Chronic Wasting Disease CWD TSE Prion Diagnostics and subclinical infection
Chronic Wasting Disease CWD TSE Prion Diagnostics and subclinical
infection
Article | Open
Prion Amplification and Hierarchical Bayesian Modeling Refine Detection of
Prion Infection
A. Christy Wyckoff , Nathan Galloway , Crystal Meyerett-Reid , Jenny Powers
, Terry Spraker , Ryan J. Monello , Bruce Pulford , Margaret Wild , Michael
Antolin , Kurt VerCauteren & Mark Zabel
Scientific Reports 5, Article number: 8358 (2015) doi:10.1038/srep08358
Download Citation Molecular ecology | Proteins | Statistics
Received:27 June 2014Accepted:19 January 2015Published online:10 February
2015 Article Tools
Abstract
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...
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.
P170
Clinical Stage of Infection is Critical in the Antemortem Diagnosis of
Chronic Wasting Disease in Deer and Elk
Chris Siepker1, Nicholas Haley1, W. David Walter2, Matteo Manca3, Laura
Hoon-Hanks4, Ryan Monello5, Jenny Powers5, Justin Greenlee6 , Bruce Thomsen7 ,
Aaron Lehmkuhl7, Gordon Mitchell8, Tracy Nichols9,Byron Caughey3, Edward
Hoover4, and Juergen Richt1.
1. Department of Diagnostic Medicine and Pathobiology, Kansas State
University, Manhattan KS USA 2. United States G e o l o g i c a l Survey, P e n
n s y l v a n i a Cooperative Fish and Wildlife Research Unit, University Park
PA USA 3. TSE/Prion Biochemistry Section, Rocky Mountain Laboratories, National
Institutes of Health, Hamilton, MT USA 3. Department of MIP, Colorado State
University, Fort Collins CO USA 4. National Park Service, Wildlife Health
Branch, Fort Collins CO USA 5. Virus and Prion Research Unit, National Animal
Disease Center, ARS, USDA, Ames IA USA 6. USDA, APHIS, VS, STAS, National
Veterinary Service Laboratories, Ames IA USA 7. National and OIE Reference
Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa ON
Canada 8. National Wildlife Research Center Wildlife Services, APHIS, USDA, Fort
Collins CO USA
Chronic wasting disease (CWD) is an e f f i c i e n t l y t r a n s mi t t
e d s p o n g i f o r m encephalopathy of cervids (e.g. deer, elk, and moose),
and is the only known prion disease affecting both free-ranging wildlife and
captive animals. The antemortem detection of CWD and other prion diseases has
proven difficult, due in part to difficulties in identifying an appropriate
peripheral tissue specimen and complications with conventional test sensitivity.
At present, biopsies of the recto-
Prion2015 Program Guide 22
anal mucosal-associated lymphoid tissues (RAMALT) have shown promising
sensitivity and are not impractical to collect in live animals. Nasal brush
collections have likewise proven both sensitive and practical for identification
of prion infections in humans. In this study, we evaluated both RAMALT and nasal
brush collections by real time quaking-induced conversion (RT-QuIC), and
compared our findings to RAMALT immu n o h i s t o c h emi s t r y a s we l l a
s conventional postmortem evaluation of obex and retropharyngeal lymph node
tissues from over 700 captive and free-ranging deer and elk in areas with
endemic CWD. We correlated our results with various clinical findings, including
pathological stage of infection as determined by obex scoring, PrP genotype,
age, and sex. While the sensitivity of RAMALT RT-QuIC analyses exceeded that of
RAMALT IHC (69-80% vs. >44%) and nasal brush collections (15-30%), the
sensitivity of both biopsy and nasal brush analyses were dependent primarily on
clinical stage of disease, although PrP genotype was also an important predictor
of sample positivity. Our findings further demonstrate the potential and
limitations of antemortem sample analyses by RT-QuIC in the identification and
management of prion diseases.
J Vet Sci. 2015 Jun; 16(2): 179–186.
