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From: TSS (
Date: September 18, 2004 at 11:35 am PST

-------- Original Message --------
Date: Sat, 18 Sep 2004 11:49:29 -0500
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

##################### Bovine Spongiform Encephalopathy #####################

UK Strategy for Research and
Development on Human and Animal
Health Aspects of Transmissible
Spongiform Encephalopathies


Since the announcement in 1996 of a possible link between BSE in cattle
and vCJD in
humans two strategy documents for research and development into
spongiform encephalopathies (TSEs) have been produced by the UK
government. TSE
research has advanced significantly during the past few years, and as
representatives of the UK public funders of TSE research, we considered
it timely to
review the current understanding of these diseases and set out a new
research strategy
that reflected the changes in knowledge.

This document represents the first UK joint human and animal health
strategy for TSE
research and addresses issues that affect livestock, the food chain and
public health. It
highlights the improved co-ordination that has taken place between
departments since the publication of the Phillips Report and reflects
the overlapping
issues of relevance to both human and animal health.

In the production of this document, we have drawn on a wide range of
advice from all of
the UK?s major funders of TSE research and from members of specialist
committees such as SEAC, which are detailed within. We have also
undertaken wide
consultation in the UK and overseas in an attempt to ensure the accuracy
of the
information included. We are particularly indebted to Professor Chris
Bostock (formerly
director of the Institute for Animal Health) for his review of the
current state of the
science, which forms the backbone of this document.

With numbers of BSE cases continuing to fall and vCJD cases apparently
stabilising in the
UK, it would be easy to be complacent. However, the increasing incidence
of chronic
wasting disease in the USA reinforces the concern that TSEs are an
international problem
and one that will need to be monitored carefully for a number of years.
In the UK, there
remain key issues that continue to pose threats to animal and human
health. Not least is
the possibility of a non-symptomatic carrier state or the emergence of
BSE-like illness in
another species such as sheep.

While every attempt is made to reduce exposure to disease it is not
always possible to
eradicate it completely. Disease management is thus often based on risk
assessments of
potential exposure and these are based on the latest scientific results.

This document aims to highlight the major scientific uncertainties
relating to TSEs and
how these will be addressed by the research and development strategies
of the major UK
funders. The necessary experience of managing BSE and producing
scientific evidence
upon which to base control policies has led to the development of an
extensive UK
research base in human and animal health aspects of TSEs.

The UK has a special responsibility to share its expertise in TSEs with
Europe and the rest
of the world to minimise the effects of TSE infection in other countries.

Although considerable progress has been made, further studies are
required to fill in the
many gaps in our basic understanding of TSEs and in our knowledge of how
epidemics can be effectively controlled. It is therefore vital that the
UK research base is
maintained and that a co-ordinated and effective funding strategy is
adopted for new
research. Science develops and inevitably this strategy will require
regular reassessment.
Professor Sir John Pattison
Director of Research
Department of Health

Dr John Bell
Chief Executive
Food Standards Agency

Professor Colin Blakemore FRS
Chief Executive
Medical Research Council

Professor Julia Goodfellow CBE
Chief Executive
Biotechnology and Biological Sciences Research Council

Professor Howard Dalton FRS
Chief Scientific Adviser
Department for Environment, Food and Rural Affairs


Executive Summary

1. Transmissible spongiform encephalopathies (TSEs) have been present in
and animal populations for centuries. Interest in them has intensified
since BSE
became significant in British cattle in the 1990s and especially since
its human
version became a hazard to the UK population.

2. The number of human fatalities from TSEs in the UK is relatively
small, in the low
hundreds, and on this criterion TSEs currently appear to present a
smaller risk to
public health risk than vascular disorders such as heart disease or
strokes, which
are the UK?s biggest killers, or cancers.

3. However, the impact of TSEs has been high and they remain important
for a
number of reasons. There may be human TSEs with longer latency periods than
those that have appeared clinically to date. This could mean a further
cost in
terms of human life and suffering and to the healthcare system, where
considerable resource has already been committed to importing blood
and improving safety measures such as the decontamination of surgical
instruments. Identification of another TSE in another species (e.g. BSE
in sheep)
would affect consumer confidence in British produce, which would add a
burden to a slowly recovering farming and meat industry.

