §
43.351. Nowhere do the statutes or regulations state that breeder deer become
the property of a permit holder.4 Regardless, even if they did give ownership of
breeder deer to permit holders, the Andertons were not permit holders when the
deer were killed.
While a permittee may have possession of the breeder deer, the deer are
only “held under a permit[.]” Id. § 43.351
Monday, August 24, 2015
Ohio wildlife officials ramp up fight against fatal deer brain disease
after 17 more positive tests CWD
Monday, August 31, 2015
Illinois Loosing Ground to Chronic Wasting Disease CWD cases mounting with
71 confirmed in 2015 and 538 confirmed cases to date
HIGHEST INFECTION RATE ON SEVERAL CWD CONFIRMED CAPTIVES
CHRONIC WASTING DISEASE CWD WISCONSIN Almond Deer (Buckhorn Flats) Farm
Update DECEMBER 2011
The CWD infection rate was nearly 80%, the highest ever in a North American
captive herd.
RECOMMENDATION: That the Board approve the purchase of 80 acres of land for
$465,000 for the Statewide Wildlife Habitat Program in Portage County and
approve the restrictions on public use of the site.
SUMMARY:
For Immediate Release Thursday, October 2, 2014
Dustin Vande Hoef 515/281-3375 or 515/326-1616 (cell) or
Dustin.VandeHoef@IowaAgriculture.gov
*** TEST RESULTS FROM CAPTIVE DEER HERD WITH CHRONIC WASTING DISEASE
RELEASED 79.8 percent of the deer tested positive for the disease
DES MOINES – The Iowa Department of Agriculture and Land Stewardship today
announced that the test results from the depopulation of a quarantined captive
deer herd in north-central Iowa showed that 284 of the 356 deer, or 79.8% of the
herd, tested positive for Chronic Wasting Disease (CWD).
*** see history of this CWD blunder here ;
On June 5, 2013, DNR conducted a fence inspection, after gaining approval
from surrounding landowners, and confirmed that the fenced had been cut or
removed in at least four separate locations; that the fence had degraded and was
failing to maintain the enclosure around the Quarantined Premises in at least
one area; that at least three gates had been opened;and that deer tracks were
visible in and around one of the open areas in the sand on both sides of the
fence, evidencing movement of deer into the Quarantined Premises.
The overall incidence of clinical CWD in white-tailed deer was 82%
Species (cohort) CWD (cases/total) Incidence (%) Age at CWD death (mo)
CWD, spreading it around...
for the game farm industry, and their constituents, to continue to believe
that they are _NOT_, and or insinuate that they have _NEVER_ been part of the
problem, will only continue to help spread cwd. the game farming industry, from
the shooting pens, to the urine mills, the antler mills, the sperm mills, velvet
mills, shooting pens, to large ranches, are not the only problem, but it is
painfully obvious that they have been part of the problem for decades and
decades, just spreading it around, as with transportation and or exportation and
or importation of cervids from game farming industry, and have been proven to
spread cwd. no one need to look any further than South Korea blunder ;
===========================================
spreading cwd around...
Between 1996 and 2002, chronic wasting disease was diagnosed in 39 herds of
farmed elk in Saskatchewan in a single epidemic. All of these herds were
depopulated as part of the Canadian Food Inspection Agency’s (CFIA) disease
eradication program. Animals, primarily over 12 mo of age, were tested for the
presence CWD prions following euthanasia. Twenty-one of the herds were linked
through movements of live animals with latent CWD from a single infected source
herd in Saskatchewan, 17 through movements of animals from 7 of the secondarily
infected herds.
***The source herd is believed to have become infected via importation of
animals from a game farm in South Dakota where CWD was subsequently diagnosed
(7,4). A wide range in herd prevalence of CWD at the time of herd depopulation
of these herds was observed. Within-herd transmission was observed on some
farms, while the disease remained confined to the introduced animals on other
farms.
spreading cwd around...
Friday, May 13, 2011
Chronic Wasting Disease (CWD) outbreaks and surveillance program in the
Republic of Korea
Hyun-Joo Sohn, Yoon-Hee Lee, Min-jeong Kim, Eun-Im Yun, Hyo-Jin Kim,
Won-Yong Lee, Dong-Seob Tark, In- Soo Cho, Foreign Animal Disease Research
Division, National Veterinary Research and Quarantine Service, Republic of Korea
Chronic wasting disease (CWD) has been recognized as an important prion
disease in native North America deer and Rocky mountain elks. The disease is a
unique member of the transmissible spongiform encephalopathies (TSEs), which
naturally affects only a few species. CWD had been limited to USA and Canada
until 2000.
On 28 December 2000, information from the Canadian government showed that a
total of 95 elk had been exported from farms with CWD to Korea. These consisted
of 23 elk in 1994 originating from the so-called “source farm” in Canada, and 72
elk in 1997, which had been held in pre export quarantine at the “source
farm”.Based on export information of CWD suspected elk from Canada to Korea, CWD
surveillance program was initiated by the Ministry of Agriculture and Forestry
(MAF) in 2001.
All elks imported in 1997 were traced back, however elks imported in 1994
were impossible to identify. CWD control measures included stamping out of all
animals in the affected farm, and thorough cleaning and disinfection of the
premises. In addition, nationwide clinical surveillance of Korean native
cervids, and improved measures to ensure reporting of CWD suspect cases were
implemented.
Total of 9 elks were found to be affected. CWD was designated as a
notifiable disease under the Act for Prevention of Livestock Epidemics in 2002.
Additional CWD cases - 12 elks and 2 elks - were diagnosed in 2004 and
2005.
