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I L S I E u ro p e
Report Series
T RANSMISSIBLE S PONGIFORM E NCEPHALOPATHY
AS A Z OONOTIC D ISEASE
R EPORT
P REPARED UNDER THE RESPONSIBILITY OF THE
ILSI E UROPE E MERGING PATHOGEN TASK F ORCE
W ITH THE E NDORSEMENT OF THE I NTERNATIONAL
F ORUM FOR TSE AND F OOD S AFETY (TAFS)
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About ILSI / ILSI Europe
The International Life Sciences Institute (ILSI) is a nonprofit, worldwide foundation established in 1978 to advance the
understanding of scientific issues relating to nutrition, food safety, toxicology, and the environment. By bringing together
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hereunder are the ILSI Europe Board of Directors and the ILSI Europe Emerging Pathogen Task Force members.
ILSI Europe Board of Directors members
Prof. N-G. Asp, SNF – Swedish Nutrition Foundation (S)
Prof. P.A. Biacs, Ministry of Agriculture and Regional Development (H)
Prof. J.W. Bridges (UK)
Prof. G. Eisenbrand, University of Kaiserslautern (D)
Prof. M.J. Gibney, Institute of European Food Studies (IRL)
Prof. A. Grynberg, INRA (F)
Dr. M.E. Knowles, Coca-Cola (B)
Dr. I. Knudsen, Danish Veterinary and Food Administration (DK)
Prof. R. Kroes (NL)
Dr. G. Malgarini, Ferrero Group (B)
Mr. J.W. Mason, Frito Lay Europe (UK)
Dr. D.J.G. Müller, Procter & Gamble Service GmbH (D)
Dr. J. O’Brien, Danone Vitapole (F)
Dr. L. Serra Majem, Grup de Recerca en Nutrició Comunitaria (E)
Drs. P.J. Sträter, Südzucker AG Mannheim/Ochsenfurt (D)
Prof. V. Tutelyan, National Nutrition Institute (RUS)
Prof. P. van Bladeren, Nestlé Research Center (CH)
Prof. W.M.J. van Gelder, Royal Numico (NL)
Drs. P.M. Verschuren, Unilever Health Institute (NL)
Dr. J. Wills, Masterfoods (UK)
ILSI Europe Emerging Pathogen Task Force member companies
Campina Melkunie BV (NL)
Danone Vitapole (F)
Friesland Coberco Dairy Foods (NL)
Kraft Foods Europe R&D (D)
Masterfoods (UK)
Nestec S.A. (CH)
Parmalat (I)
Unilever Research and Development (UK)
About TAFS
The ultimate goal of the International Forum for TSE and Food Safety (TAFS) is to contribute to the safety of food. Its mission
is to provide an international and independent platform for different sectors of the food chain including industry,
consumers, regulators, and scientists, to openly share, evaluate, and disseminate reliable information concerning the
impact of TSEs on the food chain.
TAFS member companies
Danone, Kraft Foods, Nestlé S.A., McDonald's, Migros, Unilever
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TRANSMISSIBLE SPONGIFORM
ENCEPHALOPATHY AS A
ZOONOTIC DISEASE
WRITTEN BY
PAUL BROWN
WITH CONTRIBUTIONS BY:
RAYMOND BRADLEY
LINDA DETWILER
DOMINIQUE DORMONT
NORA HUNTER
GERALD A.H. WELLS
JOHN WILESMITH
ROBERT WILL
ELIZABETH WILLIAMS
PREPARED UNDER THE RESPONSIBILITY OF THE ILSI EUROPE EMERGING PATHOGEN TASK FORCE
WITH THE ENDORSEMENT OF THE INTERNATIONAL FORUM FOR TSE AND FOOD SAFETY (TAFS)
March 2003
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© 2003 International Life Sciences Institute
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4
THE DISEASE FAMILY
5
THE ILLNESSES
7
Scrapie (sheep and goats)
7
Chronic wasting disease (deer and elk)
7
Bovine spongiform encephalopathy (cattle)
8
Variant Creutzfeldt-Jakob disease (humans)
8
ETIOLOGY AND PATHOGENESIS
11
Etiology
11
Pathogenesis
13
GENETICS AND SUSCEPTIBILITY
14
ORIGINS OF BSE: THE ANIMAL FOOD CHAIN
16
ORIGINS OF vCJD: THE HUMAN FOOD CHAIN
20
CHRONIC WASTING DISEASE
24
EVALUATION OF RISK
26
GOVERNMENT RESPONSES AND THE CURRENT SCENE
30
CONCLUSIONS AND RECOMMENDATIONS
33
REFERENCES
35
ANNEX 1: INFECTIVITY CALCULATION FOR A CATTLE FOOD PRODUCT
44
ANNEX 2: INFECTIVITY CALCULATION FOR A HUMAN FOOD PRODUCT
45
ANNEX 3: GEOGRAPHIC RISK ASSESSMENT FACTORS FOR BSE
46
3
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
INTRODUCTION
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CONTENTS
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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INTRODUCTION
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Although recycling of otherwise wasted carcass tissues makes good sense economically, the
transfer of material from one body to another carries a risk of transferring pathogenic passengers.
In this case, the passenger was an agent with formidable resistance to ordinary methods of
decontamination, including the chemicals and elevated temperatures used in rendering carcasses.
Processing changes that were introduced towards the end of the 1970s evidently were sufficient
to make the difference between destruction and survival of what must have been an irregular
presence of very small concentrations of the infectious agent.
T
ransmissible spongiform encephalopathy (TSE) is a slowly progressive,
uniformly fatal neurodegenerative disease that affects several animal species
as well as humans. The prototype TSE is scrapie, a naturally occurring
disease of sheep and goats that has been recognized in Europe since at least the middle of the 18th
century and was shown to be transmissible in 1936. The most important human TSE is
Creutzfeldt-Jakob disease (CJD), which was first described in the 1920s and shown to be
transmissible in 1968. No direct causal connection between these two diseases has ever been
documented, and the concept of any form of human TSE as a zoonotic disease lay latent in
scientific thinking for decades.