4. The five major UK funders of TSE research each have their own remits,
and approaches. The Department of Health and the Department for
Food and Rural Affairs are government departments with responsibility
for human
and animal health, the environment and the agricultural industry. The Food
Standards Agency is a public body established to protect the public from
hazards. The Medical Research Council and the Biotechnology and Biological
Sciences Research Council are non-departmental public bodies that fund
in human and animal biology and health.

5. These bodies have different but related interests in TSE research.
Although the
research councils pay attention to the applicability of the research
they fund, they
have a strong interest in pursuing excellent science independently of
its possible
use. The three government departments have responsibilities for human and
animal health, and for the environmental and economic ramifications of
TSEs, and
require sound scientific information on which to base their policies.

6. These organisations are currently (2002/03) spending approximately
£37M per
year on TSE research. The programmes supported by the five funders are
coordinated and discussed with other interested organisations, including
UK administrations, the voluntary sector, industry and organisations
outside the
UK, including the European Commission and research groups in the USA.
to this cooperation is the TSE R & D Funders Co-ordination Group.

7. Research on TSEs will continue to be necessary in part because of
their economic
consequences for agriculture, an industry that is already under pressure
from a
variety of sources. The National Scrapie Plan, which involves
eliminating scrapie
from the UK sheep flock by removing sheep with lower levels of genetic
to it, is costly and requires an adequate base of research knowledge.
Furthermore, as a large proportion of the UK population may have been
to BSE-contaminated material, the implications for public health and the
additional burden on the NHS could be severe. Consequently it is
imperative that
accurate estimates of the size of the human epidemic are obtained,
methods to
prevent its spread are continually updated and potential treatments

8. Research in this area, especially that funded by the research
councils, forms part
of the general process of advancing knowledge as well as being of
practical value.
It has been informed by, and in turn informs, advances in our
understanding of
protein and gene science, and knowledge of human and animal
epidemiology. It
illuminates important areas of knowledge such as the strength and nature of
species barriers and infection processes, including species-to-species
and possible
mother-to-offspring transmission.

9. Currently, TSEs are one of the rare disease groups where infection leads
invariably to death. For this reason, possible treatments are a target
for both
human and animal TSE research. These might take the form of drug therapy or
immunological interventions such as vaccines. If developed for the human
population, such interventions would have a high value for patients and
families despite the small number of people affected.

10. TSE research is a growing international field in which the UK has
made a
significant contribution. The UK is likely to be a valued partner in TSE
for other nations and for international organisations. TSE research has
featured in
previous European Commission framework programmes for research and is
to be further funded under the sixth framework programme, which is in
its early
stages at the time of writing. The UK is a leader in TSE science and in
the cross-
agency organisation needed to support the full range of TSE research.

11. The approach to TSE research described in this report includes a
number of short-
term targets. One of the most pressing is the need for a preclinical
diagnostic test
for humans and animals. Also important is work on the safety of medical
instruments, which is needed by the Department of Health and the UK
Health Service. Other health-related research is directed towards the
needs of
agencies such as the National Blood Service, which has an interest in
blood transfusion hazards. Research is also being pursued on methods for
monitoring food for TSE hazards, on animal feed hazards and on animal
practices. This is of interest to Defra and the FSA. Defra has a strong
interest in
research to support the National Scrapie Plan, the possibility of BSE in
sheep, and
in the risk of TSEs in other farmed animals.

12. The aim of this research is to uncover the science of TSEs, in
particular their
nature and means of transmission; to develop countermeasures at many
points in
the food chain; to protect the health of the UK population; and to
engage the
public in the research and its application.


2.5 The challenge of TSE strains
2.5.1 TSE agents are not uniformly infectious and they each cause
disease with different,
predictable and specific characteristics.

2.5.2 The existence of distinct strains was recognised when sources of
sheep scrapie were
serially transmitted to mice. A single inbred line of mice, in which all
individuals make the
same PrPC, can propagate several different strains of scrapie, each with
its own distinctive
incubation period, pattern of damage in the brain and PrPSc
properties42, 43. Strain
variation also occurs in natural scrapie, and the recent discoveries of
unidentified strains, such as 221C44 and Nor9845, suggests that the
spectrum of different
strains may change with time. Since different strains can have very
different patterns of
pathology and distribution of PrPSc, their presence may be missed by the
application of
standard sampling and testing protocols used for surveillance45.
Although a single strain of
BSE appears to be responsible for the vast majority of cases around the
world46, 47,
evidence is emerging that suggests that there may be other strains which
have different
properties when transmitted to mice48 or result in different pathology
or properties of PrPSc
in infected cattle49, 50.