Since February of 2005, when slaughtered elks were found to be positive,
all slaughtered cervid for human consumption at abattoirs were designated as
target of the CWD surveillance program. Currently, CWD laboratory testing is
only conducted by National Reference Laboratory on CWD, which is the Foreign
Animal Disease Division (FADD) of National Veterinary Research and Quarantine
Service (NVRQS).
In July 2010, one out of 3 elks from Farm 1 which were slaughtered for the
human consumption was confirmed as positive. Consequently, all cervid – 54 elks,
41 Sika deer and 5 Albino deer – were culled and one elk was found to be
positive. Epidemiological investigations were conducted by Veterinary
Epidemiology Division (VED) of NVRQS in collaboration with provincial veterinary
services.
Epidemiologically related farms were found as 3 farms and all cervid at
these farms were culled and subjected to CWD diagnosis. Three elks and 5
crossbreeds (Red deer and Sika deer) were confirmed as positive at farm 2.
All cervids at Farm 3 and Farm 4 – 15 elks and 47 elks – were culled and
confirmed as negative.
Further epidemiological investigations showed that these CWD outbreaks were
linked to the importation of elks from Canada in 1994 based on circumstantial
evidences.
In December 2010, one elk was confirmed as positive at Farm 5.
Consequently, all cervid – 3 elks, 11 Manchurian Sika deer and 20 Sika deer –
were culled and one Manchurian Sika deer and seven Sika deer were found to be
positive. This is the first report of CWD in these sub-species of deer.
Epidemiological investigations found that the owner of the Farm 2 in CWD
outbreaks in July 2010 had co-owned the Farm 5.
In addition, it was newly revealed that one positive elk was introduced
from Farm 6 of Jinju-si Gyeongsang Namdo. All cervid – 19 elks, 15 crossbreed
(species unknown) and 64 Sika deer – of Farm 6 were culled, but all confirmed as
negative.
New studies on the heat resistance of hamster-adapted scrapie agent:
Threshold survival after ashing at 600°C suggests an inorganic template of
replication
The infectious agents responsible for transmissible spongiform
encephalopathy (TSE) are notoriously resistant to most physical and chemical
methods used for inactivating pathogens, including heat. It has long been
recognized, for example, that boiling is ineffective and that higher
temperatures are most efficient when combined with steam under pressure (i.e.,
autoclaving). As a means of decontamination, dry heat is used only at the
extremely high temperatures achieved during incineration, usually in excess of
600°C. It has been assumed, without proof, that incineration totally inactivates
the agents of TSE, whether of human or animal origin.
Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel
Production
Histochemical analysis of hamster brains inoculated with the solid residue
showed typical spongiform degeneration and vacuolation. Re-inoculation of these
brains into a new cohort of hamsters led to onset of clinical scrapie symptoms
within 75 days, suggesting that the specific infectivity of the prion protein
was not changed during the biodiesel process. The biodiesel reaction cannot be
considered a viable prion decontamination method for MBM, although we observed
increased survival time of hamsters and reduced infectivity greater than 6 log
orders in the solid MBM residue. Furthermore, results from our study compare for
the first time prion detection by Western Blot versus an infectivity bioassay
for analysis of biodiesel reaction products. We could show that biochemical
analysis alone is insufficient for detection of prion infectivity after a
biodiesel process.
Detection of protease-resistant cervid prion protein in water from a
CWD-endemic area
The data presented here demonstrate that sPMCA can detect low levels of
PrPCWD in the environment, corroborate previous biological and experimental data
suggesting long term persistence of prions in the environment2,3 and imply that
PrPCWD accumulation over time may contribute to transmission of CWD in areas
where it has been endemic for decades. This work demonstrates the utility of
sPMCA to evaluate other environmental water sources for PrPCWD, including
smaller bodies of water such as vernal pools and wallows, where large numbers of
cervids congregate and into which prions from infected animals may be shed and
concentrated to infectious levels.
A Quantitative Assessment of the Amount of Prion Diverted to Category 1
Materials and Wastewater During Processing
Keywords:Abattoir;bovine spongiform encephalopathy;QRA;scrapie;TSE
In this article the development and parameterization of a quantitative
assessment is described that estimates the amount of TSE infectivity that is
present in a whole animal carcass (bovine spongiform encephalopathy [BSE] for
cattle and classical/atypical scrapie for sheep and lambs) and the amounts that
subsequently fall to the floor during processing at facilities that handle
specified risk material (SRM). BSE in cattle was found to contain the most oral
doses, with a mean of 9864 BO ID50s (310, 38840) in a whole carcass compared to
a mean of 1851 OO ID50s (600, 4070) and 614 OO ID50s (155, 1509) for a sheep
infected with classical and atypical scrapie, respectively. Lambs contained the
least infectivity with a mean of 251 OO ID50s (83, 548) for classical scrapie
and 1 OO ID50s (0.2, 2) for atypical scrapie. The highest amounts of infectivity
falling to the floor and entering the drains from slaughtering a whole carcass
at SRM facilities were found to be from cattle infected with BSE at rendering
and large incineration facilities with 7.4 BO ID50s (0.1, 29), intermediate
plants and small incinerators with a mean of 4.5 BO ID50s (0.1, 18), and
collection centers, 3.6 BO ID50s (0.1, 14). The lowest amounts entering drains
are from lambs infected with classical and atypical scrapie at intermediate
plants and atypical scrapie at collection centers with a mean of 3 × 10−7 OO
ID50s (2 × 10−8, 1 × 10−6) per carcass. The results of this model provide key
inputs for the model in the companion paper published here.