The landscape changed dramatically in 1996 with the suggestion that several cases of a variant
form of CJD (vCJD) in young people in the United Kingdom might have resulted from exposure
to bovine spongiform encephalopathy (BSE, or “mad cow disease”), itself probably the result of
cattle having been exposed to scrapie-infected sheep carcasses rendered to produce meat and
bone meal dietary supplements.
Considering the large-scale human exposure to bovine products and the extraordinary
commercial and public health consequences of BSE as a danger to humans, the suggested BSEvCJD connection was not proposed without a solid epidemiological basis, and it has since been
confirmed by biological and molecular studies.
BSE has shifted the engines of TSE research into high gear, and powered an awesome amount of
industry and government activity, to say nothing of its interest to the general public. This report
places these concerns in the historical context of TSE, and attempts to distill the essence of what
has become an intimidating volume of intermingled fact and fiction surrounding the story of
“mad cow disease” and its consequences to human and animal well-being, with particular
attention to food.
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Scrapie was recognized early on as a contagious disease of sheep that was (probably) not infectious
for humans, as illustrated in the following passage in a German manual of veterinary medicine
published in 1759: “The best solution for the shepherd who notices that one of his animals is
suffering from scrapie is to dispose of it quickly, and slaughter it away from the manorial lands for
consumption by the servants of the nobleman. A shepherd must isolate such an animal from healthy
stock immediately because it is infectious and can cause serious harm to the flock”.
Many mammalian species are susceptible to experimental infection by TSE agents, including
primates, ruminants, ungulates, felines, and laboratory rodents (Table 1). Natural infections have so
far been restricted to sheep and goats (scrapie), and to deer and elk (chronic wasting disease, or
CWD). Sheep have transmitted scrapie in “unnatural infections” to other sheep and goats via
scrapie-contaminated vaccines, and very probably to mink and cattle as a result of the practice of
feeding carcasses to mink, and rendered carcasses to livestock. Recycled BSE-infected carcass tissues
have also transmitted disease to a variety of zoo felines and exotic ungulates.
It is possible that many other species are susceptible to experimental infection, or that they
harbour natural disease, but systematic TSE strain-host species studies are incomplete. If natural
disease were to occur at the same one per million incidence as sporadic CJD in humans,
histological and immunological examination of brains from several hundred thousand members
of each species could be required to determine its presence and such an investigation is unlikely
ever to be carried out.
Figure 1
Known and suspected inter-relationships between animal and human TSEs. TME = transmissible mink
encephalopathy; BSE = bovine spongiform encephalopathy; CWD = chronic wasting disease; CJD =
Creutzfeldt-Jakob disease; GSS = Gerstmann-Sträussler-Scheinker disease; FFI = fatal familial insomnia.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
T
he family of transmissible spongiform encephalopathies (TSEs) encompasses
several entities that have different names as a result of having been first
described before their common pathogenesis was recognized. The proven or
likely interrelationships among members of the disease family are shown in Figure 1. It is important
to understand that all of these diseases are expressions of the same pathological process occurring
under different circumstances in different species, and that they share far more biological similarities
than their different names would suggest.
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THE DISEASE FAMILY
6
Sheep,
goats,
cattle,
deer,
and elk
Cattle (bovids)
includes
domestic cattle
and captive
nyala, gemsbok,
eland, kudu,
oryx spp. and
bison
Pig
Domestic cat
Other captive
felids
including
lion, tiger,
puma,
cheetah,
ocelot, and
Asian golden
cat
Domestic
dog
Mongoose
Mink, ferret,
skunk,
martens and
raccoon
Mink
(farmed)
Weasels
(Mustelids) &
Raccoons
Hamster
(BSE)
Mouse, vole
hamster, rat,
gerbil
guinea pig
*Resulting from dietary exposure or iatrogenic procedures in humans, or from husbandry of domestic or captive animals.
Unsuccessful
Successful
Apes, old/
new world
monkeys &
Microcebe
spp.
Humans
?Lemurs
“Unnatural”
disease*
Sheep and
moufflon
(ovines)
Goats
(caprines)
Deer and
elk (cervids)
Dogs
Rodents
Rabbit
Lagomorphs
Opossum
Marsupials
Chicken,
duck,
goose,
turkey,
raven
Birds
Z OONOTIC D ISEASE
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Experimental
Transmission
Humans
Cats
Ruminants
Non-ruminants
Carnivores
Ungulates
(hooved mammals)
AS A
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Natural disease
Primates
TABLE 1
Host susceptibility to TSE infection
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THE ILLNESSES
Scrapie (sheep and goats)
Pruritus may be so subtle as to go undetected or so dramatic that an animal will rub off most of
its wool, and the areas of wool loss may sometimes be rubbed raw (scrapie acquired its name from
the fact that sheep were observed to scrape themselves against fixed objects). Some sheep will pull
wool from their sides or bite at their legs or exhibit a “nibble reflex” when rubbing themselves or
when scratched by hand over the lumbar area of the back. Affected goats are less likely to rub
against fixed objects, instead scratching vigorously with their hind feet and horns.
Hypersensitivity is another characteristic of scrapie. An affected animal may appear normal if left
undisturbed at rest, but when stimulated by a sudden noise, excessive movement, or the stress of
handling, tremors may appear or the animal may even fall down in a convulsive-like state.
Scrapie-affected sheep (but not goats) also may lose weight despite retaining a normal appetite.
Motor abnormalities often include a high-stepping (trotting) gait of the forelimbs and a “bunny
hop” movement of the back legs. This gait is especially exaggerated when the animal is made to
run. As the disease progresses there may be severe ataxia of the hind limbs, causing the animal to
sway, support its hindquarters against a fence when standing, and have difficulty rising. The
duration of illness typically ranges from one to six months.