2.5.3 Since different strains can be replicated in an identical PrPC
background the
proteinaceous component of PrPSc produced by each strain must be the
same. Thus, if PrP
is the only component of the infectious agent, the distinguishing
features of strains must
be enciphered in hypothetically different shapes adopted by PrPSc. At
least one of the
properties of strains, the size of the PrP fragment remaining after
protease digestion, can
be transmitted to PrPC during conversion to PrPRes in vitro, which shows
that PrPSc can pass
on elements of its distinctive 3-D structure to PrPC (51). Whether this
structure determines
the properties of a strain, or is a consequence of it, can not be
resolved in this system
because it has not yet proved possible to produce newly formed PrPRes
that is infectious.
PrPSc isolated from brains of hamsters infected with various strains of
TSEs have different
degrees of resistance to unfolding and, by implication, different
structures52. This is
consistent with the idea that the shape of PrPSc is unique to a strain,
but at present there
is no molecular model of what these different conformations might be or
how they could
have such diverse biological effects.

2.5.4 Many strains of TSEs, even those that have been biologically
cloned by repeated
passage within a species, can change their characteristics when
transmitted from one
species to another. This is perhaps not surprising, if the prion
hypothesis is correct, given
that different species have PrPCs with different amino acid sequences.
BSE has been
transmitted to a number of different species either ?naturally?, through
contaminated food,
as with cats, exotic ruminants, and humans, or experimentally, through
injection or
feeding, for example sheep, goats and pigs. Each of these species has a
different PrPC, but
where it has been tested, the strain characteristics of their
BSE-derived TSEs are the same
as for BSE from cattle. This ?stability? of the BSE strain in different
PrP backgrounds
challenges the notion that shape of PrP in PrPSc encodes the properties
of a strain. The
shape that, according to the prion hypothesis, defines the BSE strain
must be independent
of differences, often large, between the amino acid sequence in PrPC.
Alternatively, there
may be one or more other molecules (yet to be identified) that associate
with PrP to
determine the strain properties. Knowing the molecular basis of TSE
strains is, therefore,
at the heart of understanding the nature of the TSE agent.

2.6 The role of the hosts genes in TSEs

2.6.1 A gene, Sip in sheep and Sinc in mice, that controls the
incubation period of TSEs,
has been known for several decades, long before PrP was discovered. The
recognition of
the important role that the prion protein plays in scrapie, its
purification and, later, the
isolation of the gene (Prnp) that encodes it led to the idea that that
Prnp and Sinc or Sip
may be one and the same.

2.6.2 There are two forms of Sinc, s7 which results in mice having a
short incubation
period after infection with ME7 scrapie, and p7 with which mice have a
incubation time. Similarly, mice have two forms, ?a? and ?b?, of Prnp,
which differ only by
their amino acids at coding positions 108 and 189. Sinc s7 mice always
have Prnpa,
whereas Sinc p7 mice have Prnpb. This could mean that Sinc and Prnp are
one and the
same or that they are separate, but closely linked. They were shown to
be identical by
making a transgenic mouse in which only the codons for the two amino
acids of Prnpa at
108 and 189 were changed to those of Prnpb. The mouse had also changed
and become
Sinc p7-like53.

2.6.3 Sheep also have several forms (alleles) of Prnp, some of which are
associated with the incidence of natural scrapie or susceptibility to
experimental scrapie or
BSE. Until 1997 it was a matter of debate whether scrapie was a genetic
disease, arising
as a direct consequence of a sheep carrying a particular form of Prnp,
even though it may
not have been exposed to any ?external? infection. Sheep in Australia
and New Zealand,
where there are no reported cases of scrapie, contain the same
scrapie-associated forms
of Prnp as are found in sheep in the UK, where there is a high incidence
of scrapie,
showing that scrapie is not a genetic disease, but a genetic
susceptibility to infection by an
external scrapie agent54.

