Journal of Parkinson’s Disease xx (20xx) x–xx DOI 10.3233/JPD-150691 IOS
Press
An efficient procedure for removal and inactivation of alpha-synuclein
assemblies from laboratory materials
Luc Bousseta,a∗, Patrik Brundinb, Anja Bockmannc, Beat Meierd and Ronald
Melkia
a Paris-Saclay Institute of Neurosciences, CNRS, Gif-sur-Yvette,
France
bVan Andel Research Institute, Center for Neurodegenerative Science, Grand
Rapids, Michigan, USA
cInstitut de Biologie et Chimie des Proteines, CNRS/Universite´ de Lyon 1,
Lyon, France
dPhysical Chemistry, ETH Zurich, Zurich, Switzerland
Abstract.
Background: Preformed a-synuclein fibrils seed the aggregation of soluble
a -synuclein in cultured cells and in vivo. This, and other findings, has
kindled the idea that a-synuclein fibrils possess prion-like properties.
Objective: As a-synuclein fibrils should not be considered as innocuous,
there is a need for decontamination and inactivation procedures for laboratory
benches and non-disposable laboratory material.
Methods: We assessed the effectiveness of different procedures designed to
disassemble a-synuclein fibrils and reduce their infectivity. We examined
different commercially available detergents to remove a-synuclein assemblies
adsorbed on materials that are not disposable and that are most found in
laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces).
Results: We show that methods designed to decrease PrP prion infectivity
neither effectively remove a -synuclein assemblies adsorbed to different
materials commonly used in the laboratory nor disassemble the fibrillar form of
the protein with efficiency. In contrast, both commercial detergents and SDS
detached a-synuclein assemblies from contaminated surfaces and disassembled the
fibrils.
Conclusions: We describe three cleaning procedures that effectively remove
and disassemble a-synuclein seeds. The methods rely on the use of detergents
that are compatible with most non-disposable tools in a laboratory. The
procedures are easy to implement and significantly decrease any potential risks
associated to handling a -synuclein assemblies.
24 Keywords: Alpha synuclein, cleaning procedures, detergent, fibrils,
inactivation, Parkinson’s disease, removal
SNIP...
DISCUSSION
Findings that fibrillar a -Syn can seed the aggregation of monomeric a-Syn
both in cell cultures and in animal models, coupled to observations that
injected a-Syn fibrils propagate from the olfactory bulb, intestine,muscle and
the blood to the central nervous system suggest that a-Syn fibrils should be
handled with caution in laboratories and hospital settings.
Akin to what has already been reported for prions [32] and A B [33],
aggregated a -Syn in homogenates derived from the brains of transgenic mice that
progressively develop a -synucleinopathy seeds aggregation of a-Syn when
injected into the brains of mice after formaldehyde fixation of the brain tissue
homogenate [34]. While this suggests a-synucleinopathies can be transmitted
from one mouse to another, in an artificial laboratory setting, it does not
provide proof that transmission occurs in any other paradigm or in, e.g.,
humans. A study performed on recipients of human growth hormone purified from
cadavers concluded that there is no evidence for transmission of a -Syn
aggregates between humans [35]. This retrospective study, however, does not
definitively exclude the possibility that a -synucleinopathy can transmit
between humans. The ability of protein seeds to transmit disease depends on
several factors.
First, the purification procedure of human growth hormone (based on
ammonium sulfate precipitation and size exclusion chromatography) [36, 37],
while insufficient to fully eliminate infectious prions from the preparation
might well destroy other protein assemblies and/or change their seeding
propensity.
Second, the amount of a-Syn seeds necessary to trigger
a-synucleinopathies in human might well be higher than that of the prion
protein seeding units required to trigger Creutzfeldt-Jakob disease.
Third, difference in the resistance of different types of protein seeds to
degradation following injection into a tissue may explain variations in disease
transmission/induction efficiencies. Considering that the amounts of a-Syn
fibrils that are used in the laboratory exceed by orders of magnitude the amount
of prion protein seeds that are needed to transmit disease, we recommend that
work safety conditions are based on a worst-case scenario where a-Syn
aggregates are considered infectious.
Safety rules have been implemented in facilities where autopsies are
performed to confirm Creutzfeldt- Jakob Disease diagnosis and in laboratories
where potentially infectious material is handled [38–40]. They are mostly based
on avoiding the use of material, such as needles, that can cause penetrating
wounds and on the careful collection of potentially infectious biological
material and its inactivation by well-defined procedures [28–30]. Reusable
surgical instruments decontamination protocols were also implemented to minimize
the transmission of spongiform encephalopathies after neurosurgical procedures
were suspected to allow such transmission [41]. Similarly, procedures allowing
the inactivation of A seeds have been described [42, 43].