Not all animals exhibit all signs of the disease, and there can be enormous variation in the clinical
signs among individual animals. For example, a sheep with severe pruritus may show little if any
incoordination and vice versa. There may also be differences between breeds.
The clinical signs in most cases of scrapie are quite distinct and can be easily recognized.
However, several other conditions should be considered, especially in the early stages of illness,
including lice or mite infestation, rabies and pseudorabies, listeriosis, ovine progressive
pneumonia (maedi/visna), pregnancy toxemia, and poisoning by various chemical and plant
toxins.
Chronic wasting disease (deer and elk)
Affected cervids are invariably older than 17 months of age, and the majority are three to five
years of age. The first signs of disease are usually subtle changes in behaviour that may be
recognizable only by caretakers who are familiar with the individual animals, and may involve
only changes in interactions with handlers or other herd members. Animals may show repetitive
behaviours or periods of somnolence or apparent depression, and carry their heads and ears in a
lowered position. Feed consumption is decreased, and there is a gradual loss of weight and
general condition.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Scrapie is a fatal, degenerative disease that affects the central nervous system of sheep and goats.
It is thought to spread from ewe to offspring and other lambs through contact with the placenta
and placental fluids, although disease symptoms usually do not appear until two to five years
after the animal has been infected, and may not appear until much later. The illness
characteristically begins with a slight change in behaviour; the animal becomes more nervous or
aggressive and may separate itself from the rest of the flock. Some sheep appear to be demented
and have been observed pressing their heads against objects, or “star gazing”.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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As the illness progresses, weight loss becomes increasingly evident, and many animals display
increased drinking and urination, increased salivation with slobbering or drooling, and a
progressive incoordination with stumbling, trembling, and a wide-legged stance. Uncontrollable
regurgitation, hyperexcitability, and fainting are occasionally observed. The clinical course can
vary from a few days to more than a year in unusual cases, with most animals surviving for two
to three months. Death may occur suddenly as a result of swallowing difficulty and aspiration, or
may occur only after a prolonged period of progressive wasting to a terminal emaciated state.
Other conditions that may at times be confused with CWD are internal parasites (especially
meningeal worm infections), locoism, elaeophorosis, brain abscess, pneumonia, chronic
hemorrhagic disease, trauma, toxicity, dental attrition, arthritis, and starvation.
Bovine spongiform encephalopathy (cattle)
BSE infections are thought usually to occur during the first year of life, especially from about three
weeks of age, when milk is replaced with solid food concentrates; the average age at onset of
illness is about five years. The first observable abnormalities may be subtle changes in behaviour
or mental status, such as increased nervousness, apprehension, reluctance to enter doorways, and
an unusual tendency to kick when being milked. Behavioural signs may also include a change in
ear position, teeth grinding and frenzy (the “mad cow”). Initial physical signs include changes in
sensation, posture and gait, especially locomotor ataxia.
Signs of sensory dysfunction include hyperaesthesia, excessive licking of the muzzle and flank,
and head shyness. Ataxia and body tremors are frequent, and changes in posture can be seen in a
wide-based stance, low head carriage, arched back and knuckling (falling and recumbency may
complicate the differential diagnosis). Pruritus, probably of central origin and a distinctive sign in
many scrapie-affected sheep, is not a common feature of BSE. Blindness and circling are unusual.
In addition to neurological signs, there is frequently loss of bodily condition, weight loss, and
reduced milk yield. Appetite and thirst are normally maintained, but rumination is reduced. No
animal shows all of these signs, and none of these signs alone is sufficient to make a diagnosis.
There is little clinical variation by breed, sex, geographical residence, or season.
The illness progresses over a period of days to weeks to become clearly evident to an experienced
clinician, especially if the animal is stressed by unusual restraint, light or sound. Occasionally an
animal in apparent good health suddenly exhibits typical signs after transportation or
unaccustomed and prolonged stress. Clinical signs last two weeks to more than one year but
typically last about two months. Alternative diagnoses can include metabolic disorders, hepatic
encephalopathy, cerebral listeriosis, Haemophilus somnus encephalitis, rabies, cerebrocortical
necrosis, abscesses, tumours, and a range of toxic conditions.
Variant Creutzfeldt-Jakob disease (humans)
Variant Creutzfeldt-Jakob disease (vCJD) is a clinically distinctive form of CJD that affects a much
younger age group than the sporadic form of the disease, which typically appears in adults
between 50 and 70 years of age. The usual onset of vCJD ranges from early adolescence to the
mid-30s, with infections having probably occurred 10 to 15 years earlier. The earliest symptoms
are also distinct from sporadic disease: instead of the memory loss and incoordination that herald
the onset of sporadic CJD, patients with vCJD typically present with psychiatric symptoms such
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The diagnosis of vCJD may be impossible to make in the early stages, particularly if the symptoms
are purely psychiatric. When neurological deficits develop, the diagnosis is now suspected fairly
quickly, and helpful laboratory investigations can be performed, some of which are important
because they exclude other potentially treatable disorders. One non-invasive test has a high
specificity for vCJD: magnetic resonance imaging (MRI) of the brain shows a characteristically
increased signal in the thalamic region in up to 90% of cases.
Figure 2
The pathognomonic vCJD “daisy plaque” consisting of a core of amyloid protein surrounded by vacuolar
“petals”. This unusual plaque morphology is also seen in CWD-infected mule deer and BSE-infected rhesus
monkeys, but not in BSE-infected cattle. Dr. James Ironside, CJD Surveillance Unit, Edinburgh, Scotland.
9
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
After about six months, progressive neurological problems become prominent, usually in the
form of increasing clumsiness, slurring of speech, or an unsteady gait. Involuntary movements of
the limbs, face and body develop, such as sudden jerking (myoclonus), fidgeting (chorea) or
writhing (dystonia) movements. Memory becomes increasingly affected and there is progressive
impairment of thinking processes and increasing physical disability. At the end of the illness
individuals are usually helpless, unable to get out of bed, incontinent and unable to speak. Death
is often due to a pulmonary infection. The average time from the first symptom to death is
14 months, although some individuals survive for nearly three years.