Published online 2015 Jun 17. doi: 10.4142/jvs.2015.16.2.179
PMCID: PMC4483501
Classical natural ovine scrapie prions detected in practical volumes of
blood by lamb and transgenic mouse bioassays
Rohana P. Dassanayake,corresponding author1 Thomas C. Truscott,2 Dongyue
Zhuang,2 David A. Schneider,2 Sally A. Madsen-Bouterse,1 Alan J. Young,3 James
B. Stanton,1 William C. Davis,1 Katherine I. O'Rourke1
J Virol. 2013 May; 87(10): 5895–5903. doi: 10.1128/JVI.03469-12 PMCID:
PMC3648199
Dissociation between Transmissible Spongiform Encephalopathy (TSE)
Infectivity and Proteinase K-Resistant PrPSc Levels in Peripheral Tissue from a
Murine Transgenic Model of TSE Disease
Karen Dobie and Rona Barroncorresponding author Neurobiology Division, The
Roslin Institute & R(D)SVS, Easter Bush, Midlothian, United Kingdom
corresponding authorCorresponding author. Address correspondence to Rona Barron,
Email: ku.ca.de.nilsor@norrab.anor. Author information ► Article notes ►
Copyright and License information ► Received 2012 Dec 19; Accepted 2013 Mar 7.
Copyright © 2013, American Society for Microbiology. All Rights Reserved. This
article has been cited by other articles in PMC. Go to:
Abstract
Most current diagnostic tests for transmissible spongiform encephalopathies
(TSE) rely on the presence of proteinase K (PK)-resistant PrPSc (PrP-res) in
postmortem tissues as an indication of TSE disease. However, a number of studies
have highlighted a discrepancy between TSE infectivity and PrP-res levels in
both natural and experimental cases of TSE disease. Previously, we have shown
high TSE infectivity levels in the brain tissue of mice that have a clinical TSE
disease with associated vacuolar pathology but little or no detectable PrP-res.
Here, the levels of TSE infectivity and PrP-res within a peripheral tissue of
this mouse model were investigated. Biochemical analysis showed that low levels
of PrP-res were present in the spleen tissue in comparison to the levels
observed in the spleen of mice infected with ME7 or 79A. However, upon
subpassage of brain and spleen tissue from clinically ill mice with little or no
PrP-res detectable, similar short incubation periods to disease were observed,
indicating that infectivity levels were similarly high in both tissues. Thus,
the discrepancy between PrP-res and TSE infectivity was also present in the
peripheral tissues of this disease model. This result indicates that peripheral
tissues can contain higher levels of infectivity given the correct combination
of host species, PrP genotype, and TSE agent. Therefore, the assumption that the
levels of peripheral infectivity are lower than those in the central nervous
system is not always correct, and this could have implications for current food
safety regulations.
SNIP...
Several recent studies have identified the presence of quasispecies within
individual cases of TSE disease in humans (23, 24) and animals (25) and in
cell-culture models (26). Indeed, it has been hypothesized that subvariants of
disease-associated PrP replicate preferentially in specific tissue types, with a
dependence on tissue-specific host factors (25, 27). The biochemical and
immunohistochemical analyses of the recipient mice from the 101LL/GSS subpassage
demonstrated that PrP-res deposition was lower in the brain tissue of mice that
received the spleen homogenate inocula than in those that received the brain
homogenate inocula (Fig. 6), while in contrast, the vacuolation score in mice
was greater in those that received the spleen homogenate than in those that
received the brain homogenate (Fig. 4). These results indicate that a
potentially heterogeneous population of PrP-res was present. It is hypothesized
that tissue-specific conditions supported the replication of different
subvariants that showed different replication efficiencies upon subpassage. If
heterogeneous populations of PrP-res exist that include variants that have
different replication capabilities, a variant present in peripheral tissues may
have a higher level of infectivity than the corresponding brain-derived variant.
Therefore, assessment of the peripheral infectivity levels from novel and
emerging isolates is urgently required to ensure that an accurate titer is
established to maintain food safety.