We implemented in our laboratories stringent rules to avoid the generation
of aerosols when shearing by sonication fibrillar a-Syn into short seeds. As
many of the tools we use to handle a-Syn fibrils are not disposable, we first
tested methods designed to decrease the infectivity of human and animal prion
particles regarding their ability to clean exposed surfaces and to disassemble
a-Syn fibrils. Typically, the non-disposable tools we use in the laboratory are
made of plastic, glass, stainless steel or other metals. We therefore assessed
the ability of prion inactivating procedures to remove a -Syn fibrils, ribbons
and oligomers that we had left to dry on the above listed materials.
As it is important to determine whether the fibrils have retained the
fibrillar structure prior to disposing of the cleaning solutions, we also
determined whether they remained fibrillar or had disassembled into their
constituting soluble a-Syn in the washing solutions. We found that methods
designed to decrease prion infectivity are ineffective at removing fibrillar
a-Syn adsorbed to different materials commonly used in the laboratory and do
not disassemble a-Syn fibrils (Fig. 1 & 2). The most likely explanation is
that these are different assemblies made of distinct proteins. In contrast
commercial detergents and SDS not only detach dried fibrillar a -Syn from
contaminated surfaces, even when they are scratched, but also disassemble the
fibrils. Based on our findings and experience, we have summarized good
laboratory practices for fibrillar a -Syn in Table 1. We conclude that cleaning
procedures relying on the use of detergents that are compatible with most
non-disposable tools in a laboratory are simple to implement and highly
recommended when working with fibrillar a-Syn in a laboratory setting. The
procedures we describe remove and inactivate a -Syn fibrillar assemblies to a
level where they are undetectable using the method we used. Further work is
needed to establish the infectious unit of recombinant a -Syn and the biological
efficiency of the cleaning methods we describe.
***>>>An efficient procedure for removal and inactivation of
alpha-synuclein assemblies from laboratory materials<<<***
***>>> This retrospective study, however, does not definitively
exclude the possibility that a-synucleinopathy can transmit between humans.
<<<***
An efficient procedure for removal and inactivation of alpha-synuclein
assemblies from laboratory materials ???
Original Article
Effect of heating on the stability of amyloid A (AA) fibrils and the intra-
and cross-species transmission of AA amyloidosis
DOI:10.3109/13506129.2015.1095735Saki Ogawaa, Tomoaki Murakamib, Yasuo
Inoshimaa & Naotaka Ishiguroa*
Publishing models and article dates explained
Received: 5 May 2015 Accepted: 14 Sep 2015 Published online: 20 Nov 2015
.
Abstract
Amyloid A (AA) amyloidosis is a protein misfolding disease characterized by
extracellular deposition of AA fibrils. AA fibrils are found in several tissues
from food animals with AA amyloidosis. For hygienic purposes, heating is widely
used to inactivate microbes in food, but it is uncertain whether heating is
sufficient to inactivate AA fibrils and prevent intra- or cross-species
transmission. We examined the effect of heating (at 60 °C or 100 °C) and
autoclaving (at 121 °C or 135 °C) on murine and bovine AA fibrils using Western
blot analysis, transmission electron microscopy (TEM), and mouse model
transmission experiments. TEM revealed that a mixture of AA fibrils and
amorphous aggregates appeared after heating at 100 °C, whereas autoclaving at
135 °C produced large amorphous aggregates. AA fibrils retained antigen
specificity in Western blot analysis when heated at 100 °C or autoclaved at 121
°C, but not when autoclaved at 135 °C. Transmissible pathogenicity of murine and
bovine AA fibrils subjected to heating (at 60 °C or 100 °C) was significantly
stimulated and resulted in amyloid deposition in mice. Autoclaving of murine AA
fibrils at 121 °C or 135 °C significantly decreased amyloid deposition.
Moreover, amyloid deposition in mice injected with murine AA fibrils was more
severe than that in mice injected with bovine AA fibrils. Bovine AA fibrils
autoclaved at 121 °C or 135 °C did not induce amyloid deposition in mice. These
results suggest that AA fibrils are relatively heat stable and that similar to
prions, autoclaving at 135 °C is required to destroy the pathogenicity of AA
fibrils. These findings may contribute to the prevention of AA fibril
transmission through food materials to different animals and especially to
humans.