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as depression, anxiety and personality change that often persist for many months before objective
signs of brain dysfunction appear. The first clue that the psychiatric symptoms are caused by an
underlying brain disease is the development of pain in the limbs, face or body, which may be
associated with tingling or numbness. In a minority of individuals these symptoms develop at the
same time as psychiatric symptoms or memory loss.
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Immunostaining of tonsillar biopsy tissue for the presence of pathognomonic misfolded protein
is also a reliable diagnostic method, but the procedure is invasive and entails post-operative pain
and potential bleeding complications. Therefore, tonsillar biopsy (like brain biopsy) is not
recommended unless persuasive therapeutic or public health considerations require a premortem diagnosis. A definitive diagnosis can only be made by post-mortem examination of brain
tissue, which shows the unique presence of many amyloid plaques that stain positive for prion
protein and are surrounded by petals of spongiosis (“daisy plaques”) (Figure 2).
The question has been raised as to whether the distinctive clinical, neuropathological, and
immunohistological features of vCJD identify all patients with BSE infection, and whether
atypical cases of apparently sporadic disease might be caused by BSE. Indeed, recent experiments
with “humanized” transgenic mice that showed a sporadic CJD phenotype after BSE infection
have prompted the question of whether a proportion of human cases with “typical” sporadic CJD
might be the result of BSE infection. However, even if this were true, the proportion must be very
small, because the number of UK cases diagnosed as sporadic CJD has not increased since the
appearance of vCJD in the mid-1990s.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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Etiology
However, all known biological viruses contain nucleic acid, and intensive searches during the past
half century for a disease-specific nucleic acid associated with TSE have proved fruitless. In the
interim, it was discovered that a host-encoded protein (the “prion”) was inseparable from
infectivity and that mutations in its encoding gene on chromosome 20 could cause disease.
Molecular biological studies showed that in the infected host, the soluble and proteinase-sensitive
normal protein was converted into an insoluble and partially proteinase-resistant isoform that
had the characteristics of amyloid1. Although we have now begun to think of TSE as a “misfolded
protein disease” with similarities to other amyloidoses, including Alzheimer’s disease, the unique
and distinguishing feature of TSE is that it alone is transmissible.
It is unnecessary here to recapitulate all of the accumulated evidence showing the critical
importance of the prion protein to the infectious disease process. The major question that
continues to torment scientists is: how is it possible for a protein to encode information that would
be needed to account for different strains of the infectious agent? Even allowing for the fact that
conformational differences in the protein are alone sufficient to distinguish between different
agent strains, how are these differences generated?
Theoretically, a misfolded protein could be formed either directly from its unfolded precursor or
by transformation of a normally folded mature form. The original “protein only” theory
postulated that the misfolding process in which ·-helical domains are converted into a ß-sheet
configuration (Figure 3) results from direct interaction and dimerization of the normal and
misfolded forms, with or without the help of cellular chaperones. Strain diversity of the misfolded
isoform is enciphered within the tertiary structure of the molecule, implying that several stable
abnormal conformations of the protein exist in a given genotype and that any one of these
multiple isoforms can be transmitted between affected hosts of the same or different species.
1. Over the years, “prion” terminology has accumulated a family of acronyms that can be confusing. The
term “PrP” was originally used as an abbreviation for “proteinase-resistant protein”, but later became an
acronym for “prion protein”, and it has been used by different authors to indicate all forms of the protein,
or just the misfolded form. The normal protein has been abbreviated as PrPc (for cellular) or PrPsen
(because it is sensitive to proteinase K). The misfolded protein has been designated as PrPsc (for scrapie),
PrPBSE (for BSE), PrPCJD (for CJD), etc., or PrPres (for proteinase resistance). PrPsen/PrPres is the least
ambiguous pairing, but even this terminology now requires qualification, as ongoing studies are
discovering varying degrees of proteinase resistance of non-infectious intermediate PrP species. For the
present, we prefer to use the terms “normal protein” and “misfolded protein” (the fully misfolded amyloid
form of PrP that is associated with infectivity).
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
The disease agents that cause transmissible spongiform encephalopathy were recognized from the
start as being rather “special” in that, although they behaved in many ways like a virus, they
showed a very long latent period between infection and illness, a correspondingly long duration
of illness, an extraordinary resistance to inactivation, and they could not be linked with any
identifiable morphological structure. They were for many years therefore known as “slow
viruses”, and later as “unconventional viruses”. They can still be considered to be
“unconventional viruses” if we think in terms of minimum criteria: a submicroscopic entity with
the capacity both to replicate and to transmit information, as in computer “viruses”.
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ETIOLOGY AND PATHOGENESIS
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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Figure 3
Schematic structural transformation of the normal (left) to the misfolded (right) prion protein molecule,
showing the partial conversion of α-helices (coils) to ß-sheets (arrow ribbons). The structure of the normal
protein has been confirmed by NMR analysis; the structure of the misfolded protein is speculative.
One modification proposes that the normal isoform is its own chaperone – in other words, that
the unfolded protein is either normally folded in the presence of normal protein, or misfolded in
the presence of the misfolded isoform, avoiding the need for misfolded protein to act directly on
normally folded protein. A further refinement proposes that the protein molecules aggregate by
interaction of the globular region of the protein (amino acids 121–231), which has allosteric and
regulatory control functions. The increase in ß sheet content is due entirely to misfolding of the
flexible tail that starts from the first ß sheet of the normal molecule. This hypothesis also implies
that folding is a primary phenomenon that requires no refolding process.
Another theory invokes a seeding mechanism (nucleation) similar to what is seen in inorganic
crystallizations. In vitro, freshly prepared recombinant protein is monomeric, but over time it
transforms itself into dimeric crystals in which helices are “swapped” and which could be the
basis for the formation of misfolded crystal polymers. Different protein-mineral-protein
interactions within microcellular milieux would account for strain variation.
