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28 January 2003
Transmissible Spongiform Encephalopathies
Susan Haywood, BVSc, PhD,
MRCVS, and David R. Brown, M.Sc, Ph.D.
discuss a re-evaluation of the TSE enigma and explore the role
of environmental factors in prion diseases
Volume 33, number 2
27 January 2003
Just over two decades ago the spongiform encephalopathies,
as they were then known, were confined to a disparate group that
included scrapie in sheep, the rare Creutzfeld-Jakob disease in
man (CJD) and the even more exotic Kuru in a supposedly cannibalistic
tribe in Papua, New Guinea.
All that changed in 1986 when Bovine Spongiform
Encephalopathy (BSE) was identified in UK cattle.
Very soon after, the transmissible spongiform
encephalopathies (TSEs) or prion diseases came to include
transmissible mink encephalopathy (TME), chronic wasting disease
in mule deer (CWD), reports of TSEs in zoo animals and felines
and latterly variant vCJD in young people.
All these are progressively degenerative diseases
of the central nervous system that prove ultimately fatal.
They are characterised by a long incubation period,
failure to elicit an immune response and an aetiology which involves
a hitherto unknown class of infectious agents of remarkable stability
They have in common a pathology that, in addition
to neuronal death and spongiform changes, includes the presence
of amyloid plaques in the CNS.
The TSEs have raised considerable public
concern with respect to the unknown extent of the infection in the
food chain, the possible transmissibility to humans and most particularly
the relationship of BSE to vCJD.
Despite extensive research and an equally wide-ranging
BSE Public Inquiry chaired by Lord Phillips (1),
there is much that is unanswered or mainly speculative and it is
time for a re-evaluation of the collated information, together with
more recent investigations which have an important bearing on the
pathogenesis on this unique class of diseases.
Scrapie, prion hypothesis and interraction with the host genome
Prior to 1986 most research centred on Scrapie.
This disease has been endemic in UK for over 250 years and is found
in most countries except Australia and New Zealand.
As all Vet students know scrapie is characterised
by neuronal vacuolation, reactive astrogliosis and occasional plaque
formation. The scrapie agent was deemed infectious in that it could
be passaged to other hosts, but was biologically unique in its heat
resistant properties, small size and apparent lack of nucleic acid.
As many as 22 strains have been isolated characterised
by differing incubation periods in the natural host and lesion profiles
produced in inoculated experimental mice.
Natural infection is by lateral spread and possibly
maternal transmission, otherwise it can be transmitted by intracerebral
inoculation to goats, mice and hamsters but not cats or mink.
The route of natural infection has been established
as via the gastrointestinal tract to the lymphatic tissues (Peyers
patches) then to spleen and thymus during which passage the scrapie
agent replicates principally within the follicular dendritic cells
of the lymphoid tissues.
The subsequent passage to the brain was thought
to be via the ganglia of the autonomic system - a process taking
The cause has been much disputed, but in 1982
Prusiner (2) showed that the scrapie agent was
a small proteinaceous infectious particle, lacking nucleic acid,
which he named a prion. He later demonstrated that prion protein
PrPC is a component of the normal cell and encoded by the host genome.
The scrapie-associated prion protein PrPSc has
a similar amino acid structure but an altered 3D configuration.
This proposed conformational change conferred on it a resistance
to protease digestion and the ability to convert normal PrPC to
PrPSc, by a form of chain reaction.
The abnormal isoform accumulates in the brain
in insoluble aggregates: a characteristic of prion diseases in general.
Prusiners hypothesis has become known as the protein only
or prion hypothesis, of TSE disease causation.
Genetic factors, however, can affect susceptibility
or otherwise to disease, and by 1986 it was understood that susceptibility
to scrapie was controlled by a complex interaction between host
genes and the particular strain of PrPSc.
The PrP (sinc) gene has been identified in sheep
and found to have polymorphisms at codons 136,154 and 171 which
are disease linked. In Suffolk sheep it has been found
that valine (V), arginine (R) and glutamine (Q) or (VRQ) encoded
at these sites are most susceptible to Scrapie, whilst alanine (A),
arginine (R), arginine (R) or (ARR) are most resistant.
An ARQ associated genotype is also linked with
susceptibility in the Suffolk, but not in the Cheviot, breed in
which the same pattern confers resistance (3).
