Does the Presence of Scrapie Affect the Ability of Current Statutory
Discriminatory Tests To Detect the Presence of Bovine Spongiform Encephalopathy?
M. M. Simmonsa, M. J. Chaplina, C. M. Vickerya, S. Simonc, L. Davisa, M.
Denyera, R. Lockeya*, M. J. Stackb, M. J. O'Connord, K. Bishope, K. C. Goughd,
B. C. Maddisone, L. Thorneb and J. Spiropoulosa
aDepartment of Pathology, Animal and Plant Health Agency, Weybridge,
Surrey, United Kingdom bDepartment of Virology, Animal and Plant Health Agency,
Weybridge, Surrey, United Kingdom cCEA Saclay, Service de Pharmacologie et
d'Immunoanalyse, iBiTec-S, Gif sur Yvette, France dSchool of Veterinary Medicine
and Science, The University of Nottingham, Leicestershire, United Kingdom eADAS
United Kingdom, School of Veterinary Medicine and Science, The University of
Nottingham, Leicestershire, United Kingdom B. W. Fenwick, Editor + Author
Affiliations
ABSTRACT
Current European Commission (EC) surveillance regulations require
discriminatory testing of all transmissible spongiform encephalopathy
(TSE)-positive small ruminant (SR) samples in order to classify them as bovine
spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests,
including characterization by bioassay in mouse models. Since 2005, naturally
occurring BSE has been identified in two goats. It has also been demonstrated
that more than one distinct TSE strain can coinfect a single animal in natural
field situations. This study assesses the ability of the statutory methods as
listed in the regulation to identify BSE in a blinded series of brain samples,
in which ovine BSE and distinct isolates of scrapie are mixed at various ratios
ranging from 99% to 1%. Additionally, these current statutory tests were
compared with a new in vitro discriminatory method, which uses serial protein
misfolding cyclic amplification (sPMCA). Western blotting consistently detected
50% BSE within a mixture, but at higher dilutions it had variable success. The
enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only
when it was present as 99% of the mixture, with variable success at higher
dilutions. Bioassay and sPMCA reported BSE in all samples where it was present,
down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at
0.1%. While bioassay is the only validated method that allows comprehensive
phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears
to offer a fast and cost-effective alternative for the screening of unknown
isolates when the purpose of the investigation was solely to determine the
presence or absence of BSE.
FOOTNOTES Received 23 February 2015. Returned for modification 28 March
2015. Accepted 27 May 2015. Accepted manuscript posted online 3 June 2015.
Address correspondence to M. M. Simmons, marion.simmons@apha.gsi.gov.uk. ↵*
Present address: R. Lockey, University of Southampton, Southampton, United
Kingdom.
Citation Simmons MM, Chaplin MJ, Vickery CM, Simon S, Davis L, Denyer M,
Lockey R, Stack MJ, O'Connor MJ, Bishop K, Gough KC, Maddison BC, Thorne L,
Spiropoulos J. 2015. Does the presence of scrapie affect the ability of current
statutory discriminatory tests to detect the presence of bovine spongiform
encephalopathy? J Clin Microbiol 53:2593–2604. doi:10.1128/JCM.00508-15.
Copyright © 2015, American Society for Microbiology. All Rights
Reserved.
IBNC Tauopathy or TSE Prion disease, it appears, no one is sure
Posted by flounder on 03 Jul 2015 at 16:53 GMT
HOUND STUDY
AS implied in the Inset 25 we must not _ASSUME_ that transmission of BSE to
other species will invariably present pathology typical of a scrapie-like
disease.
snip...
Wednesday, July 15, 2015
Additional BSE TSE prion testing detects pathologic lesion in unusual brain
location and PrPsc by PMCA only, how many cases have we missed?
Saturday, July 18, 2015
SPONTANEOUS TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY TSE PRION AKA MAD COW
TYPE DISEASE, DOES IT EXIST NATURALLY IN THE FIELD?