A major limitation of the prion hypothesis and probably of the other “protein only” alternatives
is our ignorance of the thermodynamics of the process by which a normal protein molecule is
converted to a misfolded isoform. Within the context of protein biochemistry, it is difficult to
imagine that the very limited number of stable conformations of a given monomeric protein could
be sufficient to account for the observed number of strains of the infectious agent. Much more
work on the details of protein misfolding, and especially on the identity of intermediate protein
species, will be necessary to decipher this mystery.
A growing body of data from both cell culture and in vivo experimental models is gradually
advancing our understanding of the intracellular trafficking of the normal protein, and
implicating pathways along which the normal protein may undergo misfolding and accumulation
in neural cells. Recent observations suggest that unglycosylated protein increases during infection
at the expense of glycosylated forms, and that under reducing conditions the unglycosylated
protein is vulnerable to misfolding, which triggers a cellular mechanism of retrograde transport
to the cell cytosol, where the protein accumulates in an aggregated form that resists catabolic
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vagus n.
blood
IV, IP, IM
spleen
splanchnic n.
blood
spinal cord
BRAIN
intestinal
mucosa & LRT
blood
PNS
Neuroinvasive pathways from peripheral sites of TSE infection. The peripheral nervous system (with and
without splenic involvement) has been well documented in experimental models. Hematogenous routes
have been inferred to occur under some circumstances. IV = intravenous; IP = intraperitoneal; IM =
intramuscular; LRT = lymphoreticular tissue; n = nerve; PNS = peripheral nervous system.
degradation by cellular proteasomes. The misfolded species has most of the chemical and
biological properties of the pathological amyloid protein, is toxic to neural cells, and is even
capable of inducing a neurological disease when inoculated into normal mice; however, the
disease is not transmissible from these mice to normal mice.
Pathogenesis
Whatever the final judgment about “prions” as the cause of TSE, there is no argument about the
crucial role of the prion protein in the infectious process, or about its value as a marker of
infectivity. Thus, the pathogenesis of TSE has been investigated using methods that either infer
infectivity from detection of the protein (Western blots or immunohistochemistry) or by direct
measurement of infectivity (bioassay by animal inoculation).
The itinerary of the infectious agent within the body depends on how infection is initiated
(Figure 4). In experimentally induced rodent infections, when the agent is introduced by
parenteral inoculation, neuroinvasion is preceded by a phase of replication within the
lymphoreticular system followed by transport via the splanchnic nerve to the spinal cord. In the
spleen, mature follicular dendritic cells play an important role in replication, and maturation
depends on the presence of B cells. When the agent is given by mouth, infection can bypass the
spleen and proceed directly from the gut to the brainstem via the vagus nerve. Based on findings
of misfolded protein or infectivity in human subjects with CJD, the optic and olfactory tracts have
also been identified as potential portals of entry.
The role of circulating blood in naturally occurring disease remains uncertain. The few reported
disease transmissions from the blood of patients with CJD are far outnumbered by transmission
failures, but blood has been repeatedly shown to be infectious in experimental models of TSE, as
well as in naturally occurring scrapie infections. Even if neuroinvasion occurs via nerves, blood is
the most plausible vehicle by which infectivity in both experimental and natural disease
ultimately finds its way to the many peripheral organs in which it is present.
It is interesting that the clinical onset of illness after infections introduced directly into the brain
(e.g. by contaminated neurosurgical instruments) are indistinguishable from the clinical features
of sporadic CJD, but different from the clinical presentations that follow peripheral routes of
infection (e.g. from contaminated growth hormone injections). This difference suggests that
sporadic CJD may arise within the brain itself, in which case infection would have no
neuroinvasive phase.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
ORAL
blood
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Figure 4
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GENETICS AND SUSCEPTIBILITY
enetic factors play an important role in susceptibility to at least some TSEs.
Indeed, during the 1960s a ferocious argument consumed proponents of
purely genetic versus contagious etiologies of scrapie. Classical genetic
studies defined a hierarchy of susceptibility to scrapie infection according to purebred breeds of
sheep, and clearly distinguished between two varieties of the infecting agent, characterized by
short and long incubation periods.
G
In the 1980s, the discovery of the prion-encoding gene made it possible to correlate the results of
classical genetics with modern molecular genetics, and the short and long incubation period
phenotypes were found to be linked to alternative genotypes of polymorphic codons both in
naturally infected sheep and experimentally infected mice. Extensive further studies of breeds of
sheep with different susceptibilities to infection have not been rewarded with the same degree of
clarity, but it appears that certain allelic combinations of three polymorphic sites within the
encoding gene can be broadly correlated with resistance or susceptibility to clinical disease, and
perhaps to infection (Table 2).
TABLE 2
Molecular genetics of sheep susceptibility or resistance to scrapie
Polymorphic coding combinations in sheep “prion” gene
Codon 136
Codon 154
Codon 171
A
A
A
A
V
R
H
R
R
R
R
Q
H
Q
Q
A = alanine, H = histidine, Q = glutamine, R = arginine, V = valine
Genotypic susceptibility of Suffolk or Cheviot sheep to scrapie
Sheep breed
Genotype
Susceptibility/resistance
Suffolk
ARQ/ARQ
ARQ/ARR
ARR/ARR
Highly susceptible
Occasional occurrence
Resistant
Cheviot
VRQ/VRQ
VRQ/ARQ
VRQ/ARR
ARQ/ARQ
ARQ/ARR
ARR/ARR
Highly susceptible
Highly susceptible
Occasional occurrence
Resistant
Resistant
Resistant
Note: Other breeds of sheep (e.g., Texel) can show still different allelic patterns of susceptibility and
resistance.