AA amyloidosis, AA fibrils, Image J software, immunohistochemistry, prion,
silver nitrate, transmission electron microscopy, Western blot analysis
***PRION2015 Ft. Collins***
Alzheimer’s disease
*** P.34: Preliminary study of Alzheimer’s disease transmission to bank
vole ***
Guido Di Donato1, Geraldina Riccardi1, Claudia D’Agostino1, Flavio
Torriani1, Maurizio Pocchiari2, Romolo Nonno1, Umberto Agrimi1, and Michele
Angelo Di Bari1
1Department of Food Safety and Veterinary Public Health Istituto Superiore
di Sanit a, Rome, Italy; 2Department of Cellular Biology and Neuroscience;
Istituto Superiore di Sanit a, Rome, Italy
Extensive experimental findings indicate that prion-like mechanisms underly
the pathogenesis of Alzheimer disease (AD). Transgenic mice have been pivotal
for investigating prionlike mechanisms in AD, still these models have not been
able so far to recapitulate the complex clinical-pathological features of AD.
Here we aimed at investigating the potential of bank vole, a wild-type rodent
highly susceptible to prions, in reproducing AD pathology upon experimental
inoculation.
Voles were intracerebrally inoculated with brain homogenate from a familial
AD patient. Animals were examined for the appearance of neurological signs until
the end of experiment (800 d post-inoculation, d.p.i.). Brains were studied by
immunohistochemistry for pTau Prion 2015 Poster Abstracts S29 (with AT180 and
PHF-1 antibodies) and b-amyloid (4G8).
Voles didn’t show an overt clinical signs, still most of them (11/16) were
found pTau positive when culled for intercurrent disease or at the end of
experiment. Interestingly, voles culled as early as 125 d.p.i. already showed
pTau aggregates. Deposition of pTau was similar in all voles and was
characterized by neuropil threads and coiled bodies in the alveus, and by rare
neurofibrillary tangles in gray matter. Conversely, b-amyloid deposition was
rather rare (2/16). Nonetheless, a single vole showed the contemporaneous
presence of pTau in the alveus and a few Ab plaque-like deposits in the
subiculum. Uninfected age-matched voles were negative for pTau and Ab.
*** These findings corroborate and extend previous evidences on the
transmissibility of pTau and Ab aggregation. Furthermore, the observation of a
vole with contemporaneous propagation of pTau and Ab is intriguing and deserves
further studies.
=================
P.155: Quantitative real-time analysis of disease specific tau amyloid
seeding activity
Davin Henderson and Edward Hoover Prion Research Center; College of
Veterinary Medicine and Biomedical Sciences; Colorado State University; Fort
Collins, CO USA
A leading hypothesis for the cause of neurodegenerative diseases is the
templated misfolding of cellular proteins to an amyloid state. Spongiform
encephalopathies were the first diseases discovered to be caused by a misfolded
amyloid-rich protein. It is now recognized that the major human
neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s
disease (PD), and chronic traumatic encephalopathy (CTE), also are associated
with amyloid formation. Moreover, AD and PD amyloids have been shown competent
to transmit disease in experimental animal models, suggesting shared mechanisms
with traditional prion diseases. Sensitive detection of prion disease has been
advanced by in vitro amplification of low levels of disease specific amyloid
seeds, e.g. serial protein misfolding amplification (sPMCA), amyloid seeding
(ASA) and real-time quaking induced conversion (RT-QuIC), thereby replicating
the disease process in vitro. In addition, measurement of the amyloid formation
rate can estimate the level of disease-associated seed by using methods
analogous to quantitative polymerase chain reaction (qPCR). In the present work,
we apply these principles to show that seeding activity of in vitro generated
amyloid tau and AD brain amyloid tau can be readily detected and
quantitated.
=============
P.83: Gerstmann-Str€aussler-Scheinker disease with F198S mutation:
Selective propagation of PrPSc and pTau upon inoculation in bank vole
Michele Angelo Di Bari1, Romolo Nonno1, Laura Pirisinu1, Claudia
D’Agostino1, Geraldina Riccardi1, Guido Di Donato1, Paolo Frassanito1,
Bernardino Ghetti2, Pierluigi Gambetti3, and Umberto Agrimi1
1Department of Veterinary Public Health and Food Safety; Istituto Superiore
di Sanit a; Rome, Italy;
2Indiana University-Purdue University Indianapolis; Department of Pathology
and Laboratory Medicine; Indianapolis, IN USA; 3Case Western Reserve University;
Cleveland, OH USA
Gerstmann-Str€aussler-Scheinker disease with F198S mutation (GSS-F198S) is
characterized by the presence of PrP amyloid plaques as well as neurofibrillary
tangles with abnormally-phosphorylated tau protein (pTau) in the brain. The
relationship between tau protein and PrP in the pathogenesis of GSS-F198S is
unknown. In a previous study, we inoculated intracerebrally 2 GSS-F198S cases in
2 lines of voles carrying either methionine (Bv109M) or isoleucine (Bv109I) at
codon 109 of PrP. GSS-F198S transmitted rather efficiently to Bv109I, but not to
Bv109M.