Friday, July 10, 2015
CANADA TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY TSE PRION UPDATE
Tuesday, June 23, 2015
Report on the monitoring and testing of ruminants for the presence of
transmissible spongiform encephalopathies (TSEs) in the EU in 2013 Final version
18 May 2015
Wednesday, January 18, 2012
Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural
Scrapie Isolates Similar to CH1641 Experimental Scrapie
Journal of Neuropathology & Experimental Neurology:
February 2012 - Volume 71 - Issue 2 - p 140–147
doi: 10.1097/NEN.0b013e3182439519
Original Articles
Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural
Scrapie Isolates Similar to CH1641 Experimental Scrapie
Vulin, Johann PhD; Beck, Katy E. PhD; Bencsik, Anna PhD; Lakhdar, Latefa
PhD; Spiropoulos, John PhD; Baron, Thierry PhD
Supplemental Author Material
Abstract
Abstract: A few cases of transmissible spongiform encephalopathies in sheep
have been described in France in which the protease-resistant prion protein
(PrPres) exhibited some features in Western blot of experimental bovine
spongiform encephalopathy in sheep. Their molecular characteristics were
indistinguishable from those produced in the CH1641 experimental scrapie
isolate. Four of these CH1641-like isolates were inoculated intracerebrally into
wild-type C57Bl/6 mice. In striking contrast to previous results in ovine
transgenic mice, CH1641 transmission in wild-type mice was efficient. Several
components of the strain signature, that is, PrPres profile, brain distribution,
and morphology of the deposits of the disease-associated prion protein, had some
similarities with “classical” scrapie and clearly differed from both bovine
spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic
mice. These results on CH1641-like isolates in wild-type mice may be consistent
with the presence in these isolates of mixed conformers with different abilities
to propagate and mediate specific disease phenotypes in different species.
Bovine spongiform encephalopathy, CH1641, Prion disease pathogenesis
© 2012 American Association of Neuropathologists, Inc
Wednesday, January 18, 2012
BSE IN GOATS CAN BE MISTAKEN FOR SCRAPIE
February 1, 2012
In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q
(AAQQ) and the disease phenotype is similar to that seen with experimental
strain CH1641.
Thursday, July 14, 2011
Histopathological Studies of "CH1641-Like" Scrapie Sources Versus Classical
Scrapie and BSE Transmitted to Ovine Transgenic Mice (TgOvPrP4)
EVIDENCE OF SCRAPIE IN SHEEP AS A RESULT OF FOOD BORNE EXPOSURE
This is provided by the statistically significant increase in the incidence
of sheep scrape from 1985, as determined from analyses of the submissions made
to VI Centres, and from individual case and flock incident studies.
........
RISK OF BSE TO SHEEP VIA FEED
Marion Simmons communicated surprising evidence for oral transmissibility
of Nor98/atypical scrapie in neonatal sheep and although bioassay is ongoing,
infectivity of the distal ileum of 12 and 24 month infected sheep is positive in
Tg338 mice.
SUMMARY REPORTS OF MAFF BSE TRANSMISSION STUDIES AT THE CVL ;
THE RISK TO HUMANS FROM SHEEP;
EXPERIMENTAL TRANSMISSION OF BSE TO SHEEP
SHEEP AND BSE
PERSONAL AND CONFIDENTIAL
SHEEP AND BSE
A. The experimental transmission of BSE to sheep.
Studies have shown that the ''negative'' line NPU flock of Cheviots can be
experimentally infected with BSE by intracerebral (ic) or oral challenge (the
latter being equivalent to 0.5 gram of a pool of four cow brains from animals
confirmed to have BSE).
RB264
BSE - TRANSMISSION STUDIES
Monday, March 21, 2011
Sheep and Goat BSE Propagate More Efficiently than Cattle BSE in Human PrP
Transgenic Mice
snip...
On the other hand, this component would not be distinguishable from
bovine-passaged BSE prions due to the current limits of the standard biological
methods and/or the molecular tools employed here to characterize prion strains.
Whatever the mechanism, the notion that a passage through an intermediate
species can profoundly alter prion virulence for the human species has important
public-health issues, regarding emerging and/or expanding TSEs, like atypical
scrapie or CWD.
snip...