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The human gene contains several polymorphic sites, one of which (at codon 129) has been shown
to influence susceptibility to all forms of human TSE. Codon 129 encodes either methionine (M)
or valine (V), and in the general Caucasian population the three alternative genotypes are
approximately distributed in the proportions of 50% MV (heterozygotes), 40% MM, and 10% VV
(homozygotes). The homozygous genotypes are nearly twice as common in human TSE –
whatever the cause – as in the general population, and the homozygous MM genotype is the only
genotype found in more than 100 tested cases of vCJD. Thus, this genotype appears to be a
requirement for susceptibility to infection by BSE, or at least for the expression of disease.
There are two caveats to this statement, however. First, it remains possible that codon 129
heterozygotes have a longer incubation period than do homozygotes, and thus would not
develop symptoms until after the epidemic was well under way (experience with kuru, also
acquired by the oral route of infection, lends support to this idea). Because we do not yet know
where we are in the epidemic curve, we cannot know whether heterozygote cases will appear, or
whether they will continue to be totally resistant to infection. Second, it is possible that some cases
of vCJD might escape detection because instead of having the characteristic vCJD phenotype, they
might look like sporadic disease. This possibility has been a kind of shadowy concern from the
beginning, and it has recently been given substance by studies of BSE in “humanized” transgenic
mice in which two distinct phenotypes were observed: one that was indistinguishable from vCJD
transmissions, and another that resembled transmission of the most common form of sporadic
CJD. However, if a sporadic-like CJD phenotype from BSE infection occurs in humans, it must be
extremely rare, given the fact that the incidence of sporadic disease in the UK has not increased
since the first case of vCJD was identified more than eight years ago.
A final point concerns mutations. It is curious that whereas in humans more than 30 pathogenic
mutations account for some 5–10% of all cases, no pathogenic mutation of any sort has ever been
identified in either sheep with scrapie or in cattle with BSE (the same can tentatively be said about
deer and elk with CWD and mink with TME, but they are less well studied). This apparent genetic
discrepancy between human and animal disease remains entirely unexplained.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Cattle have a different type of genotypic polymorphism, involving the number of octapeptide
repeats – either five or six – in the region of codons 50–91 of the prion-encoding gene. The 6-6
homozygous genotype is present in about 90% of cattle, and the 6-5 heterozygous genotype occurs
in most of the remaining 10% of cattle (the 5-5 genotype is rare). Several studies have failed to
reveal any correlation of genotype with BSE susceptibility.
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These sites are located at codons 136, 154, and 171, and the homozygous allelic combination
conferring the greatest resistance encodes the amino acid trio of alanine, arginine, and arginine
(ARR). The allelic combination that encodes valine, arginine, and glutamine (VRQ) is associated
with susceptibility, both in its homozygous state and in some heterozygous combinations. Some
genotypes are susceptible to experimentally induced scrapie but resistant to natural disease, and
a further complication arises from the range of genotypic complexity found in different breeds of
sheep. However, enough is known to guide breeding programs aimed at producing sheep that are
resistant to the development of clinical disease.
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ORIGINS OF BSE: THE ANIMAL FOOD CHAIN
T
he origin of BSE will probably never be known with certainty, but the
hypothesis of a species-crossing infection from scrapie-affected sheep is more
plausible than the leading alternative hypothesis of a spontaneous case of
BSE in cattle. Both hypotheses enlist the mechanism of contaminated carcass recycling as the
vehicle by which infection was rapidly amplified to an annual level of over 30,000 new cases
within five years of its first appearance in Britain.
Any satisfactory explanation of BSE must answer two questions: Why did BSE begin in the mid1980s, and why did it begin in Britain? Both the sheep and cattle origin hypotheses have the same
answer to the question of timing, which is that changes in the system of rendering carcasses into
meat and bone meal dietary supplements that took place around 1980 permitted infectivity to
survive the process and be recycled.
At least two such changes could have contributed to the survival of infectivity: increased use of
continuous rather than single-batch rendering (which could have created incomplete exposure to
heat), and the elimination of a final tallow extraction step consisting of exposure to hydrocarbon
solvents under steam. It has since been shown experimentally that neither of these changes had a
very great effect, but if the level of infectivity entering the rendering process were near the threshold
of transmissibility, small changes could have made the difference between destruction and survival.
For example, a simple calculation suggests that a maximum level of input carcass infectivity would
have been highly unlikely to exceed 1 log10LD50 (10 mean lethal doses) per gram of tissue, which is
the amount that the tallow extraction step was capable of eliminating (Annex 1).
More convincing than speculation and arithmetic is the fact that the prohibition of ruminant protein
in dietary supplements in 1988 was followed five years later by the start of a downturn in BSE cases,
and five years is the average incubation period between BSE infection and manifest illness (Figure
5). It has been objected that if contaminated feed were the only means of spreading disease, no BSE
infections should have occurred after the ruminant feed ban was fully in effect by the mid-1990s.
How to explain the fact that some cattle born after the ban have come down with the disease?
Although maternal transmission has been proposed, the epidemiological evidence favours a
widespread but very low-level exposure that in all likelihood represents a kind of “Continental
revenge”: the unforeseen importation of contaminated feed from countries to which UK meat and
bone meal had earlier been exported, with the result that the carcasses of newly infected cattle
began to enter their rendering plants, producing feed for both domestic and international
commerce.
Whereas the timing of the epidemic is reasonably well explained by changes in the rendering
process with consequent survival of infectivity in meat and bone meal dietary supplements, the
origin of the contamination is open to discussion: scrapie or the chance occurrence of a de novo case
of BSE?