2.6.4 Sheep that carry two copies of the Prnp allele that codes for PrPC
with amino acids
valine (V) at position 136, arginine (R) at position 154 and glutamine
(Q) at position 171
are called homozygous VRQ and are the most susceptible to scrapie. Sheep
with two
alleles encoding amino acids alanine (A) at position 136 and arginine
(R) at positions 154
and 171 (homozygous ARR) are the most resistant. With the exception of
an unconfirmed
case in Japan, clinical signs of scrapie have not been reported to occur
naturally in ARR
homozygous sheep. However, as a result of active scrapie surveillance in
Europe, based
on the post mortem application of rapid diagnostic tests, two cases of
scrapie in
homozygous ARR sheep have been diagnosed55. Furthermore, scrapie
surveillance of
sheep in Great Britain identified seven homozygous ARR sheep that tested
positive for
scrapie by the Bio-Rad Platelia ELISA test, but these could not be
confirmed as scrapie-
positive by immunohistochemistry and thus remains unclassified56.
Detection of scrapie
infections in asymptomatic ARR homozygous sheep, which rarely, if ever,
show clinical
signs of scrapie, could indicate that ARR homozygous sheep may be able
to be sub-clinical
carriers of TSE infections (see Section 2.12).

2.6.5 Homozygous ARR sheep are resistant to experimental peripheral
challenge with
scrapie or BSE - that is by a route that does not go directly into the
central nervous
system or brain, for example, via the skin, a vein, orally or via the
peritoneum - but do
succumb to an injection of BSE directly into their brain, after a very
long incubation period
57. Animals with one copy of each of the VRQ and ARR alleles
(heterozygous VRQ/ARR) or
one or two copies of the other Prnp alleles have various levels of
intermediate resistance58.
The relative susceptibility of VRQ and ARQ alleles versus the relative
resistance of the ARR
allele is also found in the in vitro conversion of PrPSen to PrPRec (34)
as well as the relative
infectability in culture of Rov cells expressing the different allelic
forms of PrP59.

2.6.6 The sheep Prnp alleles that affect scrapie (and BSE)
susceptibility have been ranked
and assigned to risk groups for scrapie. It is this that forms the basis
of the National
Scrapie Plan, which seeks to reduce and eventually eliminate Prnp
alleles associated with
susceptibility to TSEs in sheep. Over 500,000 sheep have been genotyped
as part of the
NSP and, while the vast majority fit into the known ?two allele?
genotypes, a small, but
significant, number (0.08 per cent) appear to carry three or more PrP
alleles60. It is not
clear what the genetic basis, origin and significance of these complex
PrP genotypes might

2.6.7 In contrast to sheep, very few Prnp polymorphisms have been found
in cattle, and
those that have been found, including the common allele that codes for
an extra copy of
the octapeptide repeat, are not associated with differential
susceptibility to BSE61-63.
Polymorphic microsatellites have also been identified within the bovine
(and ovine) Prnp64,
but there is no evidence that any of these are linked to differential
susceptibility to BSE.
Humans, on the other hand, have one common polymorphism (alternative
amino acids
methionine or valine at coding position 129), about 50 rare point
mutations in Prnp, of
which about half are associated with various inherited forms of human
prion disease, and
many insertions or deletions in the octapeptide repeat region, several
of which are linked
with inherited disease.

2.6.8 The methionine129/valine129 polymorphism in humans acts in a
similar way to
polymorphisms in sheep and mice. Having both alleles the same
(homozygous) increases
the risk of both sporadic65, 66 and iatrogenic67 CJD. So far all
investigated cases of vCJD
are homozygous for methionine129, indicating that individuals with this
genotype are the
most susceptible to vCJD68. But this does not necessarily mean that
individuals who carry
one or two copies of the valine129 allele will not also be susceptible
to vCJD, albeit with
longer incubation periods.

2.6.9 In addition to its effects on TSE susceptibility the human
polymorphism causes two distinct disease outcomes of a single pathogenic
mutation at
coding position 178; FFI if PrPC contains methionine129 or familial CJD
if it contains valine129
69. It would be interesting to model this common human polymorphism in
transgenic mice
to understand the mechanism of its action.