Taken all together, our results suggest that the possibility of a small
ruminant BSE prion as vCJD causal agent could not be ruled out, which has
important implications on public and animal health policies. On one hand,
although the exact magnitude and characteristic of the vCJD epidemic is still
unclear, its link with cattle BSE is supported by strong epidemiological ground
and several experimental data. On the other hand, the molecular typing performed
in our studies, indicates that the biochemical characteristics of the PrPres
detected in brains of our sheep and goat BSE-inoculated mice seem to be
indistinguishable from that observed in vCJD. Considering the similarity in
clinical manifestation of BSE- and scrapie-affected sheep [48], a masker effect
of scrapie over BSE, as well as a potential adaptation of the BSE agent through
subsequent passages, could not be ruled out. As BSE infected sheep PrPSc have
been detected in many peripheral organs, small ruminant-passaged BSE prions
might be a more widespread source of BSE infectivity compared to cattle [19],
[49], [50]. This fact is even more worrying since our transmission studies
suggest that apparently Met129 human PrP favours a BSE agent with ovine rather
than a bovine sequence. Finally, it is evident that, although few natural cases
have been described and so far we cannot draw any definitive conclusion about
the origin of vCJD, we can not underestimate the risk of a potential goat and/or
sheep BSE agent.
snip...
Technical Abstract:
Prion strains may vary in their ability to transmit to humans and animals.
Few experimental studies have been done to provide evidence of differences
between U.S. strains of scrapie, which can be distinguished by incubation times
in inbred mice, microscopic lesions, immunoreactivity to various antibodies, or
molecular profile (electrophoretic mobility and glycoform ratio). Recent work on
two U.S. isolates of sheep scrapie supports that at least two distinct strains
exist based on differences in incubation time and genotype of sheep affected.
One isolate (No. 13-7) inoculated intracerebrally caused scrapie in sheep AA at
codon 136 (AA136) and QQ at codon 171 (QQ171) of the prion protein in an average
of 19 months post-inoculation (PI) whereas a second isolate (No. x124) caused
disease in less than 12 months after oral inoculation in AV136/QQ171 sheep.
Striking differences were evident when further strain analysis was done in R111,
VM, C57Bl6, and C57Bl6xVM (F1) mice. No. 13-7 did not induce disease in any
mouse strain at any time post-inoculation (PI) nor were brain tissues positive
by western blot (WB). Positive WB results were obtained from mice inoculated
with isolate No. x124 starting at day 380 PI. Incubation times averaged 508,
559, 601, and 633 days PI for RIII, C57Bl6, VM, and F1 mice, respectively.
Further passage will be required to characterize these scrapie strains in mice.
This work provides evidence that multiple scrapie strains exist in U.S.
sheep.
One of these isolates (TR316211) behaved like the CH1641 isolate, with
PrPres features in mice similar to those in the sheep brain. From two other
isolates (O100 and O104), two distinct PrPres phenotypes were identified in
mouse brains, with either high (h-type) or low (l-type) apparent molecular
masses of unglycosylated PrPres, the latter being similar to that observed with
CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions
in the brains of the individual mice. In contrast with BSE, l-type PrPres from
"CH1641-like" isolates showed lower levels of diglycosylated PrPres. From one of
these cases (O104), a second passage in mice was performed for two mice with
distinct PrPres profiles. This showed a partial selection of the l-type
phenotype in mice infected with a mouse brain with predominant l-type PrPres,
and it was accompanied by a significant increase in the proportions of the
diglycosylated band. These results are discussed in relation to the diversity of
scrapie and BSE strains.
1: J Infect Dis 1980 Aug;142(2):205-8
Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to
nonhuman primates.
Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.
Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep
and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were
exposed to the infectious agents only by their nonforced consumption of known
infectious tissues. The asymptomatic incubation period in the one monkey exposed
to the virus of kuru was 36 months; that in the two monkeys exposed to the virus
of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the
two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively.
Careful physical examination of the buccal cavities of all of the monkeys failed
to reveal signs or oral lesions. One additional monkey similarly exposed to kuru
has remained asymptomatic during the 39 months that it has been under
observation.
snip...
The successful transmission of kuru, Creutzfeldt-Jakob disease, and scrapie
by natural feeding to squirrel monkeys that we have reported provides further
grounds for concern that scrapie-infected meat may occasionally give rise in
humans to Creutzfeldt-Jakob disease.
PMID: 6997404
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract
12/10/76 AGRICULTURAL RESEARCH COUNCIL REPORT OF THE ADVISORY COMMITTE ON
SCRAPIE Office Note CHAIRMAN: PROFESSOR PETER WILDY
snip...