Here we investigated the presence of pTau, as assessed by
immunohistochemistry with anti-pTau antibodies AT180 and PHF-1, in the same
voles previously inoculated with GSSF198S. Among these voles, most Bv109I showed
clinical signs after short survival times (»150 d.p.i.) and were positive for
PrPSc. The remaining Bv109I and all Bv109M survived for longer times without
showing prion-related pathology or detectable PrPSc. All Bv109I which were
previously found PrPSc-positive,
S54 Prion 2015 Poster Abstracts
were immunonegative for pTau deposition. In contrast, pTau deposition was
detected in 16/20 voles culled without clinical signs after long survival times
(225–804 d.p.i.). pTau deposition was characterized by neuropil threads and
coiled bodies in the alveus, and was similar in all voles analyzed.
These findings highlight that pTau from GSS-F198S can propagate in voles.
Importantly, pTau propagation was independent from PrPSc, as pTau was only found
in PrPSc-negative voles surviving longer than 225 d.p.i. Thus, selective
transmission of PrPSc and pTau proteinopathies from GSS-F198S can be
accomplished by experimental transmission in voles.
=========
I3 Aβ Strains and Alzheimer’s Disease
Lary Walker Emory University, Atlanta, GA, USA
An essential early event in the development of Alzheimer’s disease is the
misfolding and aggregation of Aβ. Enigmatically, despite the extensive
deposition of human-sequence Aβ in the aging brain, nonhuman primates do not
develop the full pathologic or cognitive phenotype of Alzheimer’s disease, which
appears to be unique to humans. In addition, some humans with marked Aβ
accumulation in the brain retain their cognitive abilities, raising the question
of whether the pathogenicity of Aβ is linked to the molecular features of the
misfolded protein. I will present evidence for strain-like molecular differences
in aggregated Aβ between humans and nonhuman primates, and among end-stage
Alzheimer patients. I will also discuss a case of Alzheimer’s disease with
atypical Aβ deposition to illustrate heterogeneity in the molecular architecture
of Aβ assemblies, and how this variability might influence the nature of the
disease. As in the case of prion diseases, strain-like variations in the
molecular architecture of Aβ could help to explain the phenotypic variability in
Alzheimer’s disease, as well as the distinctively human susceptibility to the
disorder.
This research was conducted in collaboration with Harry LeVine, Rebecca
Rosen, Amarallys Cintron, David Lynn, Yury Chernoff, Anil Mehta and Mathias
Jucker and colleagues. Supported by AG040589, RR165/OD11132, AG005119, NS077049,
the CART Foundation and MetLife.
==========
I5 Pathogenic properties of synthetically generated prions
Jiyan Ma Van Andel Research Institute, Grand Rapids, Michigan, USA
Synthetically generating prions with bacterially expressed recombinant
prion protein (recPrP) strongly supports the prion hypothesis. Yet, it remains
unclear whether the pathogenic properties of synthetically generated prions
(rec-Prion) fully recapitulate those of naturally occurring prions. A series of
analyses including intracerebral and intraperitoneal transmissions of rec-Prion
in wild-type mice were performed to determine the characteristics of rec-Prion
induced diseases. Results from these analyses demonstrated that the rec-Prion
exhibits the same pathogenic properties with naturally occurring prions,
including a titratable infectivity that can be determined by endpoint titration
assays, capability of transmitting prion disease via routes other than the
direct intra-cerebral inoculation, causing ultra-structural lesions that are
specific to prion disease, and sharing a similar manner of visceral
dissemination and neuroinvasion with naturally occurring scrapie and chronic
wasting disease. These findings confirmed that the disease caused by rec-Prion
in wild-type mice is bona fide prion disease or transmissible spongiform
encephalopathiges, and the rec-Prion contains similar pathogenic properties as
naturally occurring prions.