98 | Veterinary Record | January 24, 2015
EDITORIAL
Scrapie: a particularly persistent pathogen
Cristina Acín
Resistant prions in the environment have been the sword of Damocles for
scrapie control and eradication. Attempts to establish which physical and
chemical agents could be applied to inactivate or moderate scrapie infectivity
were initiated in the 1960s and 1970s,with the first study of this type focusing
on the effect of heat treatment in reducing prion infectivity (Hunter and
Millson 1964). Nowadays, most of the chemical procedures that aim to inactivate
the prion protein are based on the method developed by Kimberlin and
collaborators (1983). This procedure consists of treatment with 20,000 parts per
million free chlorine solution, for a minimum of one hour, of all surfaces that
need to be sterilised (in laboratories, lambing pens, slaughterhouses, and so
on). Despite this, veterinarians and farmers may still ask a range of questions,
such as ‘Is there an official procedure published somewhere?’ and ‘Is there an
international organisation which recommends and defines the exact method of
scrapie decontamination that must be applied?’
From a European perspective, it is difficult to find a treatment that could
be applied, especially in relation to the disinfection of surfaces in lambing
pens of affected flocks. A 999/2001 EU regulation on controlling spongiform
encephalopathies (European Parliament and Council 2001) did not specify a
particular decontamination measure to be used when an outbreak of scrapie is
diagnosed. There is only a brief recommendation in Annex VII concerning the
control and eradication of transmissible spongiform encephalopathies (TSE
s).
Chapter B of the regulation explains the measures that must be applied if
new caprine animals are to be introduced to a holding where a scrapie outbreak
has previously been diagnosed. In that case, the statement indicates that
caprine animals can be introduced ‘provided that a cleaning and disinfection of
all animal housing on the premises has been carried out following
destocking’.
Issues around cleaning and disinfection are common in prion prevention
recommendations, but relevant authorities, veterinarians and farmers may have
difficulties in finding the specific protocol which applies. The European Food
and Safety Authority (EFSA ) published a detailed report about the efficacy of
certain biocides, such as sodium hydroxide, sodium hypochlorite, guanidine and
even a formulation of copper or iron metal ions in combination with hydrogen
peroxide, against prions (EFSA 2009). The report was based on scientific
evidence (Fichet and others 2004, Lemmer and others 2004, Gao and others 2006,
Solassol and others 2006) but unfortunately the decontamination measures were
not assessed under outbreak conditions.
The EFSA Panel on Biological Hazards recently published its conclusions on
the scrapie situation in the EU after 10 years of monitoring and control of the
disease in sheep and goats (EFSA 2014), and one of the most interesting findings
was the Icelandic experience regarding the effect of disinfection in scrapie
control. The Icelandic plan consisted of: culling scrapie-affected sheep or the
whole flock in newly diagnosed outbreaks; deep cleaning and disinfection of
stables, sheds, barns and equipment with high pressure washing followed by
cleaning with 500 parts per million of hypochlorite; drying and treatment with
300 ppm of iodophor; and restocking was not permitted for at least two years.
Even when all of these measures were implemented, scrapie recurred on several
farms, indicating that the infectious agent survived for years in the
environment, even as many as 16 years after restocking (Georgsson and others
2006).
In the rest of the countries considered in the EFSA (2014) report,
recommendations for disinfection measures were not specifically defined at the
government level. In the report, the only recommendation that is made for sheep
is repopulation with sheep with scrapie-resistant genotypes. This reduces the
risk of scrapie recurrence but it is difficult to know its effect on the
infection.
Until the EFSA was established (in May 2003), scientific opinions about TSE
s were provided by the Scientific Steering Committee (SSC) of the EC, whose
advice regarding inactivation procedures focused on treating animal waste at
high temperatures (150°C for three hours) and high pressure alkaline hydrolysis
(SSC 2003). At the same time, the TSE Risk Management Subgroup of the Advisory
Committee on Dangerous Pathogens (ACDP) in the UK published guidance on safe
working and the prevention of TSE infection. Annex C of the ACDP report
established that sodium hypochlorite was considered to be effective, but only if
20,000 ppm of available chlorine was present for at least one hour, which has
practical limitations such as the release of chlorine gas, corrosion,
incompatibility with formaldehyde, alcohols and acids, rapid inactivation of its
active chemicals and the stability of dilutions (ACDP 2009).
In an international context, the World Organisation for Animal Health (OIE)
does not recommend a specific disinfection protocol for prion agents in its
Terrestrial Code or Manual. Chapter 4.13 of the Terrestrial Code, General
recommendations on disinfection and disinsection (OIE 2014), focuses on
foot-and-mouth disease virus, mycobacteria and Bacillus anthracis, but not on
prion disinfection. Nevertheless, the last update published by the OIE on bovine
spongiform encephalopathy (OIE 2012) indicates that few effective
decontamination techniques are available to inactivate the agent on surfaces,
and recommends the removal of all organic material and the use of sodium
hydroxide, or a sodium hypochlorite solution containing 2 per cent available
chlorine, for more than one hour at 20ºC.
The World Health Organization outlines guidelines for the control of TSE s,
and also emphasises the importance of mechanically cleaning surfaces before
disinfection with sodium hydroxide or sodium hypochlorite for one hour (WHO
1999).
Finally, the relevant agencies in both Canada and the USA suggest that the
best treatments for surfaces potentially contaminated with prions are sodium
hydroxide or sodium hypochlorite at 20,000 ppm. This is a 2 per cent solution,
while most commercial household bleaches contain 5.25 per cent sodium
hypochlorite. It is therefore recommended to dilute one part 5.25 per cent
bleach with 1.5 parts water (CDC 2009, Canadian Food Inspection Agency
2013).
So what should we do about disinfection against prions? First, it is
suggested that a single protocol be created by international authorities to
homogenise inactivation procedures and enable their application in all
scrapie-affected countries. Sodium hypochlorite with 20,000 ppm of available
chlorine seems to be the procedure used in most countries, as noted in a paper
summarised on p 99 of this issue of Veterinary Record (Hawkins and others 2015).