A The Present Position with respect to Scrapie A] The Problem Scrapie is a
natural disease of sheep and goats. It is a slow and inexorably progressive
degenerative disorder of the nervous system and it ia fatal. It is enzootic in
the United Kingdom but not in all countries. The field problem has been reviewed
by a MAFF working group (ARC 35/77). It is difficult to assess the incidence in
Britain for a variety of reasons but the disease causes serious financial loss;
it is estimated that it cost Swaledale breeders alone $l.7 M during the five
years 1971-1975. A further inestimable loss arises from the closure of certain
export markets, in particular those of the United States, to British sheep. It
is clear that scrapie in sheep is important commercially and for that reason
alone effective measures to control it should be devised as quickly as possible.
Recently the question has again been brought up as to whether scrapie is
transmissible to man. This has followed reports that the disease has been
transmitted to primates.
One particularly lurid speculation (Gajdusek 1977) conjectures that the
agents of scrapie, kuru, Creutzfeldt-Jakob disease and transmissible
encephalopathy of mink are varieties of a single "virus". The U.S. Department of
Agriculture concluded that it could "no longer justify or permit scrapie-blood
line and scrapie-exposed sheep and goats to be processed for human or animal
food at slaughter or rendering plants" (ARC 84/77)" The problem is emphasised by
the finding that some strains of scrapie produce lesions identical to the once
which characterise the human dementias" Whether true or not. the hypothesis that
these agents might be transmissible to man raises two considerations. First, the
safety of laboratory personnel requires prompt attention. Second, action such as
the "scorched meat" policy of USDA makes the solution of the acrapie problem
urgent if the sheep industry is not to suffer grievously.
snip...
76/10.12/4.6
Nature. 1972 Mar 10;236(5341):73-4.
Transmission of scrapie to the cynomolgus monkey (Macaca
fascicularis).
Gibbs CJ Jr, Gajdusek DC. Nature 236, 73 - 74 (10 March 1972);
doi:10.1038/236073a0
Transmission of Scrapie to the Cynomolgus Monkey (Macaca
fascicularis)
C. J. GIBBS jun. & D. C. GAJDUSEK National Institute of Neurological
Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
SCRAPIE has been transmitted to the cynomolgus, or crab-eating, monkey
(Macaca fascicularis) with an incubation period of more than 5 yr from the time
of intracerebral inoculation of scrapie-infected mouse brain. The animal
developed a chronic central nervous system degeneration, with ataxia, tremor and
myoclonus with associated severe scrapie-like pathology of intensive astroglial
hypertrophy and proliferation, neuronal vacuolation and status spongiosus of
grey matter. The strain of scrapie virus used was the eighth passage in Swiss
mice (NIH) of a Compton strain of scrapie obtained as ninth intracerebral
passage of the agent in goat brain, from Dr R. L. Chandler (ARC, Compton,
Berkshire).
Wednesday, February 16, 2011
IN CONFIDENCE SCRAPIE TRANSMISSION TO CHIMPANZEES
IN CONFIDENCE
Wednesday, February 16, 2011 IN CONFIDENCE SCRAPIE TRANSMISSION TO
CHIMPANZEES
IN CONFIDENCE
reference...
RB3.20
TRANSMISSION TO CHIMPANZEES
1. Kuru and CJD have been successfully transmitted to chimpanzees but
scrapie and TME have not.
2. We cannot say that scrapie will not transmit to chimpanzees. There are
several scrapie strains and I am not aware that all have been tried (that would
have to be from mouse passaged material). Nor has a wide enough range of field
isolates subsequently strain typed in mice been inoculated by the appropriate
routes (i/c, ilp and i/v) :
3. I believe the proposed experiment to determine transmissibility, if
conducted, would only show the susceptibility or resistance of the chimpanzee to
infection/disease by the routes used and the result could not be interpreted for
the predictability of the susceptibility for man. Proposals for prolonged oral
exposure of chimpanzees to milk from cattle were suggested a long while ago and
rejected.
4. In view of Dr Gibbs' probable use of chimpazees Mr Wells' comments
(enclosed) are pertinent. I have yet to receive a direct communication from Dr
Schellekers but before any collaboration or provision of material we should
identify the Gibbs' proposals and objectives.
5. A positive result from a chimpanzee challenged severely would likely
create alarm in some circles even if the result could not be interpreted for
man. I have a view that all these agents could be transmitted provided a large
enough dose by appropriate routes was given and the animals kept long enough.
Until the mechanisms of the species barrier are more clearly understood it might
be best to retain that hypothesis.