2.6.10 It is possible that the methionine129 homozygous individuals who
have so far
succumbed to vCJD have in common other, non-Prnp, genes that contribute
to the
shortening of the incubation period. Genetic studies in mice are
revealing several non-
Prnp genes that influence incubation period70-72. As yet their
identities or how they operate
are not known, but they are likely to code for some of the many host
proteins involved in
the replication, transport and effects of the infectious agent. It is
important that these
mouse genes be identified and characterised so that their equivalents
can be assessed for
pathogenic effects in humans and other species.

2.6.11 Susceptibility-linked mutations in Prnp can be mapped to the
molecular structure of
PrP to give pointers to the pathogenic roles played by different parts
of the molecule. The
mutation that is associated with human GSS changes the amino acid
proline to leucine at
position 102, but cannot be mapped to a structure because it is in the
disordered N-
terminal tail. This mutation has been studied in transgenic mice with
mixed results.
Transgenic mice that over-expressed the mutant PrP succumbed to a
neurodegenerative disease and, although little PrPSc was present in the
brains of the
affected mice, the disease was transmissible to other transgenic mice
expressing low
amounts of the same mutant PrP, and to hamsters73. At the time this was
an exciting
result, because it seemed to provide experimental evidence in direct
support of the basic
tenet of the prion hypothesis ? the spontaneous creation of an
infectious form of the prion
protein. Recent attempts to repeat this result using ?gene replacement?
transgenic mice
have not resulted in mice which go down with a spontaneous spongiform
or which have infectious material in their brains74. Compared to their
littermates, however, they have dramatically altered response to a
number of TSE
strains75, and the mutation that they carry extends the incubation
periods of four different
strains of murine scrapie, although the extension varies considerably
between the different
strains76. It is not known how this important mutation exerts its many
effects, but it does
show the importance of the unstructured N-terminal tail of PrP in
affecting the behaviour of
PrP and disease outcome. As noted above, there are many other mutations
with inherited forms of human TSEs. The ability to study them
individually in transgenic
mice or cell culture, and map them on the structures of PrP, provides an
opportunity to
understand how they affect the behaviour of PrPC and result in disease.

2.6.12 The N-terminal tail is an important part of PrPC in its normal
function and in terms
of the pathological consequences of mutations within it. It is not
essential for the
infectiousness of PrPSc, once PrPSc has been formed, since it can be
removed without the
loss of infectivity. Nor is most of it essential for the formation of
PrPSc, although its
presence does accelerate the accumulation of PrPSc (77). Normally the
N-terminal region
contains five tandemly arranged copies of the octapeptide repeat.
Changes in the number
of repeats above or below the normal five copies are often associated
with inherited forms
of human prion disease78. In these cases disease probably results from
mutant PrPC taking
the wrong path through the cell and being accumulated as an incorrectly
folded form
resembling PrPSc, ultimately triggering the death of nerve cells in the

2.6.13 Genes have extensive non-coding regions, parts of which control
their timing and
level of expression. Mutations in control regions will affect how a gene
is expressed. In
general, the more PrPC that is made, the shorter will be the incubation
period for a
particular strain of TSE80-83, so a mutation in the control regions of
Prnp, which alters the
expression of PrPC, could affect the susceptibility of the animal that
carries it. Several
polymorphisms, representing such mutations, have been identified in the
control regions of
the human Prnp. Some have been associated with the occurrence of spCJD
(although not
vCJD or iatrogenic CJD), indicating that altered levels of PrPC
expression may be a risk
factor for the disease84, 85. It is important to understand how the cell
controls PrPC
expression and how mutations or other treatments may affect it, since
shutting down PrPC
expression may have potential as a strategy for treatment (see Section

2.7 Inactivation of TSE infectivity

2.7.1 TSE agents are notoriously difficult to inactivate and are largely
resistant to the
conditions normally used to kill viruses and bacteria. Of all TSE
strains, BSE is amongst
the most resistant, a characteristic perhaps selected by its past need
to have survived
multiple rounds of rendering in the production of meat and bone meal.