But are we totally sure of its effectiveness as a preventive measure in a
scrapie outbreak? Would an in-depth study of the recurrence of scrapie disease
be needed?
What we can conclude is that, if we want to fight prion diseases, and
specifically classical scrapie, we must focus on the accuracy of diagnosis,
monitoring and surveillance; appropriate animal identification and control of
movements; and, in the end, have homogeneous and suitable protocols to
decontaminate and disinfect lambing barns, sheds and equipment available to
veterinarians and farmers. Finally, further investigations into the resistance
of prion proteins in the diversity of environmental surfaces are required.
References
snip...
98 | Veterinary Record | January 24, 2015
Persistence of ovine scrapie infectivity in a farm environment following
cleaning and decontamination
Steve A. C. Hawkins, MIBiol, Pathology Department1, Hugh A. Simmons, BVSc
MRCVS, MBA, MA Animal Services Unit1, Kevin C. Gough, BSc, PhD2 and Ben C.
Maddison, BSc, PhD3 + Author Affiliations
1Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey
KT15 3NB, UK 2School of Veterinary Medicine and Science, The University of
Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK 3ADAS
UK, School of Veterinary Medicine and Science, The University of Nottingham,
Sutton Bonington, Loughborough, Leicestershire LE12 5RD, UK E-mail for
correspondence: ben.maddison@adas.co.uk Abstract Scrapie of sheep/goats and
chronic wasting disease of deer/elk are contagious prion diseases where
environmental reservoirs are directly implicated in the transmission of disease.
In this study, the effectiveness of recommended scrapie farm decontamination
regimens was evaluated by a sheep bioassay using buildings naturally
contaminated with scrapie. Pens within a farm building were treated with either
20,000 parts per million free chorine solution for one hour or were treated with
the same but were followed by painting and full re-galvanisation or replacement
of metalwork within the pen. Scrapie susceptible lambs of the PRNP genotype
VRQ/VRQ were reared within these pens and their scrapie status was monitored by
recto-anal mucosa-associated lymphoid tissue. All animals became infected over
an 18-month period, even in the pen that had been subject to the most stringent
decontamination process. These data suggest that recommended current guidelines
for the decontamination of farm buildings following outbreaks of scrapie do
little to reduce the titre of infectious scrapie material and that environmental
recontamination could also be an issue associated with these premises.
SNIP...
Discussion
Thorough pressure washing of a pen had no effect on the amount of
bioavailable scrapie infectivity (pen B). The routine removal of prions from
surfaces within a laboratory setting is treatment for a minimum of one hour with
20,000 ppm free chlorine, a method originally based on the use of brain
macerates from infected rodents to evaluate the effectiveness of decontamination
(Kimberlin and others 1983). Further studies have also investigated the
effectiveness of hypochlorite disinfection of metal surfaces to simulate the
decontamination of surgical devices within a hospital setting. Such treatments
with hypochlorite solution were able to reduce infectivity by 5.5 logs to lower
than the sensitivity of the bioassay used (Lemmer and others 2004). Analogous
treatment of the pen surfaces did not effectively remove the levels of scrapie
infectivity over that of the control pens, indicating that this method of
decontamination is not effective within a farm setting. This may be due to the
high level of biological matrix that is present upon surfaces within the farm
environment, which may reduce the amount of free chlorine available to
inactivate any infectious prion. Remarkably 1/5 sheep introduced into pen D had
also became scrapie positive within nine months, with all animals in this pen
being RAMALT positive by 18 months of age. Pen D was no further away from the
control pen (pen A) than any of the other pens within this barn. Localised hot
spots of infectivity may be present within scrapie-contaminated environments,
but it is unlikely that pen D area had an amount of scrapie contamination that
was significantly different than the other areas within this building.
Similarly, there were no differences in how the biosecurity of pen D was
maintained, or how this pen was ventilated compared with the other pens. This
observation, perhaps, indicates the slower kinetics of disease uptake within
this pen and is consistent with a more thorough prion removal and
recontamination. These observations may also account for the presence of
inadvertent scrapie cases within other studies, where despite stringent
biosecurity, control animals have become scrapie positive during challenge
studies using barns that also housed scrapie-affected animals (Ryder and others
2009). The bioassay data indicate that the exposure of the sheep to a farm
environment after decontamination efforts thought to be effective in removing
scrapie is sufficient for the animals to become infected with scrapie. The main
exposure routes within this scenario are likely to be via the oral route, during
feeding and drinking, and respiratory and conjunctival routes. It has been
demonstrated that scrapie infectivity can be efficiently transmitted via the
nasal route in sheep (Hamir and others 2008), as is the case for CWD in both
murine models and in white-tailed deer (Denkers and others 2010, 2013).
Recently, it has also been demonstrated that CWD prions presented as dust when
bound to the soil mineral montmorillonite can be infectious via the nasal route
(Nichols and others 2013). When considering pens C and D, the actual source of
the infectious agent in the pens is not known, it is possible that biologically
relevant levels of prion survive on surfaces during the decontamination regimen
(pen C). With the use of galvanising and painting (pen D) covering and sealing
the surface of the pen, it is possible that scrapie material recontaminated the
pens by the movement of infectious prions contained within dusts originating
from other parts of the barn that were not decontaminated or from other areas of
the farm.
Given that scrapie prions are widespread on the surfaces of affected farms
(Maddison and others 2010a), irrespective of the source of the infectious prions
in the pens, this study clearly highlights the difficulties that are faced with
the effective removal of environmentally associated scrapie infectivity. This is
likely to be paralleled in CWD which shows strong similarities to scrapie in
terms of both the dissemination of prions into the environment and the facile
mode of disease transmission. These data further contribute to the understanding
that prion diseases can be highly transmissible between susceptible individuals
not just by direct contact but through highly stable environmental reservoirs
that are refractory to decontamination.