I6 Transmissible protein toxins in neurodegenerative disease
Jacob Ayers, David Borchelt University of Florida, Gainesville, FL,
USA
Amyotrophic lateral sclerosis (ALS) is an obvious example of
neurodegenerative disease that seems to spread along anatomical pathways. The
spread of symptoms from the site of onset (e.g. limb) to the respiratory
musculature drives the rate of disease progression. In cognitive disorders, such
as Alzheimer’s disease, one can find similarly find evidence of spreading
dysfunction and pathology. One mechanism to account for this spread of disease
from one neural structure to another is by evoking prion-like propagation of a
toxic misfolded protein from cell to cell. Recent studies in animals that model
aspects of Alzheimer’s Disease, Parkinson’s Disease, and Tauopathy, have
bolstered the arguments in favor of prion-like, although in most of these models
the mice do not develop overt “clinical” symptoms. Recently, Jacob Ayers
demonstrated that the symptoms of ALS can be transmitted from a strain of mice
that expresses mutant SOD1-G93A at high levels to a second transgenic strain
that expresses mutant SOD1 at low, nontoxic, levels. This model showed many
prion-like features including evidence of host-adaptation (earlier and more
penetrant disease upon second passage). Interestingly, homogenates from
paralyzed mice expressing the G37R variant of SOD1 transmitted poorly, a finding
suggestive that different SOD1 variants may exhibit strain-like properties.
These “ i n d u c i b l e ” m o d e l s o f h u m a n neurodegenerative disease
enable the generation of models that do not require extraordinary levels of
transgene expression and provide a more precise means of initiating the disease
process, advances that may translate into more predictive pre-clinical
models.
=======
P188 Transmission of amyloid pathology by peripheral administration of
misfolded Aβ
Javiera Bravo-Alegria1 ,2, Rodrigo Morales2, Claudia Duran-Aniotz3, Claudio
Soto2 1University of Los Andes, Santiago, Chile, 2Mitchell Center for
Alzheimer’s Disease and Related Brain Disorders, Department of Neurology,
University of Texas Medical School, Houston, Texas, USA, 3University of Chile,
Santiago, Chile
Misfolding and aggregation of Amyloid-β (Aβ) is one of the primary events
involved in the pathogenesis of Alzheimer's disease (AD). Recently, it has been
proposed that Aβ aggregates can transmit and spread the pathology following a
prion-like mechanism. Prions can be exogenously transmitted by many different
routes of administration. In the case of Aβ, previous studies showed that
intraperitoneal (i.p.) injection of seeds can accelerate cerebral amyloidosis in
mouse models. However, other potential routes have not yet been studied. The
goal of this work was to assess whether Aβ amyloidosis can be seeded in the
brain of a transgenic mouse model of AD by peripheral administration of
misfolded particles.
Young tg2576 animals (50 days old) were inoculated with a pool of brain
extract coming from old Tg2576 animals (10%w/v) by different routes: i.p.
(100μL), eye drops (5μL each eye, 3 times), intramuscular (i.m., 50μL), and per
os (p.o., 1000μL). Animals were sacrificed at 300 days old, and brain samples
were analyzed for amyloid pathology by IHC and ELISA.
The i.p., i.m., and eye drops administration of Aβ seeds significantly
accelerated pathological features in tg2576. Regardless of the higher volume
administered, p.o. treated animals did not show any pathological changes when
compared to untreated controls. Differences in the proportion of diffuse, core
and vascular deposition was observed within experimental groups. Our data show
that peripheral administration of Aβ seeds could accelerate pathological changes
in the brain and suggest that an orchestrated cross-talk between the brain and
peripheral tissues occurs in AD.
==========
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
*** These results suggest that AA fibrils are relatively heat stable and
that similar to prions, autoclaving at 135 °C is required to destroy the
pathogenicity of AA fibrils.
*** These findings may contribute to the prevention of AA fibril
transmission through food materials to different animals and especially to
humans.
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.
*** Infectious agent of sheep scrapie may persist in the environment for
at least 16 years ***
Gudmundur Georgsson1, Sigurdur Sigurdarson2 and Paul Brown3
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.