2.7.2 Much of the research into TSE inactivation has been pragmatic,
exposing infected
material to harsh physical (heat, pressure) or chemical (acid, alkali,
conditions and measuring the amount of infectivity that survives. From
this have
developed several effective methods86, but understanding why TSEs are
resistant to
inactivation should also give insight to the nature of the causative
agent. Different TSE
strains vary enormously in their resistance to inactivation, which is
consistent with the idea
that strains adopt different PrP conformations. The relative resistance
of a strain to heat is
unaffected by the amino acid sequence of the PrP of the infectious
material87. Thus if heat
resistance is determined by PrP structure, it must, like the other
properties of the BSE
strain in different species, be independent of the sequence of PrP in
PrPSc. There are three
phases to the heat inactivation of a TSE agent in wet conditions. In the
first there is no
loss of infectivity as the temperature rises to a threshold, which
varies widely between
strains, but is characteristic for a strain. The second phase follows
this threshold and
involves rapid loss of some but not all infectivity. The third phase is
prolonged with little
further inactivation88.

2.7.3 The survival of this ?resistant? fraction presents the biggest
challenge to ensuring
the safe disposal of TSE infectivity. The amount of surviving TSE
infectivity can be greatly
influenced by prior conditions, for example drying, that may ?fix? the
infectious agent in a
dehydrated structure that is stable to disruptive physical or chemical
conditions89. This
retention of an infectious state through dehydration is reminiscent of
the survival of
infectivity with unaltered strain properties in material chemically
fixed by formaldehyde for
the preservation of fine biological structure for analysis under the
microscope90 (see also
Section 2.8). The ultimate example of a resistant core is the reported
survival of a small
fraction of infectivity following exposure to dry heat at 6000C91.

2.7.4 The notion that fixed or dehydrated material can be infectious
suggests that, under
some conditions, a ?dead? or ?dried? template can initiate an infection.
If so, complete
inactivation of a TSE will only be achieved under conditions that allow
the inactivating
agent access to the core of the infectious agent. It is important to
determine what the
resistant material is, both to develop effective methods of inactivation
as well as to
understand the chemical and physical forms of this infectious material.


2.11.8 The involvement of the LRS in the uptake and replication of the
scrapie agent
explains the widespread peripheral distribution of PrPSc and infectivity
in many breeds and
Prnp genotypes of scrapie-infected sheep190. Since BSE infection is also
widely distributed
in the peripheral organs and blood of orally dosed sheep191-193 it is
surprising that little
infectivity or PrPSc have been found in the peripheral organs of
BSE-infected cattle, either
in clinical cases from the field or at all stages of an experimental
infection194. Peripheral
infectivity in orally dosed BSE-infected cattle is largely restricted to
the distal ileum, in
keeping with an initial uptake of infectivity from the gut by the
Peyer?s patch. PrPSc has
also been detected in some of the follicles of the Peyer?s patch in
experimentally exposed
animals, but not in those of naturally occurring clinical cases of BSE,
and, sparsely, in the
neurons associated with the distal ileum195. A low level of infectivity
has also been
detected in the tonsil, another lymphoid organ associated with the
alimentary tract, 10
months after experimental oral infection196. This suggests that in
cattle the infection
travels directly from the lymphoid tissue within the Peyer?s patch (and
perhaps tonsil) to
the CNS via peripheral nerves rather than spreading further within the
LRS. It may
therefore be difficult to find a non-CNS based test for BSE in cattle,
but, if at all possible, a
suitable accessible target tissue should be sought to make large scale
surveillance on live
animals a reality. Fundamental to this is the question of what
determines whether a strain
of TSE will replicate widely in the LRS or restrict itself to the
nervous system. This remains
a mystery that needs to be solved. The assumption that TSE infection
only results in loss
of function from damage in the CNS should also be questioned with, for
example, studies
to establish whether TSEs affect the functioning of an infected immune
system in which
they reside and replicate.

2.11.9 As with scrapie and experimental BSE infections in sheep,
infectivity and PrPSc
appear to be widely distributed in peripheral tissues and CNS in vCJD
patients197, 198,
whereas they were thought to be restricted to the CNS in cases of
spCJD199. However, the
application of detection methods with greater sensitivity has identified
the presence of
PrPSc in the spleen and skeletal muscle of about one third of a group of
patients who died
of spCJD in Switzerland between 1996 and 2002. Patients with peripheral
PrPSc also had
longer duration of disease200. In view of the potential for ante mortem
diagnosis, it will be
important to establish, given sufficient sensitivity of test, whether
peripheral PrPSc can be
detected in patients with all forms of CJD.