The presence of these environmentally associated prions in farm buildings
make the control of these diseases a considerable challenge, especially in
animal species such as goats where there is lack of genetic resistance to
scrapie and, therefore, no scope to re-stock farms with animals that are
resistant to scrapie.
Scrapie Sheep Goats Transmissible spongiform encephalopathies (TSE)
Accepted October 12, 2014. Published Online First 31 October 2014
Monday, November 3, 2014
Persistence of ovine scrapie infectivity in a farm environment following
cleaning and decontamination
PPo3-22:
Detection of Environmentally Associated PrPSc on a Farm with Endemic
Scrapie
Ben C. Maddison,1 Claire A. Baker,1 Helen C. Rees,1 Linda A. Terry,2 Leigh
Thorne,2 Susan J. Belworthy2 and Kevin C. Gough3 1ADAS-UK LTD; Department of
Biology; University of Leicester; Leicester, UK; 2Veterinary Laboratories
Agency; Surry, KT UK; 3Department of Veterinary Medicine and Science; University
of Nottingham; Sutton Bonington, Loughborough UK
Key words: scrapie, evironmental persistence, sPMCA
Ovine scrapie shows considerable horizontal transmission, yet the routes of
transmission and specifically the role of fomites in transmission remain poorly
defined. Here we present biochemical data demonstrating that on a
scrapie-affected sheep farm, scrapie prion contamination is widespread. It was
anticipated at the outset that if prions contaminate the environment that they
would be there at extremely low levels, as such the most sensitive method
available for the detection of PrPSc, serial Protein Misfolding Cyclic
Amplification (sPMCA), was used in this study. We investigated the distribution
of environmental scrapie prions by applying ovine sPMCA to samples taken from a
range of surfaces that were accessible to animals and could be collected by use
of a wetted foam swab. Prion was amplified by sPMCA from a number of these
environmental swab samples including those taken from metal, plastic and wooden
surfaces, both in the indoor and outdoor environment. At the time of sampling
there had been no sheep contact with these areas for at least 20 days prior to
sampling indicating that prions persist for at least this duration in the
environment. These data implicate inanimate objects as environmental reservoirs
of prion infectivity which are likely to contribute to disease transmission.
*** Infectious agent of sheep scrapie may persist in the environment for at
least 16 years ***
Gudmundur Georgsson1, Sigurdur Sigurdarson2 and Paul Brown3
P.97: Scrapie transmits to white-tailed deer by the oral route and has a
molecular profile similar to chronic wasting disease and distinct from the
scrapie inoculum
Justin Greenlee1, S Jo Moore1, Jodi Smith1, M Heather West Greenlee2, and
Robert Kunkle1 1National Animal Disease Center; Ames, IA USA; 2Iowa State
University; Ames, IA USA
The purpose of this work was to determine susceptibility of white-tailed
deer (WTD) to the agent of sheep scrapie and to compare the resultant PrPSc to
that of the original inoculum and chronic wasting disease (CWD). We inoculated
WTD by a natural route of exposure (concurrent oral and intranasal (IN); n D 5)
with a US scrapie isolate. All scrapie-inoculated deer had evidence of PrPSc
accumulation. PrPSc was detected in lymphoid tissues at preclinical time points,
and deer necropsied after 28 months post-inoculation had clinical signs,
spongiform encephalopathy, and widespread distribution of PrPSc in neural and
lymphoid tissues. Western blotting (WB) revealed PrPSc with 2 distinct molecular
profiles. WB on cerebral cortex had a profile similar to the original scrapie
inoculum, whereas WB of brainstem, cerebellum, or lymph nodes revealed PrPSc
with a higher profile resembling CWD. Homogenates with the 2 distinct profiles
from WTD with clinical scrapie were further passaged to mice expressing cervid
prion protein and intranasally to sheep and WTD. In cervidized mice, the 2
inocula have distinct incubation times. Sheep inoculated intranasally with WTD
derived scrapie developed disease, but only after inoculation with the inoculum
that had a scrapie-like profile. The WTD study is ongoing, but deer in both
inoculation groups are positive for PrPSc by rectal mucosal biopsy. In summary,
this work demonstrates that WTD are susceptible to the agent of scrapie, 2
distinct molecular profiles of PrPSc are present in the tissues of affected
deer, and inoculum of either profile readily passes to deer.
PRION 2015 CONFERENCE FT. COLLINS CWD RISK FACTORS TO HUMANS
*** LATE-BREAKING ABSTRACTS PRION 2015 CONFERENCE ***
O18
Zoonotic Potential of CWD Prions
Liuting Qing1, Ignazio Cali1,2, Jue Yuan1, Shenghai Huang3, Diane Kofskey1,
Pierluigi Gambetti1, Wenquan Zou1, Qingzhong Kong1 1Case Western Reserve
University, Cleveland, Ohio, USA, 2Second University of Naples, Naples, Italy,
3Encore Health Resources, Houston, Texas, USA
*** These results indicate that the CWD prion has the potential to infect
human CNS and peripheral lymphoid tissues and that there might be asymptomatic
human carriers of CWD infection.
==================
***These results indicate that the CWD prion has the potential to infect
human CNS and peripheral lymphoid tissues and that there might be asymptomatic
human carriers of CWD infection.***
==================
P.105: RT-QuIC models trans-species prion transmission
Kristen Davenport, Davin Henderson, Candace Mathiason, and Edward Hoover
Prion Research Center; Colorado State University; Fort Collins, CO USA
Conversely, FSE maintained sufficient BSE characteristics to more
efficiently convert bovine rPrP than feline rPrP. Additionally, human rPrP was
competent for conversion by CWD and fCWD.