Wednesday, December 16, 2015
Objects in contact with classical scrapie sheep act as a reservoir for
scrapie transmission
Objects in contact with classical scrapie sheep act as a reservoir for
scrapie transmission
Timm Konold1*, Stephen A. C. Hawkins2, Lisa C. Thurston3, Ben C. Maddison4,
Kevin C. Gough5, Anthony Duarte1 and Hugh A. Simmons1
1 Animal Sciences Unit, Animal and Plant Health Agency Weybridge,
Addlestone, UK, 2 Pathology Department, Animal and Plant Health Agency
Weybridge, Addlestone, UK, 3 Surveillance and Laboratory Services, Animal and
Plant Health Agency Penrith, Penrith, UK, 4 ADAS UK, School of Veterinary
Medicine and Science, University of Nottingham, Sutton Bonington, UK, 5 School
of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington,
UK
Classical scrapie is an environmentally transmissible prion disease of
sheep and goats. Prions can persist and remain potentially infectious in the
environment for many years and thus pose a risk of infecting animals after
re-stocking. In vitro studies using serial protein misfolding cyclic
amplification (sPMCA) have suggested that objects on a scrapie affected sheep
farm could contribute to disease transmission. This in vivo study aimed to
determine the role of field furniture (water troughs, feeding troughs, fencing,
and other objects that sheep may rub against) used by a scrapie-infected sheep
flock as a vector for disease transmission to scrapie-free lambs with the prion
protein genotype VRQ/VRQ, which is associated with high susceptibility to
classical scrapie. When the field furniture was placed in clean accommodation,
sheep became infected when exposed to either a water trough (four out of five)
or to objects used for rubbing (four out of seven). This field furniture had
been used by the scrapie-infected flock 8 weeks earlier and had previously been
shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of
23) through exposure to contaminated field furniture placed within pasture not
used by scrapie-infected sheep for 40 months, even though swabs from this
furniture tested negative by PMCA. This infection rate decreased (1 out of 12)
on the same paddock after replacement with clean field furniture. Twelve grazing
sheep exposed to field furniture not in contact with scrapie-infected sheep for
18 months remained scrapie free. The findings of this study highlight the role
of field furniture used by scrapie-infected sheep to act as a reservoir for
disease re-introduction although infectivity declines considerably if the field
furniture has not been in contact with scrapie-infected sheep for several
months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental
contamination.
snip...
Discussion
Classical scrapie is an environmentally transmissible disease because it
has been reported in naïve, supposedly previously unexposed sheep placed in
pastures formerly occupied by scrapie-infected sheep (4, 19, 20). Although the
vector for disease transmission is not known, soil is likely to be an important
reservoir for prions (2) where – based on studies in rodents – prions can adhere
to minerals as a biologically active form (21) and remain infectious for more
than 2 years (22). Similarly, chronic wasting disease (CWD) has re-occurred in
mule deer housed in paddocks used by infected deer 2 years earlier, which was
assumed to be through foraging and soil consumption (23).
Our study suggested that the risk of acquiring scrapie infection was
greater through exposure to contaminated wooden, plastic, and metal surfaces via
water or food troughs, fencing, and hurdles than through grazing. Drinking from
a water trough used by the scrapie flock was sufficient to cause infection in
sheep in a clean building. Exposure to fences and other objects used for rubbing
also led to infection, which supported the hypothesis that skin may be a vector
for disease transmission (9). The risk of these objects to cause infection was
further demonstrated when 87% of 23 sheep presented with PrPSc in lymphoid
tissue after grazing on one of the paddocks, which contained metal hurdles, a
metal lamb creep and a water trough in contact with the scrapie flock up to 8
weeks earlier, whereas no infection had been demonstrated previously in sheep
grazing on this paddock, when equipped with new fencing and field furniture.
When the contaminated furniture and fencing were removed, the infection rate
dropped significantly to 8% of 12 sheep, with soil of the paddock as the most
likely source of infection caused by shedding of prions from the
scrapie-infected sheep in this paddock up to a week earlier.
This study also indicated that the level of contamination of field
furniture sufficient to cause infection was dependent on two factors: stage of
incubation period and time of last use by scrapie-infected sheep. Drinking from
a water trough that had been used by scrapie sheep in the predominantly
pre-clinical phase did not appear to cause infection, whereas infection was
shown in sheep drinking from the water trough used by scrapie sheep in the later
stage of the disease. It is possible that contamination occurred through
shedding of prions in saliva, which may have contaminated the surface of the
water trough and subsequently the water when it was refilled. Contamination
appeared to be sufficient to cause infection only if the trough was in contact
with sheep that included clinical cases. Indeed, there is an increased risk of
bodily fluid infectivity with disease progression in scrapie (24) and CWD (25)
based on PrPSc detection by sPMCA. Although ultraviolet light and heat under
natural conditions do not inactivate prions (26), furniture in contact with the
scrapie flock, which was assumed to be sufficiently contaminated to cause
infection, did not act as vector for disease if not used for 18 months, which
suggest that the weathering process alone was sufficient to inactivate prions.
PrPSc detection by sPMCA is increasingly used as a surrogate for
infectivity measurements by bioassay in sheep or mice. In this reported study,
however, the levels of PrPSc present in the environment were below the limit of
detection of the sPMCA method, yet were still sufficient to cause infection of
in-contact animals. In the present study, the outdoor objects were removed from
the infected flock 8 weeks prior to sampling and were positive by sPMCA at very
low levels (2 out of 37 reactions). As this sPMCA assay also yielded 2 positive
reactions out of 139 in samples from the scrapie-free farm, the sPMCA assay
could not detect PrPSc on any of the objects above the background of the assay.