Together, these results indicate that a form of the infectious agent may be
present in this disease model that remains undetectable by current standard
analysis. Given the increasing costs of bioassay to identify the presence of TSE
infectivity, the majority of disease cases are being confirmed by biochemical
techniques specific for the presence of PrP-res, without confirmation of the
presence of infectivity. This current reliance on PrP-res as indicative of TSE
disease may not detect all cases of TSE disease, with the possible emergence of
cases with high infectivity levels associated with low levels or an absence of
PrP-res. Indeed, the discovery of significant levels of TSE infectivity despite
the absence of PrP-res in spleens from sheep infected with atypical scrapie (13)
indicates that this disease phenomenon can occur in natural cases of TSE disease
present in the environment. Furthermore, a recent study by Gonzalez and
colleagues highlighted the discrepancy between levels of PrP-res and TSE
infectivity in sheep scrapie and sheep BSE and indicated that quantitative
laboratory tests to detect disease-associated PrP could not be used to
accurately predict infectious titers (28). While TSEs remain in the environment,
the emergence of novel isolates or the possibility that a known isolate could
infect a different host species remains. Our data show that a combination of
host species, PrP genotype, and TSE isolate that could produce a novel disease
phenotype with high levels of TSE infectivity in the absence of PrP-res has the
potential to occur. Therefore, if the infectivity levels in the peripheral
tissues of disease cases with low levels of PrP-res are higher than originally
hypothesized from previous research into classical isolates and current
biochemical tests, the emergence of a novel isolate could pose a major risk to
food safety if tissues were able to enter the food chain. Together with the
discrepancy between PrP-res and TSE infectivity levels presented here, the
estimation of infectious titer should not rely on the detection of PrP-res as
the sole indicator of TSE disease.
Sireesha Manne1,*, Naveen Kondru1, Nicholas Haley2, Tracy Nichols3, Bruce
Thomsen4, Roger Main5, Patrick Halbur5, Arthi Kanthasamy1, and Anumantha
Kanthasamy1 1Biomedical Sciences; Iowa State University; Ames, IA USA; 2Kansas
State University; Manhattan, KS USA; 3United States Department of Agriculture;
Fort Collins, CO USA; 4National Veterinary Service Laboratories; Ames, IA USA;
5VDPAM; Iowa State University; Ames, IA USA
Prion diseases are transmissible spongiform encephalopathies (TSEs)
characterized by an always fatal, progressive neuronal degeneration in the brain
due to infectious misfolded prion proteins whose prolonged incubation periods
often make ante-mortem diagnosis difficult. Chronic wasting disease (CWD) is a
TSE affecting both wild and captive populations of mule deer, whitetailed deer,
elk and moose. CWD in cervids was first identified in Rocky Mountain States and
has recently spread to several other states including Iowa. In this current
study, we attempted to independently confirm the results of a Real-Time
Quaking-Induced Conversion (RT-QuIC) assay to diagnose CWD using rectal biopsy
sections from farmed white-tailed deer. First, we generated recombinant prion
protein substrate and then validated the quality of protein for RT-QuIC using a
reference prion protein kindly provided by Dr. Caughey’s lab. After validating
the assay, we blindly evaluated approximately 350 rectal biopsy samples analyzed
previously by another institution. All assay plates included positive and
negative controls and were analyzed in triplicate. Samples were analyzed using
the Biotek Cytation-3 multimode plate reader for 24-hrs duration. Our RT-QuIC
assays showed 55% positivity for 356 rectal samples analyzed. Comparison of
RT-QuIC results with the immunohistochemical results of obex revealed 93%
sensitivity (95% confidence limits: 88.05–95.78%) and 96% specificity (95% CL:
91–99%), confirming that the RT-QuIC assay may be one of the most promising
rapid assays for detecting CWD prions. We are currently working on applying the
RT-QuIC assay to other test samples (ISU Presidential Wildlife initiative,
ISU-CVM Diagnostic lab and ES10586).