***This insinuates that, at the level of protein:protein interactions, the
barrier preventing transmission of CWD to humans is less robust than previously
estimated.
================
***This insinuates that, at the level of protein:protein interactions, the
barrier preventing transmission of CWD to humans is less robust than previously
estimated.***
================
PRION2013 CONGRESSIONAL ABSTRACTS CWD
Sunday, August 25, 2013
HD.13: CWD infection in the spleen of humanized transgenic mice
Liuting Qing and Qingzhong Kong
Case Western Reserve University; Cleveland, OH USA
Chronic wasting disease (CWD) is a widespread prion disease in free-ranging
and captive cervid species in North America, and there is evidence suggesting
the existence of multiple CWD strains. The susceptibility of human CNS and
peripheral organs to the various CWD prion strains remains largely unclear.
Current literature suggests that the classical CWD strain is unlikely to infect
human brain, but the potential for peripheral infection by CWD in humans is
unknown. We detected protease-resistant PrpSc in the spleens of a few humanized
transgenic mice that were intracerebrally inoculated with natural CWD isolates,
but PrpSc was not detected in the brains of any of the CWD-inoculated mice. Our
ongoing bioassays in humanized Tg mice indicate that intracerebral challenge
with such PrpSc-positive humanized mouse spleen already led to prion disease in
most animals. ***These results indicate that the CWD prion may have the
potential to infect human peripheral lymphoid tissues.
Oral.15: Molecular barriers to zoonotic prion transmission: Comparison of
the ability of sheep, cattle and deer prion disease isolates to convert normal
human prion protein to its pathological isoform in a cell-free system
Marcelo A.Barria,1 Aru Balachandran,2 Masanori Morita,3 Tetsuyuki
Kitamoto,4 Rona Barron,5 Jean Manson,5 Richard Kniqht,1 James W. lronside1 and
Mark W. Head1
1National CJD Research and Surveillance Unit; Centre for Clinical Brain
Sciences; School of Clinical Sciences; The University of Edinburgh; Edinburgh,
UK; 2National and OIE Reference Laboratory for Scrapie and CWD; Canadian Food
Inspection Agency; Ottawa Laboratory; Fallowfield. ON Canada; 3Infectious
Pathogen Research Section; Central Research Laboratory; Japan Blood Products
Organization; Kobe, Japan; 4Department of Neurological Science; Tohoku
University Graduate School of Medicine; Sendai. Japan; 5Neurobiology Division;
The Roslin Institute and R(D)SVS; University of Edinburgh; Easter Bush;
Midlothian; Edinburgh, UK
Background. Bovine spongiform encephalopathy (BSE) is a known zoonotic
prion disease, resulting in variant Creurzfeldt- Jakob disease (vCJD) in humans.
In contrast, classical scrapie in sheep is thought to offer little or no danger
to human health. However, a widening range of prion diseases have been
recognized in cattle, sheep and deer. The risks posed by individual animal prion
diseases to human health cannot be determined a priori and are difficult to
assess empirically. The fundamemal event in prion disease pathogenesis is
thought to be the seeded conversion of normal prion protein (PrPC) to its
pathological isoform (PrPSc). Here we report the use of a rapid molecular
conversion assay to test whether brain specimens from different animal prion
diseases are capable of seeding the conversion of human PrPC ro PrPSc.
Material and Methods. Classical BSE (C-type BSE), H-type BSE, L-type BSE,
classical scrapie, atypical scrapie, chronic wasting disease and vCJD brain
homogenates were tested for their ability to seed conversion of human PrPC to
PrPSc in protein misfolding cyclic amplification (PMCA) reactions. Newly formed
human PrPSc was detected by protease digestion and western blotting using the
antibody 3F4.
Results. C-type BSE and vCJD were found to efficiently convert PrPC to
PrPSc. Scrapie failed to convert human PrPC to PrPSc. Of the other animal prion
diseases tested only chronic wasting disease appeared to have the capability ro
convert human PrPC to PrPSc. The results were consistent whether the human PrPC
came from human brain, humanised transgenic mouse brain or from cultured human
cells and the effect was more pronounced for PrPC with methionine at codon 129
compared with that with valine.
Conclusion. Our results show that none of the tested animal prion disease
isolates are as efficient as C-type BSE and vCJD in converting human prion
protein in this in vitro assay. ***However, they also show that there is no
absolute barrier ro conversion of human prion protein in the case of chronic
wasting disease.
PRION2013 CONGRESSIONAL ABSTRACTS CWD
Sunday, August 25, 2013
***Chronic Wasting Disease CWD risk factors, humans, domestic cats, blood,
and mother to offspring transmission
From: Terry S. Singeltary Sr.
Sent: Saturday, November 15, 2014 9:29 PM
To: Terry S. Singeltary Sr.
Subject: THE EPIDEMIOLOGY OF CREUTZFELDT-JAKOB DISEASE R. G. WILL 1984
THE EPIDEMIOLOGY OF CREUTZFELDT-JAKOB DISEASE
R. G. WILL
1984
*** The association between venison eating and risk of CJD shows similar
pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK
OF CJD (p = 0.04). (SEE LINK IN REPORT HERE...TSS) PLUS, THE CDC DID NOT PUT
THIS WARNING OUT FOR THE WELL BEING OF THE DEER AND ELK ;
snip...