6. A negative result would take a lifetime to determine but that would be a
shorter period than might be available for human exposure and it would still not
answer the question regarding mans' susceptibility. In the meantime no doubt the
negativity would be used defensively. It would however be counterproductive if
the experiment finally became positive. We may learn more about public reactions
following next Monday' s meeting.
R. Bradley
23 September 1990
CVO (+Mr Wells' comments)
Dr T W A Little
Dr B J Shreeve
90/9.23/1.1.
IN CONFIDENCE CHIMPANZEES
CODE 18-77 Reference RB3.46
Some further information that may assist in decision making has been gained
by discussion with Dr Rosalind Ridley.
She says that careful study of Gajdusek's work shows no increased
susceptibility of chimpanzees over New World Monkeys such as Squirrel Monkeys.
She does not think it would tell you anything about the susceptibility to man.
Also Gajdusek did not, she believes, challenge chimpanzees with scrapie as
severely as we did pigs and we know little of that source of scrapie.
Comparisons would be difficult. She also would not expect the Home Office to
sanction such experiments here unless there was a very clear and important
objective that would be important for human health protection. She doubted such
a case could be made. If this is the case she thought it would be unethical to
do an experiment abroad because we could not do it in our own country.
Retrospectively she feels they should have put up more marmosets than they
did. They all remain healthy. They would normally regard the transmission as
negative if no disease resulted in five years.
We are not being asked for a decision but I think that before we made one
we should gain as much knowledge as we can. If we decided to proceed we would
have to bear any criticisms for many years if there was an adverse view by
scientists ormedia. This should not be undertaken lightly. There is already
some adverse comment here, I gather, on the pig experiment though that will
subside.
The Gibbs' (as' distinct from Schellekers') study is somewhat different. We
are merely supplying material for comparative studies in a laboratory with the
greatest experience of human SEs in the world and it has been sanctioned by USDA
(though we do not know for certain yet if chimpanzees specifically will be
used). This would keep it at a lower profile than if we conducted such an
experiment in the UK or Europe.
I consider we must have very powerful and defendable objectives to go
beyond Gibbs' proposed experiments and should not initiate others just because
an offer has been made.
Scientists have a responsibility to seek other methods of investigative
research other than animal experimentation. At present no objective has
convinced me we need to do research using Chimpanzees - a species in need of
protection. Resisting such proposals would enable us to communicate that
information to the scientist and the public should the need arise. A line would
have been drawn.
CVO cc Dr T Dr B W A Little Dr B J Shreeve
R Bradley
26 September 1990
90/9.26/3.2
SNIP...SEE FULL TEXT ;
PRION 2015 ORAL AND POSTER CONGRESSIONAL ABSTRACTS
THANK YOU PRION 2015 TAYLOR & FRANCIS, Professor Chernoff, and
Professor Aguzzi et al, for making these PRION 2015 Congressional Poster and
Oral Abstracts available freely to the public. ...Terry S. Singeltary Sr.
O.05: Transmission of prions to primates after extended silent incubation
periods: Implications for BSE and scrapie risk assessment in human populations
Emmanuel Comoy, Jacqueline Mikol, Val erie Durand, Sophie Luccantoni,
Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys
Atomic Energy Commission; Fontenay-aux-Roses, France
Prion diseases (PD) are the unique neurodegenerative proteinopathies
reputed to be transmissible under field conditions since decades. The
transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that
an animal PD might be zoonotic under appropriate conditions. Contrarily, in the
absence of obvious (epidemiological or experimental) elements supporting a
transmission or genetic predispositions, PD, like the other proteinopathies, are
reputed to occur spontaneously (atpical animal prion strains, sporadic CJD
summing 80% of human prion cases). Non-human primate models provided the first
evidences supporting the transmissibiity of human prion strains and the zoonotic
potential of BSE. Among them, cynomolgus macaques brought major information for
BSE risk assessment for human health (Chen, 2014), according to their
phylogenetic proximity to humans and extended lifetime. We used this model to
assess the zoonotic potential of other animal PD from bovine, ovine and cervid
origins even after very long silent incubation periods. We recently observed the
direct transmission of a natural classical scrapie isolate to macaque after a
10-year silent incubation period, with features similar to some reported for
human cases of sporadic CJD, albeit requiring fourfold longe incubation than
BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014), is the third
potentially zoonotic PD (with BSE and L-type BSE), ***thus questioning the
origin of human sporadic cases. We will present an updated panorama of our
different transmission studies and discuss the implications of such extended
incubation periods on risk assessment of animal PD for human health.
