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From: TSS ()
Subject: Isolation from Cattle of a Prion StrainDistinct from That Causing Bovine Spongiform Encephalopathy
Date: October 24, 2006 at 11:33 am PST

Isolation from Cattle of a Prion StrainDistinct from That Causing Bovine Spongiform Encephalopathy

Vincent Be´ringue1, Anna Bencsik2[, Annick Le Dur1[, Fabienne Reine1, Thanh Lan Laý¨1, Nathalie Chenais3, Gae¨ lle Tilly3,

Anne-Gae¨ lle Biacabe´2, Thierry Baron2, Jean-Luc Vilotte3, Hubert Laude1*

1 Institut National de la Recherche Agronomique, Virologie Immunologie Mole´ culaires, Jouy-en-Josas, France, 2 Agence Franc¸aise de Se´curite´ Sanitaire des Aliments, Unite´

Agents Transmissibles Non Conventionnels, Lyon, France, 3 Institut National de la Recherche Agronomique, Ge´ne´tique Biochimique, et Cytoge´ne´tique, Jouy-en-Josas, France

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have

been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical

profile of abnormal protease-resistant prion protein (PrPres) isolated from brains of affected animals or humans.

However, alternate PrPres signatures in cattle have recently been discovered through large-scale screening. To test

whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrPres of

higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice

developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of

infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent

inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie

isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various

prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals.

Citation: Be´ringue V, Bencsik A, Le Dur A, Reine F, Laý¨ TL, et al. (2006) Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy.

PLoS Pathog 2(10): e112. DOI: 10.1371/journal.ppat.0020112

snip...

Discussion

In this study we show that cattle brain samples positive for

abnormal PrP with a distinct molecular pattern, called Htype,

consistently produces a fatal, TSE-like disease upon

inoculation to both bovine and ovine PrP transgenic mice.

These results, corroborating the recent transmission to wildtype

mice [18], formally establish that such cases involve an

authentic TSE infectious agent. Importantly, we provide

detailed evidence that this newly recognised agent differs

from epizootic BSE agent derived from cattle or other

species.

Both molecular and biological criteria support the conclusion

that H-type and BSE agents are distinct prion strains.

First, the incubation periods upon transmission to mice

expressing either bovine or ovine PrP produced different

patterns. Thus, while primary transmission to tgOv mice led

to longer survival times for both agents, the increase relative

to tgBov mice was significantly less for H-type than for BSEtype

agents (Figure 1). Second, the molecular profiles of the

PrPres fragments detected in the brain of diseased mice were

clearly distinguishable in either line. Strikingly, differences

observed in terms of fragment size and glycoform ratio were

essentially the same as in cattle brain. Third, unlike that for

BSE agents, no PrPres signal could be seen in the spleen of Htype–

infected tgOv mice, indicative of a stronger neurotropism

at least in this host. Fourth, histopathological

examination of tgBov mice revealed a contrasting picture.

Typically, severe spongiosis and diffuse PrP deposition were

present in several areas of H-type–infected brains, while the

same areas of BSE-infected brains showed limited spongiosis

together with marked PrP deposition. Such discrepancies are

unlikely to result from unequal survival times since they were

also observed on secondary passage, where the two agents had

comparable incubation duration (unpublished data).

The isolation from cattle of a prion strain distinct from the

one implicated in the BSE epidemics raises several concerns.

One is whether H-type isolates might result from an exposure

to prions of small ruminants via alimentary or environmental

sources, since cattle have been shown to be susceptible to

experimental infection by sheep scrapie agent [19]. In this

regards, the better compatibility between ovine PrP sequence

and H-type as compared to BSE was intriguing (Figure 1).

However, our investigations do not support this. Among the

five groups of natural isolates we have identified so far in

tgOv mice ([13,14] and our unpublished data), only one

group, made up mostly of SSit isolates, proposed to be of

iatrogenic origin [20], showed an incubation time as

prolonged as for H-type cases. However, the PrPres molecular

profile, nature of deposits, and distribution within the brain

as well as the differential accumulation in the spleen strongly

distinguish H-type and SSit isolates. In addition, the latter

failed so far to transmit to tgBov mice.