Accurate figures for the international prevalence of scrapie are not available, but scrapie is at least
as prevalent among sheep in the UK as in most other countries of the world, and the UK has a
comparatively large proportion of sheep to cattle. Thus, a widespread potential source of infection
was already in existence, whereas BSE was unknown until 1986, when the epidemic began. The
argument that unrecognized “spontaneous” cases of BSE could have been occurring in cattle for
decades at the same one per million annual frequency as sporadic cases of CJD in humans, and that
one such case was a “founder” of the BSE epidemic has a formidable obstacle: sporadic BSE cases
cannot have been occurring only in the UK, yet no coincident epidemics occurred in several other
countries (for example, the US) in which rendering processes were changed at about the same time
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Figure 5
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Chronology of the BSE epidemic in the UK.
as in the UK. Moreover, the distribution of early BSE cases in the UK (and subsequently in other
countries that unwittingly imported BSE from the UK) is more consistent with multiple initiation
points than with a single point source of infection. This would favour scrapie as the source of
infection, because scrapie was widespread enough to have entered rendering plants throughout the
UK, whereas it would have required simultaneous cases of sporadic BSE to achieve the same effect.
On balance, the evidence favours scrapie, but it is only fair to point out that the sporadic BSE
hypothesis does perhaps more easily explain two curious features of BSE: strain uniformity and
human pathogenicity. Like most infectious pathogens, the scrapie agent has many experimentally
distinguishable strains. Although rodent models of scrapie provide ample precedent for single
strain selection on passage to different rodent species, a bovine origin of BSE eliminates the need
for this kind of selection process. As for pathogenicity, all evidence to date indicates that scrapie is
not a human pathogen, and if scrapie does not infect humans, why should scrapie passaged
through cattle behave any differently? Precedents exist for a strain of TSE in one species that is
unable to transmit disease to a different species unless it has passed through an intermediate
species; however, because this phenomenon is exceptional, it is not so satisfactory as the
explanation implicit in the BSE hypothesis – a new strain of TSE arising in cattle could as easily as
not be pathogenic for humans.
Cattle were not the only target for dietary supplements: other livestock species, such as sheep,
pigs, and chickens, as well as several kinds of zoo animals, laboratory animals, and pets also
received feed to which meat and bone meal had been added. Examples of disease transmission to
non-livestock species have been reported in ungulates, felines, lemurs in zoos and pet cats. The
spectre of BSE-infected sheep has excited a growing concern, because in the process of “backcrossing” to sheep, the agent might carry its newly acquired ability to infect humans, and at the
moment there is no practical way to distinguish natural scrapie from BSE in sheep. (The mouse
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Figure 6
Worldwide distribution of UK exports of live cattle during the period 1988–1993. Data obtained from the
WHO, courtesy of Dr. Maura Ricketts.
Figure 7
Worldwide distribution of UK exports of meat and bone meal (MBM) during the period 1986–1990. Data
obtained from the WHO, courtesy of Dr. Maura Ricketts.
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TABLE 3
Indigenous cases of BSE arranged by country and date of first occurrence as of 1 January, 2003
Country
1985–89
1990–94
1995–99
United Kingdom
Ireland
10,188
10
136,574
78
32,640
342
10
12
118
France
Portugal
Switzerland
Belgium
Netherlands
Liechtenstein
Luxembourg
Germany
Spain
Denmark
Italy
Slovakia
Japan
Czech Republic
Slovenia
Austria
Finland
Greece
Poland
Israel
2000
2001
2002
Total
1443
149
1202
246
755
333
182,802
1,158
69
362
215
161
149
33
274
110
42
239
86
24
753
719
432
10
6
2
1
9
2
0
0
46
20
0
0
38
24
0
1
103
52
2
2
7
2
1
125
82
6
106
127
2
238
211
9
48
5
3
2
1
1
1
1
38
6
2
2
1
0
0
0
86
11
5
4
2
1
1
1
4
1
4
1
Note: For some countries, numbers are still incomplete for the year 2002; regular updates of global BSE
figures are available on the OIE website: www.oie.int
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Bad as it was, the seriousness of the situation was compounded by the continued exportation of
live cattle and of meat and bone meal dietary supplements to virtually every country in Europe
and a number of non-European countries (Figures 6 and 7). Sometimes, animals sick with BSE
were identified before slaughter and did not result in disease outbreaks. However, in countries
that imported large numbers of cattle and thousands of tons of meat and bone meal, BSE was able
to spread through carcass recycling of both imported and newly infected domestic cattle. By
means of systematic epidemiological surveillance and, more recently, immunological testing of
the brains of slaughtered animals, most European countries have discovered affected animals
within their cattle populations during the past few years, and new countries are added each year.
Fortunately, these secondary outbreaks are mere brushfires compared with the original UK
conflagration (Table 3).
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panel incubation period and lesion topography “signatures” of BSE require over a year to
perform). Pigs are not susceptible to oral BSE infection, and chickens are resistant to infection by
any route of administration. Dogs are also apparently resistant to TSE infection, as several animals
inoculated intracerebrally with human strains of CJD and kuru did not become ill, and in
countries with BSE no dogs have been identified with spongiform encephalopathy, despite the
certainty that some dog food, like cat food, would have included bovine tissue.
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ORIGINS OF vCJD:
THE HUMAN FOOD CHAIN
I
f ideas about the origin of BSE are less secure than those about the vehicle of
infection, vCJD presents the opposite situation of a quite certain origin but a stillspeculative vehicle of infection: dietary exposure to beef products contaminated
with central nervous system tissue.
The fact that unusual cases of spongiform encephalopathy in young people were appearing in a
country still recovering from a vast epidemic of spongiform encephalopathy in cattle was the first
clue to the possibility of a connection between the two diseases. Systematic study of the first 10
cases seen within the space of two years (1994–1996) revealed that they did in fact have a
distinctive form of illness that had not previously been seen in the UK (and was not occurring
anywhere outside the country), and that they had no mutations or recognized environmental
causes (such as contaminated growth hormone) to account for the disease. With this evidence, a
connection between BSE and the human cases was strongly suggested, and it has since been
confirmed by two independent experimental methods.