===============
*** Scrapie, as recently evoked in humanized mice (Cassard, 2014), is the
third potentially zoonotic PD (with BSE and L-type BSE), ***thus questioning the
origin of human sporadic cases.
===============
*** 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
Chronic wasting disease (CWD) is a widespread and expanding prion disease
in free-ranging and captive cervid species in North America. The zoonotic
potential of CWD prions is a serious public health concern. Current literature
generated with in vitro methods and in vivo animal models (transgenic mice,
macaques and squirrel monkeys) reports conflicting results. The susceptibility
of human CNS and peripheral organs to CWD prions remains largely unresolved. In
our earlier bioassay experiments using several humanized transgenic mouse lines,
we detected protease-resistant PrPSc in the spleen of two out of 140 mice that
were intracerebrally inoculated with natural CWD isolates, but PrPSc was not
detected in the brain of the same mice. Secondary passages with such
PrPSc-positive CWD-inoculated humanized mouse spleen tissues led to efficient
prion transmission with clear clinical and pathological signs in both humanized
and cervidized transgenic mice. Furthermore, a recent bioassay with natural CWD
isolates in a new humanized transgenic mouse line led to clinical prion
infection in 2 out of 20 mice. ***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.***
==================
Saturday, May 30, 2015
PRION 2015 ORAL AND POSTER CONGRESSIONAL ABSTRACTS
Transmission of scrapie prions to primate after an extended silent
incubation period
Emmanuel E. Comoy1 , Jacqueline Mikol1 , Sophie Luccantoni-Freire1 ,
Evelyne Correia1 , Nathalie Lescoutra-Etchegaray1 , Valérie Durand1 , Capucine
Dehen1 , Olivier Andreoletti2 , Cristina Casalone3 , Juergen A. Richt4 n1 ,
Justin J. Greenlee4 , Thierry Baron5 , Sylvie L. Benestad6 , Paul Brown1 […]
& Jean-Philippe Deslys1 - Show fewer authors Scientific Reports 5, Article
number: 11573 (2015) doi:10.1038/srep11573 Download Citation
Epidemiology | Neurological manifestations | Prion diseases Received: 16
February 2015 Accepted: 28 May 2015 Published online: 30 June 2015 ABSTRACT
Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion
disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD)
in humans and having guided protective measures for animal and human health
against animal prion diseases. Recently, partial transmissions to humanized mice
showed that the zoonotic potential of scrapie might be similar to c-BSE. We here
report the direct transmission of a natural classical scrapie isolate to
cynomolgus macaque, a highly relevant model for human prion diseases, after a
10-year silent incubation period, with features similar to those reported for
human cases of sporadic CJD. Scrapie is thus actually transmissible to primates
with incubation periods compatible with their life expectancy, although fourfold
longer than BSE. Long-term experimental transmission studies are necessary to
better assess the zoonotic potential of other prion diseases with high
prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98
scrapie.
snip...
Discussion
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.
The second possibility is a laboratory cross-contamination. Three facts
make this possibility equally unlikely. First, handling of specimens in our
laboratory is performed with fastidious attention to the avoidance of any such
cross-contamination. Second, no laboratory cross-contamination has ever been
documented in other primate laboratories, including the NIH, even between
infected and uninfected animals housed in the same or adjacent cages with daily
intimate contact (P. Brown, personal communication). Third, the cerebral lesion
profile is different from all the other prion diseases we have studied in this
model19, with a correlation between cerebellar lesions (massive spongiform
change of Purkinje cells, intense PrPres staining and reactive gliosis26) and
ataxia. The iron deposits present in the globus pallidus are a non specific
finding that have been reported previously in neurodegenerative diseases and
aging27. Conversely, the thalamic lesion was reminiscent of a metabolic disease
due to thiamine deficiency28 but blood thiamine levels were within normal limits
(data not shown). The preferential distribution of spongiform change in cortex
associated with a limited distribution in the brainstem is reminiscent of the
lesion profile in MM2c and VV1 sCJD patients29, but interspecies comparison of
lesion profiles should be interpreted with caution. It is of note that the same
classical scrapie isolate induced TSE in C57Bl/6 mice with similar incubation
periods and lesional profiles as a sample derived from a MM1 sCJD
patient30.