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.
cwd diagnostics
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 ;
The overall incidence of clinical CWD in white-tailed deer was 82%
Species (cohort) CWD (cases/total) Incidence (%) Age at CWD death (mo)
J Vet Diagn Invest 20:698–703 (2008)
Chronic wasting disease in a Wisconsin white-tailed deer farm
Delwyn P. Keane,1 Daniel J. Barr, Philip N. Bochsler, S. Mark Hall, Thomas
Gidlewski, Katherine I. O’Rourke, Terry R. Spraker, Michael D. Samuel
Abstract.
In September 2002, chronic wasting disease (CWD), a prion disorder of
captive and wild cervids, was diagnosed in a white-tailed deer (Odocoileus
virginianus) from a captive farm in Wisconsin. The facility was subsequently
quarantined, and in January 2006 the remaining 76 deer were depopulated. Sixty
animals (79%) were found to be positive by immunohistochemical staining for the
abnormal prion protein (PrPCWD) in at least one tissue; the prevalence of
positive staining was high even in young deer. Although none of the deer
displayed clinical signs suggestive of CWD at depopulation, 49 deer had
considerable accumulation of the abnormal prion in the medulla at the level of
the obex. Extraneural accumulation of the abnormal protein was observed in 59
deer, with accumulation in the retropharyngeal lymph node in 58 of 59 (98%), in
the tonsil in 56 of 59 (95%), and in the rectal mucosal lymphoid tissue in 48 of
58 (83%). The retina was positive in 4 deer, all with marked accumulation of
prion in the obex. One deer was considered positive for PrPCWD in the brain but
not in the extraneural tissue, a novel observation in white-tailed deer. The
infection rate in captive deer was 20- fold higher than in wild deer. Although
weakly related to infection rates in extraneural tissues, prion genotype was
strongly linked to progression of prion accumulation in the obex. Antemortem
testing by biopsy of recto– anal mucosal-associated lymphoid tissue (or other
peripheral lymphoid tissue) may be a useful adjunct to tonsil biopsy for
surveillance in captive herds at risk for CWD infection.
Key words: Cervids; chronic wasting disease; prion; transmissible
spongiform encephalopathy.
> the prevalence of positive staining was high even in young deer
Approximately 4,200 fawns, defined as deer under 1 year of age, were sampled from the eradication zone over the last year. The majority of fawns sampled were between the ages of 5 to 9 months, though some were as young as 1 month. Two of the six fawns with CWD detected were 5 to 6 months old. All six of the positive fawns were taken from the core area of the CWD eradication zone where the highest numbers of positive deer have been identified.
"This is the first intensive sampling for CWD in fawns anywhere," said Dr.
Julie Langenberg, Department of Natural Resources wildlife veterinarian, "and we
are trying to learn as much as we can from these data".
Saturday, February 04, 2012
Wisconsin 16 MONTH age limit on testing dead deer Game Farm CWD Testing
Protocol Needs To Be Revised
Articles of Significant Interest Selected from This Issue by the Editors Next Section Prions in the Blood of Infected Hosts: Early and Persistent Prions circulate in the blood of prion-infected hosts, including humans with variant Creutzfeldt-Jakob disease. Determining the parameters of blood-borne prions during the long asymptomatic phase of disease characteristic of all prion diseases has been a long-standing problem in prion biology. Elder et. al (p. 7421–7424) have demonstrated amyloid formation, a biomarker for prions, in the blood of prion-infected rodent and cervid hosts as early as 15 minutes post-mucosal or -intravenous infection. This prionemia persists throughout the disease course, indicating a role for hematogenous prions throughout the preclinical stage of illness.
***Immediate and Ongoing Detection of Prions in the Blood of Hamsters and Deer following Oral, Nasal, or Blood Inoculations
Alan M. Eldera, Davin M. Hendersona, Amy V. Nallsa, Edward A. Hoovera,
Anthony E. Kincaidb,c, Jason C. Bartzb and Candace K. Mathiasona aDepartment of
Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,
Colorado, USA bMedical Microbiology and Immunology, Creighton University, Omaha,
Nebraska, USA cDepartment of Pharmacy Sciences, Creighton University, Omaha,
Nebraska, USA S. Perlman, Editor + Author Affiliations
TSS
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