H-type and BSE agents might be related despite their

distinguishable phenotypes. The isolation of an additional

strain upon exposure of transgenic or wild-type mice to the

epizootic BSE agent has been reported [21], thus questioning

its strain homogeneity. Also, molecular typing studies have

revealed the presence of a minor, non–BSE-type PrPres

component in BSE- and vCJD-infected brains [22]. Hence,

H-type isolates could arise from the preferential amplification

in certain individuals of a subcomponent present in BSE

infectious sources. Comparing H-type and BSE-derived

variant prions identified in mice might be informative in

that respect.

Alternatively, such unusual cases could reflect the existence

of a natural, sporadic disease in cattle. Although it is unclear

yet if such infections may lead to a clinical disease in the

natural host, they seem to occur at a low frequency, which is

reminiscent of the situation known for sporadic CJD in

humans [23,24]. Of note, the disparities between intensity of

PrP deposition and severity of vacuolation in the brains of Htype–

inoculated tgBov mice have also been observed with

sporadic CJD both in human or mouse infected brains [21,25].

These data, however, need to be consolidated through further

investigations, including epidemiological analysis. Indeed, an

implication of this latter scenario is that such bovine

‘‘atypical’’ cases could occur in countries free of BSE

exposure. The acquisition of novel properties by an endogenous,

sporadic cattle TSE agent, as occasioned on passage

through an intermediary host or a physicochemical treatment

such as that applied to carcass-derived products, has been

invoked as one possible origin for the emergence of BSE

epidemics [7]. With the isolation of such agents, we can now

address this issue experimentally.

In conclusion, our findings support the view that at least

two and potentially three [10] distinct prion strains may be

present in cattle. The current uncertainties regarding the

origin, prevalence, and potential risk for humans of a strain

of TSE agent unrecognised until recently should support

continued efforts to characterise it in vivo and uphold the

surveillance exerted on cattle.

snip...

http://pathogens.plosjournals.org/archive/1553-7374/2/10/pdf/10.1371_journal.ppat.0020112-S.pdf

PLEASE NOTE, if spontaneous scrapie or CWD does not occur, then why is it that now only atypical BSE and sproadic CJD are capable of spontaneous mutation $$$ seems the UK study just posted about different strains of UK scrapie cause different strains of TSE when transmitted to UK cattle in the lab would dispute the spontaneous, natural, sporadic, lame duck excuse for any TSE, and how could these few 'unusual cases' _scientifically_ explain 85%+ of all sCJD, of which there are 6 documented phenotypes, or 5, depending whom you believe ? bottom line, they can't......TSS

Science 24 September 2004:
Vol. 305. no. 5692, pp. 1918 - 1921
DOI: 10.1126/science.1103581

Perspectives
BIOMEDICINE:
A Fresh Look at BSE
Bruce Chesebro*
Mad cow disease, or bovine spongiform encephalopathy (BSE), is the cattle
form of a family of progressive brain diseases. These diseases include
scrapie in sheep, Creutzfeldt-Jakob disease (CJD) in humans, and chronic
wasting disease (CWD) in deer and elk. They are also known as either "prion
diseases" because of the association of a misfolded cellular prion protein
in pathogenesis or "transmissible spongiform encephalopathies" (TSEs)
because of the spongelike nature of the damaged brain tissue (1).

The recent discovery of two BSE-infected cows, one in Canada and one in the
United States, has dramatically increased concern in North America among
meat producers and consumers alike over the extent to which BSE poses a
threat to humans as well as to domestic and wild animals. The European BSE
epidemic of the late-1980s seems to have been initiated a decade earlier in
the United Kingdom by changes in the production of meat and bone meal (MBM)
from rendered livestock, which led to contamination of MBM with the BSE
infectious agent. Furthermore, the fact that UK farmers fed this rendered
MBM to younger animals and that this MBM was distributed to many countries
may have contributed to the ensuing BSE epidemic in the United Kingdom and
internationally (2).

Despite extensive knowledge about the spread of BSE through contaminated
MBM, the source of BSE in Europe remains an unsolved mystery (2). It has
been proposed that BSE could be derived from a cross-species infection,
perhaps through contamination of MBM by scrapie-infected sheep tissues (see
the figure). Alternatively, BSE may have been an endemic disease in cattle
that went unnoticed because of its low level of horizontal transmission.
Lastly, BSE might have originated by "spontaneous" misfolding of the normal
cellular prion protein into the disease-associated abnormal isoform (3),
which is postulated to be the infectious agent or "prion."