We are therefore confident that the illness in this cynomolgus macaque
represents a true transmission of a sheep c-scrapie isolate directly to an
old-world monkey, which taxonomically resides in the primate subdivision
(parvorder of catarrhini) that includes humans. With an homology of its PrP
protein with humans of 96.4%31, cynomolgus macaque constitutes a highly relevant
model for assessing zoonotic risk of prion diseases. Since our initial aim was
to show the absence of transmission of scrapie to macaques in the worst-case
scenario, we obtained materials from a flock of naturally-infected sheep,
affecting animals with different genotypes32. This c-scrapie isolate exhibited
complete transmission in ARQ/ARQ sheep (332 ± 56 days) and Tg338 transgenic mice
expressing ovine VRQ/VRQ prion protein (220 ± 5 days) (O. Andreoletti, personal
communication). From the standpoint of zoonotic risk, it is important to note
that sheep with c-scrapie (including the isolate used in our study) have
demonstrable infectivity throughout their lymphoreticular system early in the
incubation period of the disease (3 months-old for all the lymphoid organs, and
as early as 2 months-old in gut-associated lymph nodes)33. In addition, scrapie
infectivity has been identified in blood34, milk35 and skeletal muscle36 from
asymptomatic but scrapie infected small ruminants which implies a potential
dietary exposure for consumers.
Two earlier studies have reported the occurrence of clinical TSE in
cynomolgus macaques after exposures to scrapie isolates. In the first study, the
“Compton” scrapie isolate (derived from an English sheep) and serially
propagated for 9 passages in goats did not transmit TSE in cynomolgus macaque,
rhesus macaque or chimpanzee within 7 years following intracerebral challenge1;
conversely, after 8 supplementary passages in conventional mice, this “Compton”
isolate induced TSE in a cynomolgus macaque 5 years after intracerebral
challenge, but rhesus macaques and chimpanzee remained asymptomatic 8.5 years
post-exposure8. However, multiple successive passages that are classically used
to select laboratory-adapted prion strains can significantly modify the initial
properties of a scrapie isolate, thus questioning the relevance of zoonotic
potential for the initial sheep-derived isolate. The same isolate had also
induced disease into squirrel monkeys (new-world monkey)9. A second historical
observation reported that a cynomolgus macaque developed TSE 6 years
post-inoculation with brain homogenate from a scrapie-infected Suffolk ewe
(derived from USA), whereas a rhesus macaque and a chimpanzee exposed to the
same inoculum remained healthy 9 years post-exposure1. This inoculum also
induced TSE in squirrel monkeys after 4 passages in mice. Other scrapie
transmission attempts in macaque failed but had more shorter periods of
observation in comparison to the current study. Further, it is possible that
there are differences in the zoonotic potential of different scrapie
strains.
The most striking observation in our study is the extended incubation
period of scrapie in the macaque model, which has several implications. Firstly,
our observations constitute experimental evidence in favor of the zoonotic
potential of c-scrapie, at least for this isolate that has been extensively
studied32,33,34,35,36. The cross-species zoonotic ability of this isolate should
be confirmed by performing duplicate intracerebral exposures and assessing the
transmissibility by the oral route (a successful transmission of prion strains
through the intracerebral route may not necessarily indicate the potential for
oral transmission37). However, such confirmatory experiments may require more
than one decade, which is hardly compatible with current general management and
support of scientific projects; thus this study should be rather considered as a
case report.
Secondly, transmission of c-BSE to primates occurred within 8 years post
exposure for the lowest doses able to transmit the disease (the survival period
after inoculation is inversely proportional to the initial amount of infectious
inoculum). The occurrence of scrapie 10 years after exposure to a high dose (25
mg) of scrapie-infected sheep brain suggests that the macaque has a higher
species barrier for sheep c-scrapie than c-BSE, although it is notable that
previous studies based on in vitro conversion of PrP suggested that BSE and
scrapie prions would have a similar conversion potential for human PrP38.
Thirdly, prion diseases typically have longer incubation periods after oral
exposure than after intracerebral inoculations: since humans can develop Kuru 47
years after oral exposure39, an incubation time of several decades after oral
exposure to scrapie would therefore be expected, leading the disease to occur in
older adults, i.e. the peak age for cases considered to be sporadic disease, and
making a distinction between scrapie-associated and truly sporadic disease
extremely difficult to appreciate.