The underlying rationale of these sometimes conflicting observations
is unclear and indeed Phillips warns that understanding of
these polymorphisms is fundamental to efforts to breed scrapie resistant
BSE not a form of scrapie
Identified in 1986, BSE rapidly spread to affect
UK herds although the incidence was very limited within individual
The source was assumed, on epidemiological grounds,
to be from commercial feed which contained rendered animal protein.
Certainly, cases dramatically declined subsequent to the time when
the ruminant-derived protein ban came into force in
1988, establishing this theory beyond reasonable doubt.
Initially, the infectious prion was thought to
be a modified scrapie prion which had crossed species, but this
was later dismissed by Phillips on the ground that BSE differed
essentially from scrapie in disease-profile, incubation and transmissibility.
The report states with confidence that the
BSE agent is not an unmodified form of scrapie. Rather, it seems
to be a novel TSE agent that arose from a prion mutation in cattle,
sheep or another species in the 1970s or earlier (since
zoo animals had contracted the disease ).
The report goes on to say: The infectious
agent is a post-translationally modified prion protein, a self replicating
protein. Other as yet unknown factors may contribute to the development
of BSE in infected animals.
The source of the supposedly infected feed, however,
was never identified and the disease never reproduced experimentally
in this way. Also the limited nature of the infection, localised
often to just one animal in a herd, was puzzling, the more especially
since this could not be explained by host predisposition, as with
sheep to scrapie, since no variant polymorphisms have been identified
The explanation rests on the unsatisfactory packet
theory of infection whereby single high titre doses were unevenly
dispersed in the feed; but this has never been confirmed and is
at odds with the known high infectivity of PrPSc affected tissues.
Once again, and referred to by Phillips, other
environmental factors may play an aetiological role.
Link between BSE, and vCJD remains to be confirmed
Human prion disease, including CJD, had been recognised
by this time as falling into 3 categories: sporadic (85%), familial
(<15%) as a result of a point mutation in PrP gene and iatrogenic
(<1% due to medical introduction as a result of vaccines and
Epidemiological studies from around the world
had moreover failed to identify a causal link between scrapie and
CJD. With the emergence of BSE a CJD Surveillance Unit was established
with the remit of studying cases of CJD that could have been linked
In 1995, two cases of CJD were reported in young
people. By March 1996 that had risen to 10 cases. Neuropathological
findings revealed the presence of large amyloid plaques in the brains
of these unfortunate people, more akin to Kuru than sporadic CJD,
and which was now renamed variant CJD .
Several studies showed similarities between BSE,
vCJD and TSEs in zoo animals and felines. Another study showed
that in transgenic mice in which the host PrP gene was replaced
by that of human PrP gene (effectively bypassing the species barrier),
when challenged with either BSE or vCJD it showed similar patterns
of disease distinct from sporadic or iatrogenic CJD (4).
This is supportive evidence to the effect that
BSE and vCJD are caused by a similar strain of prion but does not
conclude that vCJD is caused by BSE, as Phillips implies.
Circumstantial evidence linking the consumption
of beefburgers by young people in support of the transinfection
theory, whilst persuasive, has never been proven, in that the putative
infectious burgers have never been identified, nor indeed
fed, to experimental animals.
Groups supposedly more at risk such as farmers,
vets, abattoir workers and butchers have not shown an increased
risk of vCJD.
It is quite surprising that the one experiment
that would confirm a link between BSE and vCJD has not been carried
out. If BSE and vCJD are the same strain of disease and take on
different characteristics dependent on the host organism, then infecting
cows with vCJD should lead to the cows developing BSE.
This would prove BSE and vCJD to be the same disease.
However, those who could have carried out the experiments have classed
them as unethical because of the need to inject human
brain into an animal.
It is incontrovertible that, the experiments that
are the main support for the hypothesis that vCJD and BSE are the
same disease also require that vCJD be injected into the brain of
the putative source host animal.
Until this is performed, hypotheses of the causal
relationship of BSE and other TSEs, including vCJD, remain
conjectural and the role of other, possibly environmental, factors
must be reconsidered.
The prion protein only hypothesis may require modification
The prion hypothesis suggests that an abnormal
isoform of the prion protein PrPSc alone causes the TSEs. This hypothesis
still remains controversial because of the lack of formal proof.
In the UK in particular, many scientists still dont accept
that this protein is the sole cause of the disease.