2.11.10 Exactly how a TSE agent enters the CNS from the LRS is also not
understood, and
there may be several routes. One is likely to be through the nerves that
innervate the
lymphoid organs, either through direct contact with FDCs or indirectly
through other
lymphoid cells. Chemical or immunological removal of the sympathetic
nervous system
delays or prevents scrapie infection, whereas an over-supply of
sympathetic nerves to
lymphoid organs shortens incubation periods201, suggesting that the
supply of nerves to
lymphoid organs can be a bottleneck to an infection gaining entry to the
Furthermore, the distance between FDCs and splenic nerves controls the
rate at which
neuroinvasion occurs, being faster the closer the two cell types are


2.12 The species barrier and the carrier state

2.12.1 The ?species barrier? refers to the observation that it is
usually more difficult to
transmit a TSE between two species than it is within a species.
Transmission to a new
species can result in a low proportion of animals succumbing to disease,
often after long
incubation periods. Other features of the species barrier include a
shortening of the
incubation period for successive transmissions within the new species,
the selection of
variant strains and altered pathogenesis. Cattle BSE transmits readily
to mice, which
allowed them to be used for measuring BSE infectivity, but there is a
significant species
barrier because mice are approximately 500-fold less susceptible to BSE
than cattle221 and
there is significant shortening of the incubation period of BSE between
primary and
secondary transmission in mice44. This species barrier appears to be
removed by the
expression of high levels of bovine PrP in transgenic mice222, although
no direct
comparison of susceptibility with cattle has been reported using the
same preparation of
infectious material, as was the case for the species barrier between
cattle and wild type
mice221. The basis of the cattle-to-mouse species barrier may reside in
the difficulty cattle
BSE has in initiating an infection directly in the mouse?s brain,
needing instead to be
processed through the lymphoid system. Unlike mouse-to-mouse transmissions,
intracerebral inoculation of mice with cattle BSE results in longer
incubation periods than
does intraperitoneal injection90 and SCID mice, which are defective in
peripheral replication
of TSEs, are relatively resistant to cattle BSE, even after
intracerebral injection223.

2.12.2 One of the favoured experimental models of an extreme species
barrier has been
the inability to produce clinical signs of TSE disease in mice following
infection with the
263K or Sc237 strains of hamster scrapie, and it had been assumed by
many that this
represented a complete block to the infection of mice. However, an
absence of clinical
signs does not necessarily mean that the infectious agent has not
replicated and spread
within the host, even causing recognisable lesions within the brain. For
transmission of six different sources of sporadic CJD to four strains of
mice produced no
clinical signs of disease and no significant differences in survival
relative to uninfected
controls, but did result in characteristic TSE-related brain pathology
in the majority of such
mice surviving beyond 500 days224.

2.12.3 The hamster/mouse species barrier and the nature of the resultant
carrier state
have recently been revisited225-227. As expected, transmission of
hamster scrapie to mice
produced no overt disease and no evidence of TSE replication for about a
year, after which
there was active replication and adaptation of new strains capable of
causing disease in
mice, with limited evidence of PrPSc accumulation except after about 600
days. During the
first pass in mice, strains retained their virulence for hamsters, but
after three or four
successive sub-passages in mice, mouse-trophic strains emerged.

2.12.4 Subclinical infection indicative of a carrier state can also be
established after within-
species transmissions. Serially diluted, orally administered, 263K
hamster scrapie resulted
in the detection of PrPSc in some healthy animals which survived to be
culled at the end of
the experiment, some 239 days after the last clinical case. However, the
small difference
between the calculated LD50 (the ?lethal? dose which results in 50
percent of the animals
dying of clinical scrapie) and the ID50 (the dose which results in 50
percent of the animals
showing some sign of infection, which includes both scrapie deaths and
animals showing no clinical signs) indicates that the number of carrier
animals would be
unlikely to exceed greatly the number of clinical cases228.

2.12.5 The potential existence of asymptomatic infected ?carrier?
animals or humans is of
great relevance to the effectiveness of surveillance and control
programmes for BSE,
scrapie and vCJD and needs to be pursued. Lack of detectable PrPSc would
detection by current diagnostic tests and such ?carrier? animals or
humans would
represent hidden reservoirs of infectivity, implying a risk of onward
infection to others
through natural (scrapie) or iatrogenic (human) transmission.