False positive reactions with sPMCA at a low frequency associated with de novo
formation of infectious prions have been reported (27, 28). This is in contrast
to our previous study where we demonstrated that outdoor objects that had been
in contact with the scrapie-infected flock up to 20 days prior to sampling
harbored PrPSc that was detectable by sPMCA analysis [4 out of 15 reactions
(12)] and was significantly more positive by the assay compared to analogous
samples from the scrapie-free farm. This discrepancy could be due to the use of
a different sPMCA substrate between the studies that may alter the efficiency of
amplification of the environmental PrPSc. In addition, the present study had a
longer timeframe between the objects being in contact with the infected flock
and sampling, which may affect the levels of extractable PrPSc. Alternatively,
there may be potentially patchy contamination of this furniture with PrPSc,
which may have been missed by swabbing. The failure of sPMCA to detect
CWD-associated PrP in saliva from clinically affected deer despite confirmation
of infectivity in saliva-inoculated transgenic mice was associated with as yet
unidentified inhibitors in saliva (29), and it is possible that the sensitivity
of sPMCA is affected by other substances in the tested material. In addition,
sampling of amplifiable PrPSc and subsequent detection by sPMCA may be more
difficult from furniture exposed to weather, which is supported by the
observation that PrPSc was detected by sPMCA more frequently in indoor than
outdoor furniture (12). A recent experimental study has demonstrated that
repeated cycles of drying and wetting of prion-contaminated soil, equivalent to
what is expected under natural weathering conditions, could reduce PMCA
amplification efficiency and extend the incubation period in hamsters inoculated
with soil samples (30). This seems to apply also to this study even though the
reduction in infectivity was more dramatic in the sPMCA assays than in the sheep
model. Sheep were not kept until clinical end-point, which would have enabled us
to compare incubation periods, but the lack of infection in sheep exposed to
furniture that had not been in contact with scrapie sheep for a longer time
period supports the hypothesis that prion degradation and subsequent loss of
infectivity occurs even under natural conditions.
In conclusion, the results in the current study indicate that removal of
furniture that had been in contact with scrapie-infected animals should be
recommended, particularly since cleaning and decontamination may not effectively
remove scrapie infectivity (31), even though infectivity declines considerably
if the pasture and the field furniture have not been in contact with
scrapie-infected sheep for several months. As sPMCA failed to detect PrPSc in
furniture that was subjected to weathering, even though exposure led to
infection in sheep, this method may not always be reliable in predicting the
risk of scrapie infection through environmental contamination. These results
suggest that the VRQ/VRQ sheep model may be more sensitive than sPMCA for the
detection of environmentally associated scrapie, and suggest that extremely low
levels of scrapie contamination are able to cause infection in susceptible sheep
genotypes.
Keywords: classical scrapie, prion, transmissible spongiform
encephalopathy, sheep, field furniture, reservoir, serial protein misfolding
cyclic amplification
Congress is all set to give NIH it's largest increase in 12 years.
Included in the bill: $350 million increase for Alzheimer’s research and an
$85 million increase for the BRAIN Initiative, the project to map the human
brain.
Full story at: http://ow.ly/VYKBv
great news, with not a minute to spare...
Evidence for human transmission of amyloid-β pathology and cerebral amyloid
angiopathy
07 02:27 AM
Terry S. Singeltary Sr. said:
re-Evidence for human transmission of amyloid-? pathology and cerebral
amyloid angiopathy
Nature 525, 247?250 (10 September 2015) doi:10.1038/nature15369 Received 26
April 2015 Accepted 14 August 2015 Published online 09 September 2015 Updated
online 11 September 2015 Erratum (October, 2015)
I would kindly like to comment on the Nature Paper, the Lancet reply, and
the newspaper articles.
snip...see full text ;
Subject: 1992 IN CONFIDENCE TRANSMISSION OF ALZHEIMER TYPE PLAQUES TO
PRIMATES POSSIBILITY ON A TRANSMISSIBLE PRION REMAINS OPEN
BSE101/1 0136
IN CONFIDENCE
CMO
From: . Dr J S Metiers DCMO
4 November 1992
TRANSMISSION OF ALZHEIMER TYPE PLAQUES TO PRIMATES
1. Thank you for showing me Diana Dunstan's letter. I am glad that MRC have
recognised the public sensitivity of these findings and intend to report them in
their proper context. 'This hopefully will avoid misunderstanding and possible
distortion by the media to portray the results as having more greater
significance than the findings so far justify.