Indeed manufactured PrPSc failed
to induce prion disease in mice (5). It is clear,
however, that conversion of the normal brain PrPC to the disease
specific PrPSc is necessary for the establishment of the characteristic
neuropathology of the TSEs.
PrP is a copper-binding protein with a role in copper metabolism
On the contrary it is almost universally accepted
that the normal protein, PrPc is a metal binding protein.
PrPc was first suggested to be a copper binding
protein in the early 1990s and confirmed by David Brown and
colleagues in 1997 (6) This has been followed by
close to a 100 publications that have reafffirmed that PrPc is a
copper binding protein.
Further work by Brown and others showed that PrPc
displays SOD-like activity and indeed, it has been proposed that
neurodegeneration in prion disease is a direct consequence of a
failure of neuronal antioxidant activity. PrPc has a role in cellular
copper transport and the sequestration of copper (6).
Early work proposed a link between copper metabolism
and prion disease in that the copper chelator cuprizone causes neuropathological
changes in rodents similar to that seen in experimental scrapie.
In this context it is interesting that the abnormal isoform, PrPSc,
is almost devoid of copper and SOD-like activity is severely reduced.
PrPSc binds manganese in place of copper
A number of researchers have shown that the isoform
of the protein associates with other metals. Brown and co-workers
have shown that manganese can replace copper in native PrPC to
create the isoform PrPSc, which is protease resistant and lacks
antioxidant function, (although there is no evidence that it is
Most particularly, manganese has been shown to
be associated with PrPSc in the brains of both human CJD patients
(7) and mice inoculated with scrapie (8).
There is additional evidence that there are changes
in metal metabolism in the brain with a loss of brain copper (but
an increase in the liver) coincident with the increase of brain
manganese in the CJD patients and the scrapie infected mice.
Additionally, manganese elevations in rodent scrapie
are both presymptomatic and systemic. The cause of this remains
unclear, but it is currently being investigated as to whether elevated
blood manganese can be used as a diagnostic test for TSEs.
All in all, there is a solid and expanding amount
of literature showing that metal imbalance and TSEs are linked.
TSEs may be an environmental/industrial disease
Coming at these diseases from another direction,
Mark Purdey, a farmer from Somerset, has published evidence that
hotspots of TSEs exist in regions of the world where there is environmental
imbalance between copper and manganese.
Farms in Iceland prone to scrapie have soils with
dramatically increased manganese levels. A similar situation exists
in Colorado where deer develop chronic wasting disease (9).
These findings led both Mark Purdey and David
Brown to hypothesise that sporadic TSEs might be a result of animals
becoming exposed to conditions where manganese in their diets is
elevated and copper is deficient.
Manganese and copper are ubiquitous metals and
it is hard to imagine how the changes in these metals might initiate
such diseases. Since the mid 20th Century, however, industrialisation
has been fairly intensive, especially in the ferromanganese industry
and considerable amounts of manganese are present in pollution.
Indeed lead fuel replacements also use manganese.
In Slovakia, there are high levels of both inherited and sporadic
forms of CJD around areas of intense ferromanganese industry. Therefore
there is the potential for new disease to arise as a result of intense
One issue with BSE and vCJD remains unanswered:
why such a high incidence in the UK? One possibility is that the
UK is just the first, and others will follow. BSE that was once
thought to be specific to UK is now Europe wide.
In the UK, the majority of BSE developed as a
result of the feeding of offal from BSE-infected animals back to
other animals. This unfortunately has masked the possibility of
tracing the origins of BSE back to any source.
Similarly, the mobility of humans around the UK
also makes it difficult to trace vCJD back to any factor, especially
not one that might be environmental.
Although the BSE Inquiry clearly stated that alternative
possibilities for the origin of BSE should be investigated and given
support, there has been little support forthcoming, despite the
fact that most of the evidence linking TSEs and manganese has appeared
since that time.
This is in part as a result of the subsequent
Horn report Review of the origin of BSE (10).
This report failed to note that comparing maps of BSE incidence
to a map of manganese hotspots across the UK when the epidemic was
well established was inappropriate, since BSE was clearly spread
at this stage by recycling infected offal.
A more detailed analysis, looking at the location
of the very first cases of where BSE were reported (as viewed on
the DEFRA web page,) and the map provided in the Horn report, indicates
that the original BSE farms lie directly in a manganese hot spot!
Others, however, have not allowed themselves to
be side tracked in this way but concentrated on the scientific evidence.