*** These results would seem to suggest that CWD does indeed have zoonotic
potential, at least as judged by the compatibility of CWD prions and their human
PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests
that if zoonotic CWD occurred, it would most likely effect those of the PRNP
codon 129-MM genotype and that the PrPres type would be similar to that found in
the most common subtype of sCJD (MM1).***
*** The potential impact of prion diseases on human health was greatly
magnified by the recognition that interspecies transfer of BSE to humans by beef
ingestion resulted in vCJD. While changes in animal feed constituents and
slaughter practices appear to have curtailed vCJD, there is concern that CWD of
free-ranging deer and elk in the U.S. might also cross the species barrier.
Thus, consuming venison could be a source of human prion disease. Whether BSE
and CWD represent interspecies scrapie transfer or are newly arisen prion
diseases is unknown. Therefore, the possibility of transmission of prion disease
through other food animals cannot be ruled out. There is evidence that vCJD can
be transmitted through blood transfusion. There is likely a pool of unknown size
of asymptomatic individuals infected with vCJD, and there may be asymptomatic
individuals infected with the CWD equivalent. These circumstances represent a
potential threat to blood, blood products, and plasma supplies. ***
*** IF CWD is not a risk factor for humans, then I guess the FDA et al
recalled all this CWD tainted elk tenderloin (2009 Exotic Meats USA of San
Antonio, TX) for the welfare and safety of the dead elk. ...tss
Exotic Meats USA Announces Urgent Statewide Recall of Elk Tenderloin
Because It May Contain Meat Derived From An Elk Confirmed To Have Chronic
Wasting Disease
Contact: Exotic Meats USA 1-800-680-4375
FOR IMMEDIATE RELEASE -- February 9, 2009 -- Exotic Meats USA of San
Antonio, TX is initiating a voluntary recall of Elk Tenderloin because it may
contain meat derived from an elk confirmed to have Chronic Wasting Disease
(CWD). The meat with production dates of December 29, 30 and 31, 2008 was
purchased from Sierra Meat Company in Reno, NV. The infected elk came from Elk
Farm LLC in Pine Island, MN and was among animals slaughtered and processed at
USDA facility Noah’s Ark Processors LLC.
Chronic Wasting Disease (CWD) is a fatal brain and nervous system disease
found in elk and deer. The disease is caused by an abnormally shaped protein
called a prion, which can damage the brain and nerves of animals in the deer
family. Currently, it is believed that the prion responsible for causing CWD in
deer and elk is not capable of infecting humans who eat deer or elk contaminated
with the prion, but the observation of animal-to-human transmission of other
prion-mediated diseases, such as bovine spongiform encephalopathy (BSE), has
raised a theoretical concern regarding the transmission of CWD from deer or elk
to humans. At the present time, FDA believes the risk of becoming ill from
eating CWD-positive elk or deer meat is remote. However, FDA strongly advises
consumers to return the product to the place of purchase, rather than disposing
of it themselves, due to environmental concerns.
Exotic Meats USA purchased 1 case of Elk Tenderloins weighing 16.9 lbs. The
Elk Tenderloin was sold from January 16 – 27, 2009. The Elk Tenderloins was
packaged in individual vacuum packs weighing approximately 3 pounds each. A
total of six packs of the Elk Tenderloins were sold to the public at the Exotic
Meats USA retail store. Consumers who still have the Elk Tenderloins should
return the product to Exotic Meats USA at 1003 NE Loop 410, San Antonio, TX
78209. Customers with concerns or questions about the Voluntary Elk Recall can
call 1-800-680-4375. The safety of our customer has always been and always will
be our number one priority.
Exotic Meats USA requests that for those customers who have products with
the production dates in question, do not consume or sell them and return them to
the point of purchase. Customers should return the product to the vendor. The
vendor should return it to the distributor and the distributor should work with
the state to decide upon how best to dispose. If the consumer is disposing of
the product he/she should consult with the local state EPA office.
#
RSS Feed for FDA Recalls Information11 [what's this?12]
Thursday, May 26, 2011
Travel History, Hunting, and Venison Consumption Related to Prion Disease
Exposure, 2006-2007 FoodNet Population Survey Journal of the American Dietetic
Association Volume 111, Issue 6 , Pages 858-863, June 2011.
now, let’s see what the authors said about this casual link, personal
communications years ago. see where it is stated NO STRONG evidence. so, does
this mean there IS casual evidence ???? “Our conclusion stating that we found no
strong evidence of CWD transmission to humans”
From: TSS (216-119-163-189.ipset45.wt.net)
Subject: CWD aka MAD DEER/ELK TO HUMANS ???
Date: September 30, 2002 at 7:06 am PST
From: "Belay, Ermias"
To: Cc: "Race, Richard (NIH)" ; ; "Belay, Ermias"
Sent: Monday, September 30, 2002 9:22 AM
Subject: RE: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Dear Sir/Madam,
In the Archives of Neurology you quoted (the abstract of which was attached
to your email), we did not say CWD in humans will present like variant CJD. That
assumption would be wrong. I encourage you to read the whole article and call me
if you have questions or need more clarification (phone: 404-639-3091). Also, we
do not claim that "no-one has ever been infected with prion disease from eating
venison." Our conclusion stating that we found no strong evidence of CWD
transmission to humans in the article you quoted or in any other forum is
limited to the patients we investigated.
Ermias Belay, M.D. Centers for Disease Control and Prevention
-----Original Message-----
From: Sent: Sunday, September 29, 2002 10:15 AM
To: rr26k@nih.gov; rrace@niaid.nih.gov; ebb8@CDC.GOV
Subject: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Sunday, November 10, 2002 6:26 PM ......snip........end..............TSS
Thursday, April 03, 2008
A prion disease of cervids: Chronic wasting disease 2008 1: Vet Res. 2008
Apr 3;39(4):41 A prion disease of cervids: Chronic wasting disease Sigurdson CJ.
snip...