2. Using a highly unusual route of transmission (intra-cerebral injection)
the researchers have demonstrated the transmission of a pathological process
from two cases one of severe Alzheimer's disease the other of
Gerstmann-Straussler disease to marmosets. However they have not demonstrated
the transmission of either clinical condition as the "animals were behaving
normally when killed". As the report emphasises the unanswered question is
whether the disease condition would have revealed itself if the marmosets had
lived longer. They are planning further research to see if the conditions, as
opposed to the partial pathological process, is transmissible.
what are the implications for public health?
3. The route 'of transmission is very specific and in the natural state of
things highly unusual. However it could be argued that the results reveal a
potential risk, in that brain tissue from these two patients has been shown to
transmit a pathological process. Should therefore brain tissue from such cases
be regarded as potentially infective? Pathologists, morticians, neuro surgeons
and those assisting at neuro surgical procedures and others coming into contact
with "raw" human brain tissue could in theory be at risk. However, on a priori
grounds given the highly specific route of transmission in these experiments
that risk must be negligible if the usual precautions for handling brain tissue
are observed.
1
92/11.4/1.1
BSE101/1 0137
4. The other dimension to consider is the public reaction. To some extent
the GSS case demonstrates little more than the transmission of BSE to a pig by
intra-cerebral injection. If other prion diseases can be transmitted in this way
it is little surprise that some pathological findings observed in GSS were also
transmissible to a marmoset. But the transmission of features of Alzheimer's
pathology is a different matter, given the much greater frequency of this
disease and raises the unanswered question whether some cases are the result of
a transmissible prion. The only tenable public line will be that "more research
is required’’ before that hypothesis could be evaluated. The possibility on a
transmissible prion remains open. In the meantime MRC needs carefully to
consider the range and sequence of studies needed to follow through from the
preliminary observations in these two cases. Not a particularly comfortable
message, but until we know more about the causation of Alzheimer's disease the
total reassurance is not practical.
J S METTERS Room 509 Richmond House Pager No: 081-884 3344 Callsign: DOH
832 llllYc!eS 2 92/11.4/1.2
>>> The only tenable public line will be that "more research is
required’’ <<<
>>> possibility on a transmissible prion remains
open<<<
O.K., so it’s about 23 years later, so somebody please tell me, when is
"more research is required’’ enough time for evaluation ?
Self-Propagative Replication of Ab Oligomers Suggests Potential
Transmissibility in Alzheimer Disease
Received July 24, 2014; Accepted September 16, 2014; Published November 3,
2014
*** Singeltary comment PLoS ***
Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion
disease, Iatrogenic, what if ?
Posted by flounder on 05 Nov 2014 at 21:27 GMT
Tuesday, December 1, 2015
Sorting Out Release, Uptake and Processing of Alpha-Synuclein During
Prion-Like Spread of Pathology
Thursday, December 3, 2015
Transmission of Soluble and Insoluble α-Synuclein to Mice
Tuesday, September 1, 2015
Evidence for α-synuclein prions causing multiple system atrophy in humans
with parkinsonism
Wednesday, September 2, 2015
Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses
in an Alzheimer’s Disease Database
Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes
contaminated during neurosurgery.
Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC.
Laboratory of Central Nervous System Studies, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Stereotactic multicontact electrodes used to probe the cerebral cortex of a
middle aged woman with progressive dementia were previously implicated in the
accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger
patients. The diagnoses of CJD have been confirmed for all three cases. More
than two years after their last use in humans, after three cleanings and
repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were
implanted in the cortex of a chimpanzee. Eighteen months later the animal became
ill with CJD. This finding serves to re-emphasise the potential danger posed by
reuse of instruments contaminated with the agents of spongiform
encephalopathies, even after scrupulous attempts to clean them.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8006664&dopt=Abstract
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 ;
Tuesday, May 26, 2015
*** Minimise transmission risk of CJD and vCJD in healthcare settings
***
Last updated 15 May 2015
Saturday, December 12, 2015
CREUTZFELDT JAKOB DISEASE CJD TSE PRION REPORT DECEMBER 14, 2015
Thursday, December 24, 2015
Revisiting the Heidenhain Variant of Creutzfeldt-Jakob Disease: Evidence
for Prion Type Variability Influencing Clinical Course and Laboratory Findings
Article type: Research Article
Wednesday, January 06, 2016
CREUTZFELDT JAKOB DISEASE SURVEILLANCE IN THE U.K. 23rd ANNUAL REPORT 2014
(published 18th November 2015)
Terry S. Singeltary Sr.