Five possible sources of BSE in North American cattle. Sheep, deer, and elk
could spread prion diseases (TSEs) to cattle through direct animal contact
or contamination of pastures. Endemic BSE has not been proven to exist
anywhere in the world, but it is difficult to exclude this possibility
because of the inefficient spread of BSE infectivity between individual
animals (2). BSE caused by spontaneous misfolding of the prion protein has
not been proven.
CREDIT: KATHARINE SUTLIFF/SCIENCE

Spontaneous protein misfolding is not a new phenomenon as proteins are known
to sometimes misfold after synthesis. Cells in turn have devised ingenious
ways to deal with this problem. These include molecular chaperone proteins
that bind to misfolded proteins and help them to unfold, and organelles
called proteosomes that degrade misfolded or unwanted proteins. However,
although misfolded prion proteins have been generated in test tubes as well
as in cultured cells, it has been difficult to demonstrate that such
misfolded abnormal prion proteins are infectious (4, 5). Even the most
recent data do not prove conclusively that infectivity has been generated in
vitro because misfolded synthetic prion proteins were not able to transfer
disease directly to wild-type mice (6). To obtain infectivity and subsequent
prion disease, the misfolded proteins had to be inoculated and incubated for
1 to 2 years in transgenic mice that overexpressed a mutant version of the
prion protein. Previous data from this group showed that transgenic mice
expressing high amounts of prion protein developed neurological disease
without inoculation of misfolded prion protein (7). Thus, at the biochemical
level, the critical attributes of the misfolded prion protein required for
infectivity are not known, and misfolding of prion protein alone may not be
sufficient to generate an infectious agent (.
Nevertheless, the idea that BSE might originate due to the spontaneous
misfolding of prion proteins has received renewed interest in the wake of
reports suggesting the occurrence of atypical BSE (9-11). These results
imply that new strains of cattle BSE might have originated separately from
the main UK outbreak. Where and how might such strains have originated?
Although such rare events cannot be studied directly, any number of sources
of the original BSE strain could also explain the discovery of additional
BSE strains in cattle (see the figure). However, it would be worrisome if
spontaneous BSE were really a valid etiology because such a mechanism would
be impossible to prevent--unlike other possible scenarios that could be
controlled by large-scale eradication of TSE-positive animals.

Another way to look at this problem is to examine evidence for possible
spontaneous TSE disease in other animals besides cattle. Spontaneous BSE
would be extremely difficult to detect in cattle, where horizontal spread is
minimal. However, in the case of the sheep TSE disease, scrapie, which
spreads from ewes to lambs at birth as well as between adults, spontaneous
disease should be detectable as new foci of clinical infection. In the early
1950s scrapie was eradicated in both Australia and New Zealand, and the
mainland of both these countries has remained scrapie-free ever since. This
scrapie-free status is not the result of selection of sheep resistant to
scrapie because sheep from New Zealand are as susceptible as their UK
counterparts to experimental scrapie infection (12). These experiments of
man and nature appear to indicate that spontaneous clinical scrapie does not
occur in sheep. Similarly, because CWD is known to spread horizontally, the
lack of CWD in the deer or elk of eastern North America but its presence in
western regions would also argue against a spontaneous disease mechanism.
This is particularly noteworthy in New Zealand, where there are large
numbers of deer and elk farms and yet no evidence of spontaneous CWD. If
spontaneous scrapie does not occur in sheep or deer, this would suggest that
spontaneous forms of BSE and sporadic Creutzfeldt-Jakob disease (sCJD) are
unlikely to be found in cattle or humans. The main caveat to this notion is
that spontaneous disease may arise in some animal species but not others. In
humans, sCJD--which is considered by some researchers to begin by
spontaneous misfolding of the prion protein--usually takes more than 50
years to appear. Thus, in animals with a shorter life-span, such as sheep,
deer, and cattle, an analogous disease mechanism might not have time to
develop.