*** twenty-seven CJD patients who regularly consumed venison were reported
to the Surveillance Center***,
snip... full text ;
==============================
*** These results would seem to suggest that CWD does indeed have zoonotic
potential, at least as judged by the compatibility of CWD prions and their human
PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests
that if zoonotic CWD occurred, it would most likely effect those of the PRNP
codon 129-MM genotype and that the PrPres type would be similar to that found in
the most common subtype of sCJD (MM1).***
*** The potential impact of prion diseases on human health was greatly
magnified by the recognition that interspecies transfer of BSE to humans by beef
ingestion resulted in vCJD. While changes in animal feed constituents and
slaughter practices appear to have curtailed vCJD, there is concern that CWD of
free-ranging deer and elk in the U.S. might also cross the species barrier.
Thus, consuming venison could be a source of human prion disease. Whether BSE
and CWD represent interspecies scrapie transfer or are newly arisen prion
diseases is unknown. Therefore, the possibility of transmission of prion disease
through other food animals cannot be ruled out. There is evidence that vCJD can
be transmitted through blood transfusion. There is likely a pool of unknown size
of asymptomatic individuals infected with vCJD, and there may be asymptomatic
individuals infected with the CWD equivalent. These circumstances represent a
potential threat to blood, blood products, and plasma supplies.
FRANCE HAVE AN EPIDEMIC OF SPONTANEOUS ATYPICAL BSE ‘’LOL’’
spontaneous atypical BSE ???
if that's the case, then France is having one hell of an epidemic of
atypical BSE, probably why they stopped testing for BSE, problem solved $$$
As of December 2011, around 60 atypical BSE cases have currently been
reported in 13 countries, *** with over one third in France.
so 20 cases of atypical BSE in France, compared to the remaining 40 cases
in the remaining 12 Countries, divided by the remaining 12 Countries, about 3+
cases per country, besides Frances 20 cases. you cannot explain this away with
any spontaneous BSe. ...TSS
Sunday, October 5, 2014
France stops BSE testing for Mad Cow Disease
spontaneous TSE prion, that's wishful thinking. on the other hand, if
spontaneous did ever happen (never once documented in the field), it would be
our worst nightmare, due to feed. just saying.
*** We describe the transmission of spongiform encephalopathy in a
non-human primate inoculated 10 years earlier with a strain of sheep c-scrapie.
Because of this extended incubation period in a facility in which other prion
diseases are under study, we are obliged to consider two alternative
possibilities that might explain its occurrence. We first considered the
possibility of a sporadic origin (like CJD in humans). Such an event is
extremely improbable because the inoculated animal was 14 years old when the
clinical signs appeared, i.e. about 40% through the expected natural lifetime of
this species, compared to a peak age incidence of 60–65 years in human sporadic
CJD, or about 80% through their expected lifetimes.
***Moreover, sporadic disease has never been observed in breeding colonies
or primate research laboratories, most notably among hundreds of animals over
several decades of study at the National Institutes of Health25, and in nearly
twenty older animals continuously housed in our own facility.***
>>> Moreover, sporadic disease has never been observed in breeding
colonies or primate research laboratories, most notably among hundreds of
animals over several decades of study at the National Institutes of Health25,
and in nearly twenty older animals continuously housed in our own facility.
<<<
Evidence That Transmissible Mink Encephalopathy Results from Feeding
Infected Cattle
Over the next 8-10 weeks, approximately 40% of all the adult mink on the
farm died from TME.
snip...
The rancher was a ''dead stock'' feeder using mostly (>95%) downer or
dead dairy cattle...
In Confidence - Perceptions of unconventional slow virus diseases of
animals in the USA - APRIL-MAY 1989 - G A H Wells
3. Prof. A. Robertson gave a brief account of BSE. The US approach was to
accord it a very low profile indeed. Dr. A Thiermann showed the picture in the
''Independent'' with cattle being incinerated and thought this was a fanatical
incident to be avoided in the US at all costs. ...
*** Spraker suggested an interesting explanation for the occurrence of CWD.
The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr.
Bob Davis. At or abut that time, allegedly, some scrapie work was conducted at
this site. When deer were introduced to the pens they occupied ground that had
previously been occupied by sheep.
Tuesday, August 4, 2015
*** FDA U.S. Measures to Protect Against BSE ***
Thursday, September 10, 2015
25th Meeting of the Transmissible Spongiform Encephalopathies Advisory
Committee Food and Drug Administration Silver Spring, Maryland June 1, 2015
Monday, August 17, 2015
FDA Says Endoscope Makers Failed to Report Superbug Problems OLYMPUS
I told Olympus 15 years ago about these risk factors from endoscopy
equipment, disinfection, even spoke with the Doctor at Olympus, this was back in
1999. I tried to tell them that they were exposing patients to dangerous
pathogens such as the CJD TSE prion, because they could not properly clean them.
even presented my concern to a peer review journal GUT, that was going to
publish, but then it was pulled by Professor Michael Farthing et al... see ;
Sunday, December 14, 2014
ALERT new variant Creutzfeldt Jakob Disease nvCJD or vCJD, sporadic CJD
strains, TSE prion aka Mad Cow Disease United States of America Update December
14, 2014 Report
Sunday, August 11, 2013
Creutzfeldt-Jakob Disease CJD cases rising North America updated report
August 2013
Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing
an extreme increase of 48% between 2008 and 2010
*** These results would seem to suggest that CWD does indeed have zoonotic
potential, at least as judged by the compatibility of CWD prions and their human
PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests
that if zoonotic CWD occurred, it would most likely effect those of the PRNP
codon 129-MM genotype and that the PrPres type would be similar to that found in
the most common subtype of sCJD (MM1).***
Terry S. Singeltary Sr.
posted by Terry S. Singeltary Sr. at
8:37 PM
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