Fourthly, epidemiologic evidence is necessary to confirm the zoonotic
potential of an animal disease suggested by experimental studies. A relatively
short incubation period and a peculiar epidemiological situation (e.g., all the
first vCJD cases occurring in the country with the most important ongoing c-BSE
epizootic) led to a high degree of suspicion that c-BSE was the cause of vCJD.
Sporadic CJD are considered spontaneous diseases with an almost stable and
constant worldwide prevalence (0.5–2 cases per million inhabitants per year),
and previous epidemiological studies were unable to draw a link between sCJD and
classical scrapie6,7,40,41, even though external causes were hypothesized to
explain the occurrence of some sCJD clusters42,43,44. However, extended
incubation periods exceeding several decades would impair the predictive values
of epidemiological surveillance for prion diseases, already weakened by a
limited prevalence of prion diseases and the multiplicity of isolates gathered
under the phenotypes of “scrapie” and “sporadic CJD”.
Fifthly, considering this 10 year-long incubation period, together with
both laboratory and epidemiological evidence of decade or longer intervals
between infection and clinical onset of disease, no premature conclusions should
be drawn from negative transmission studies in cynomolgus macaques with less
than a decade of observation, as in the aforementioned historical transmission
studies of scrapie to primates1,8,9. Our observations and those of others45,46
to date are unable to provide definitive evidence regarding the zoonotic
potential of CWD, atypical/Nor98 scrapie or H-type BSE. The extended incubation
period of the scrapie-affected macaque in the current study also underscores the
limitations of rodent models expressing human PrP for assessing the zoonotic
potential of some prion diseases since their lifespan remains limited to
approximately two years21,47,48. This point is illustrated by the fact that the
recently reported transmission of scrapie to humanized mice was not associated
with clinical signs for up to 750 days and occurred in an extreme minority of
mice with only a marginal increase in attack rate upon second passage13. The low
attack rate in these studies is certainly linked to the limited lifespan of mice
compared to the very long periods of observation necessary to demonstrate the
development of scrapie. Alternatively, one could estimate that a successful
second passage is the result of strain adaptation to the species barrier, thus
poorly relevant of the real zoonotic potential of the original scrapie isolate
of sheep origin49. The development of scrapie in this primate after an
incubation period compatible with its lifespan complements the study conducted
in transgenic (humanized) mice; taken together these studies suggest that some
isolates of sheep scrapie can promote misfolding of the human prion protein and
that scrapie can develop within the lifespan of some primate species.
In addition to previous studies on scrapie transmission to primate1,8,9 and
the recently published study on transgenic humanized mice13, our results
constitute new evidence for recommending that the potential risk of scrapie for
human health should not be dismissed. Indeed, human PrP transgenic mice and
primates are the most relevant models for investigating the human transmission
barrier. To what extent such models are informative for measuring the zoonotic
potential of an animal TSE under field exposure conditions is unknown. During
the past decades, many protective measures have been successfully implemented to
protect cattle from the spread of c-BSE, and some of these measures have been
extended to sheep and goats to protect from scrapie according to the principle
of precaution. Since cases of c-BSE have greatly reduced in number, those
protective measures are currently being challenged and relaxed in the absence of
other known zoonotic animal prion disease. We recommend that risk managers
should be aware of the long term potential risk to human health of at least
certain scrapie isolates, notably for lymphotropic strains like the classical
scrapie strain used in the current study. Relatively high amounts of infectivity
in peripheral lymphoid organs in animals infected with these strains could lead
to contamination of food products produced for human consumption. Efforts should
also be maintained to further assess the zoonotic potential of other animal
prion strains in long-term studies, notably lymphotropic strains with high
prevalence like CWD, which is spreading across North America, and atypical/Nor98
scrapie (Nor98)50 that was first detected in the past two decades and now
represents approximately half of all reported cases of prion diseases in small
ruminants worldwide, including territories previously considered as scrapie
free. Even if the prevailing view is that sporadic CJD is due to the spontaneous
formation of CJD prions, it remains possible that its apparent sporadic nature
may, at least in part, result from our limited capacity to identify an
environmental origin.
Thursday, July 24, 2014
*** Protocol for further laboratory investigations into the distribution of
infectivity of Atypical BSE SCIENTIFIC REPORT OF EFSA New protocol for Atypical
BSE investigations
Saturday, March 21, 2015
Canada and United States Creutzfeldt Jakob TSE Prion Disease Incidence
Rates Increasing
Terry S. Singeltary S.
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