What can we conclude so far about BSE in North America? Is the BSE detected
in two North American cows sporadic or spontaneous or both? "Sporadic"
pertains to the rarity of disease occurrence. "Spontaneous" pertains to a
possible mechanism of origin of the disease. These are not equivalent terms.
The rarity of BSE in North America qualifies it as a sporadic disease, but
this low incidence does not provide information about cause. For the two
reported North American BSE cases, exposure to contaminated MBM remains the
most likely culprit. However, other mechanisms are still possible, including
cross-infection by sheep with scrapie or cervids with CWD, horizontal
transmission from cattle with endemic BSE, and spontaneous disease in
individual cattle. Based on our understanding of other TSEs, the spontaneous
mechanism is probably the least likely. Thus, "idiopathic" BSE--that is, BSE
of unknown etiology--might be a better term to describe the origin of this
malady.

What does all this imply about testing cattle for BSE in North America?
Current testing in the United States indicates that BSE is rare (one
positive result in 40,000 cattle tested). However, additional testing of
200,000 head of slaughtered cattle over the next 1 to 2 years, as recently
proposed by the U.S. Department of Agriculture (USDA), should tell us the
incidence more precisely. Nevertheless, if any rare cases are detected, we
may still not know their origin. If evidence arises of a focal occurrence of
BSE, we might gain important insight into unexpected sources of
contamination. However, because current tests do not seem to be able to
detect BSE in infected animals less than 30 months of age, even more
extensive testing will not completely guarantee the negative status of
younger animals in the food chain. Therefore, the alternative option of
testing all slaughtered cattle, as implemented in some countries such as
Japan, would appear to provide little additional benefit. This fact has been
acknowledged as the basis for a new agreement between the United States and
Japan aimed at reestablishing the beef trade between the two countries.

One problem with the current U.S. testing program was the announcement a few
months ago of unconfirmed positive BSE tests in two additional North
American animals that were subsequently found to be negative when tested
with the more accurate method of Western blotting. The public release of
information about unconfirmed positive tests detected by the rapid test used
for mass screening may be a good idea in the interest of openness, but it ha
s the potential to create unwarranted anxiety. If unconfirmed positives are
a frequent occurrence, it would seem reasonable to follow a more cautious
approach and wait until confirmatory testing is complete before publicly
announcing the details.

Based on the experience of many European countries, the mainstays of
controlling BSE in cattle and avoiding spread to humans are threefold:
first, eliminate feeding of ruminant tissues to ruminants; second, remove
high-risk cattle tissues from human food; and third, continue to test for
BSE in cattle in order to monitor progress with the elimination of the
disease on a local and national basis. In the next 12 months, after
extensive USDA test results are available, the extent of any possible BSE
spread in the United States will be better documented. But, in fact, the
United States and Canada have already instituted the most important steps to
prevent the spread of cattle BSE in advance of the results--that is, a ban
on feeding ruminant MBM to other ruminants and removal of high-risk tissues
from meat for human consumption. It is hoped that the new data will not
deviate enough from previous predictions to require further measures for
management of this problem. The most important line of defense against any
possible spread of BSE will be to maintain strict vigilance in the
implementation of the current regulations.

References


S. B. Prusiner, Proc. Natl. Acad. Sci. U.S.A 95, 13363 (1998) [Medline].
P. G. Smith, R. Bradley, Br. Med. Bull. 66, 185 (2003) [Medline].
C. Weissmann, A. Aguzzi, Curr. Opin. Neurobiol. 7, 695 (1997) [Medline].
A. F. Hill et al., J. Gen. Virol. 80, 11 (1999) [Medline].
R. Chiesa et al., J. Virol. 77, 7611 (2003) [Medline].
G. Legname et al., Science 305, 673 (2004).
D. Westaway et al., Cell 76, 117 (1994) [Medline].
B. Chesebro, Science 279, 42 (1998).
A. G. Biacabe et al., EMBO Rep. 5, 110 (2004) [Medline].
Y. Yamakawa et al., Jpn. J. Infect. Dis. 56, 221 (2003) [Medline].
C. Casalone et al., Proc. Natl. Acad. Sci. U.S.A. 101, 3065 (2004)
[Medline].
E. F. Houston et al., J. Gen. Virol. 83, 1247 (2002) [Medline].
http://www.sciencemag.org/cgi/content/full/305/5692/1918

TSS



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