2.13.6 Using these approaches to detect PrPSc in tonsil biopsies,
scrapie has been
diagnosed in live susceptible sheep as early as one third of the way
through an incubation
period and over one and a half years before the onset of clinical
signs243. The lymphoid
tissue that is present in the accessible ?third eyelid? of sheep also
contains PrPSc in scrapie
infected sheep, which can be used as a diagnostic test in scrapie
surveillance244. Lymphoid
tissues from vCJD patients, but not those with sporadic or inherited
forms of CJD, were
found to contain PrPSc (199) and tonsil biopsy is now used as an aid to
positive diagnosis of
vCJD. Recently, small amounts of PrPSc have been found in lymphoid
tissues and muscle
suitable for biopsy in about one third of patients in Switzerland with
spCJD200 and
deposited in the neuroepithelium on the inner surface of the nose245.
These may
eventually provide accessible tissues for confirmatory ante mortem
diagnosis of spCJD.


3.1.2 The challenge of TSE strains In sheep, where prion disease has been established for many
decades, scrapie is
found as distinct strains that have different patterns of pathology,
different resistance to
inactivation and different PrPSc distribution. We need to understand the
significance of
different strains of TSE and why some are capable of producing disease
in some host
species but not in others. Such knowledge will form the basis for
molecular models of
the infectious agent, its propagation and transmission, and may offer
novel therapeutic
targets. Understanding animal strains will inform risk assessment and
thus help establish
proportionate and cost-effective policies. Currently, panels of mice are used to determine strain type, but
this is a long
process, and in the case of scrapie, transmission on first passage to
mice is often
variable and can be unsuccessful. To improve this process, Defra is
funding work that is
applying statistical and modelling methods to analyse mouse bioassay
data. The
sensitivity of strain typing methods may be improved by using lines of
mice that express
sheep and bovine PrP alleles as transgenes. As mouse host genetics play
an important
role in characterising strain types, there is a danger that what is
observed in the mice
(particularly in subsequent passages) will bear little resemblance to
field cases of TSEs.
Some research is addressing this issue by investigating whether scrapie
strains defined in
mice can be distinguished in sheep and whether the strain
characteristics are preserved
after passaging though sheep. Another research priority for Defra is the
development and
characterisation of molecular methods that discriminate between TSE
strains. The relative diversity, in quantitative and qualitative terms,
of scrapie strains
circulating in the UK and their significance in the expression and
incidence of disease is
not known. Defra continues to fund research and surveillance work that
addresses these
issues. There is a concern over whether BSE, if present in sheep, could
be distinguished
from scrapie when present in a single, or in a mixed, infection and this
is also being
addressed. Studies are also in progress to determine the effect of agent
strain on the
distribution of infectivity in tissues by investigating different sheep
breeds and genotypes
that have been naturally infected with scrapie. Recently atypical forms of BSE have been described in cattle but
until the results
from transmission studies in mice are known, it is uncertain whether
these represent
novel strains of BSE. Although the pathology of the BSE strain in cattle
is considered to
have remained stable over time, ongoing studies are investigating the
strain stability of
BSE within UK in comparison with an isolate found in Switzerland. In
recent months
atypical cases of BSE have been reported in other countries (notably
Italy and Japan).
The Veterinary Laboratory Agency has quality control functions and is
both the
Community Reference Laboratory (CRL) and the National Reference
Laboratory for the
UK. The CRL has set up an Expert Group on strain typing, which met for
the first time in
June 2003. This Group is responsible for putting in place protocols for
unusual samples (involving a number of EU Reference Laboratories), as
well as defining
what constitutes an atypical sample. However, diagnostic uncertainties
suggest that
confirmation of apparently different strains of BSE in cattle will be
dependent upon a
better understanding of molecular diagnostic techniques. In humans vCJD, thought to arise from exposure to BSE, is seen
with a different
presentation from sporadic CJD. The MRC funds a programme of work in its
Prion Unit
that is undertaking a molecular and phenotypic analysis of human prion
strains. As part
of this work, it has been shown that infecting two different strains of
inbred mice with the
same BSE isolate can produce two different types of PrPSc. Work is now
ongoing to see
whether these differences will be maintained on passage through the same
mouse line
and thus whether the host genes play a role in strain formation. Both DH
and the MRC
will continue to support studies in this area.




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