In particular, authorities in the Environmental Protection Agency
in Colorado have begun investigating the link between manganese,
copper and chronic wasting disease incidence in deer.
This disease was originally thought to be a copper
deficiency disease before the prion hypothesis came to be recognised
and CWD was recognised as a TSE.
Although manganese might not be the cause
it is clear from the biochemical studies that have been carried
out that metals do play a role in the pathogenesis of TSEs. Therefore,
even if manganese is just a risk factor, it is important that this
factor be kept in the equation, because it might just be the key
that unlocks the truth about these diseases.
Finally, the most tantalising question remains:
how does the PrPSc isoform propagate and, even more so, become infectious?
It has been shown that the manganese-bound isoform PrPSc has a configuration
that makes it protease resistant and that this particular conformation
tends to form spontaneous amyloid aggregates.
It is suggested that when this self-propagation
reaches an optimum size, it recruits normal cellular PrPC expression
to convert to PrPSc which then replicates uncontrollably:
a sort of hijacking of the synthetic machinery which then operates
in a runaway fashion, free of normal checks and balances.
It is interesting that metalloprotein chemistry
of this nature (but involving metals other than manganese) is now
being implicated for the analogous plaque formation in Alzheimers
and certain other neurodegenerative conditions. If so, then the
prion diseases are not a biological freak of nature but a subclass
of a category of diseases relating to dysregulation of metalloprotein
chemistry associated with metal imbalance.
1. The BSE Inquiry Report (2000).
2. Prusiner S. (1982). Novel Infectious
Agents Cause Scrapie. Science, 216: 136-144.
3. Hunter, N., Foster, J., Goldman,
W., Stern, M., Hope, J. and Bostock, C. (1996). Natural Scrapie
in a Closed Flock of Cheviot Sheep Occurs Only in Specific PrP Genotypes.
Archives of Virology, 141: 809-824.
4. Hill, A. F., Debruslais, M.,
Joiner, S., Sidle. K. C., Gowand, I., Collinge, J., Doey, L. J.,
Lantos, P. (1997). The same prion strain causes vCJD and BSE. Nature,
5. Hill, A. F., Antoniou, M. and
Collinge, J. (1999). Protease resistant prion protein produced in
vitro lacks detectable infectivity. J. Gen. Virol., 80: 11-14.
6. Brown, D. R. (2001). Copper
and prion disease. Brain Research Bulletin, 55 (2): 165-173.
7. Wong, B. S., Chen, S. G., Colucci,
M., Xie, Z., Pan, T., Liu, T., Li, R., Gambetti, P., Sy M. S. and
Brown, D. R. (2001). Aberrant metal binding by prion protein in
human prion disease. Journal of Neurochemistry, 78 (6): 1400-1408.
8. Thackrey. A. M., Knight, R.,
Haswell, S. J., Bujdoso, R. and Brown, D. R. (2002). Metal imbalance
and compromised antioxidant function are early changes in prion
disease. Biochem J., 362: 253-258.
9. Purdey, M. (2000). Ecosystems
supporting clusters of sporadic TSEs demonstrate excesses
of the radical generating divalent cation manganese and deficiencies
of antioxidant co-factors; Cu, Se, Fe, Zn. Does a foreign
cation substitution at ar PrPs Cu domain initiate TSE? Med.
Hypoth., 54: 278-306.
10. Horn, G. (2001) Review of
the Origin of BSE.
Dr Susan Haywood is a Senior Fellow in the Department of Veterinary
Pathology, University of Liverpool with a Wellcome Trust-funded
research project into copper pathobiology and proteomics in sheep.
Dr David Brown is a Lecturer in biochemistry at the Department
of Biology and Biochemistry, Bath University and BBSRC Senior
Fellow who has been researching prion diseases for the last ten
years. He has a distinguished international reputation in the
prion disease field and is invited to lecture on the subject around
He is also the editor of a forthcoming book on the subject Prion
Diseases and Copper Metabolism to be published soon by Horwood
Further Reading Recommended by Land-Care
Snape, William (2003). Does Benzene Poisoning Cause Scrapie? Land-Care,
(Filed 9 January 2003, www.land-care.org.uk,
here to view).
Ebringer, Alan (2003). Benzene Poisoning Unlikely to Cause Scrapie.
(Filed 27 January 2003, www.land-care.org.uk,
here to view).