One method used Western blots to show that the misfolded protein extracted from the brains of
both BSE-affected cattle and vCJD-affected humans had an identical pattern that was different
from the pattern seen in all other TSEs, including sporadic CJD (Figure 8). The other method used
inoculations of a panel of inbred mouse strains to show that brain tissue from cases of BSE and
vCJD produced the same patterns of latency (incubation period) and brain lesion topography,
which also were different from other TSEs, including sporadic CJD and scrapie (Figure 9). These
experimental studies, together with the earlier clinical and epidemiological observations, proved
the link between BSE and vCJD beyond a shadow of a doubt.
Figure 8
Western blots of amyloid protein extracted from the brains of 2 cases of sporadic CJD (type 1 and 2A), and
a case of variant CJD (type 2B). Types 1 and 2 differ in their molecular weights; types 2A and 2B differ in
the relative amounts of each glycoform. This combination of molecular weight and glycoform pattern
distinguishes vCJD from all other TSEs. Courtesy of Dr. Mark Head, CJD Surveillance Unit, Western
General Hospital, Edinburgh.
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Figure 9
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Incubation periods (A) and brain lesion topography (B) in different strains of inbred mice inoculated with
scrapie, BSE, FSE (cat infected with BSE), vCJD, and sporadic CJD. Courtesy of Dr. Moira Bruce,
Neuropathogenesis Unit, BBSRC, Edinburgh.
The source of the human infection was cattle, but by what means was infection acquired? The
answer has come chiefly from a process of elimination, and, plausible though it is, it still lacks the
kind of laboratory evidence that clinched the identification of BSE-infected cattle as the source of
disease. Epidemiological study did not uncover any convincing disease clusters, or point to any
regional peculiarities or vectors that might have linked cattle with BSE to human disease, and
investigation of “high risk” contact groups such as farmers, slaughterhouse workers, or butchers
did not identify any cases of vCJD (although a few cases of sporadic CJD did occur among
workers in these professions).
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Figure 10
Age at onset of illness in all cases of vCJD and sporadic CJD seen in the UK during the period 1994
through 2000.
If physical contact with infected cattle could not be implicated, the next logical link to explore was
exposure to cattle products. This posed an immediate (and continuing) problem because virtually
the entire British population was exposed in one way or another to a huge number of bovinederived products, which left little likelihood of implicating any given product as the cause of
vCJD. Consumption of meat and dairy products, and exposure to products containing tallow or
gelatin (or their derivatives) are universal, and no correlations could be established between vCJD
and exposure to any given product. It was particularly important to investigate the consumption
of brain by patients with vCJD, because the distribution of infectivity in BSE-infected cows is
almost entirely limited to nervous system tissues, but no association was found.
The key lay with slaughterhouse practices and meat preparation, about which the scientific
community was completely naïve. Before the appearance of BSE, vertebral columns were
routinely included in the remains of carcasses from which as much meat as possible had been
manually removed. Spinal cords were usually removed, but cord fragments and paraspinal
ganglia were certain to remain. The truncated carcasses were then subjected to a process of
compression to yield bone fragments (used for gelatin or meat and bone meal) and a paste of
“mechanically recovered meat” (MRM) that was permitted to be added to a variety of packaged
meat products such as hot dogs, sausages, beef patties, luncheon meats, beef stews, etc., in
proportions as high as 30% (but usually in the range of 5–10%). It is now abundantly clear that
central nervous system tissues were entering the human food chain through this “unadvertised”
vehicle, and that they were the most likely cause of human infection.
That said, two very curious questions remain unanswered by any of the hypotheses that have
been advanced to explain the phenomenon of vCJD. The most puzzling question concerns the age
at which disease appears – there is no satisfactory answer as to why vCJD affects such a young
age group (Figure 10). Information about commercial food distribution and consumption, which
might provide the best clues, is almost as unreliable as a dietary history obtained from the relative
of a patient with vCJD. Thus, although comparatively inexpensive products (including school
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Figure 11
cafeteria offerings) that contained MRM have been suspected of being favoured by children and
adolescents, hard evidence is lacking. Alternatively, the preference for youth may simply be a trait
of the infecting strain of BSE – some other infectious diseases also show preferences for one or
another age group – but the word “simply” underscores our ignorance of the cause.
The second curiosity involves the relatively small number of cases (currently about 130) in a British
population of 60 million that has presumably been ubiquitously exposed to potentially
contaminated beef products, and only a handful of cases have appeared in other countries where
BSE has been exported (Figure 11). Have only these few people ingested large enough doses of
infectivity to overcome the species barrier and an inefficient route of infection? It is hard to imagine
that any product would have contained more than a trace of infectivity (Annex 2), and equally hard
to imagine that any high-dose product would only infect a random individual rather than produce
disease clusters, which have not been seen (in particular, no clusters have occurred in families
sharing the same diet). Could it be a question of genetic susceptibility? We know of only one such
example – methionine homozygosity at polymorphic codon 129 of the prion gene – and it cannot
be the sole answer, because nearly 40% of healthy humans have the same genotype.
The most reasonable explanation would seem to be the same phenomenon that operated to cause
the limited outbreak of iatrogenic CJD from contaminated human growth hormone – a kind of
epidemiological Russian roulette. Threshold doses causing rare and randomly distributed cases
were almost surely the explanation for the fact that in the US, for example, only 26 cases of CJD
have occurred in a total treated population of over 7000 hormone recipients, which corresponded
very nicely with the experimental demonstration that CJD transmitted to only one of two
monkeys inoculated with one batch among more than 70 different inoculated batches of growth
hormone. If in fact the dose of a TSE agent is usually below but exquisitely close to the threshold
of transmissibility, only the rare individual will become infected, and this may be conditioned by
numerous factors about which we have little knowledge and less control, as obviously important
as an undiscovered “susceptibility” gene, or as apparently trivial as minor inflammations of the
oral cavity or gastrointestinal tract.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Chronology of vCJD occurrence (French and Italian cases had not visited the UK). Data for 2002 are still
incomplete.
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CHRONIC WASTING DISEASE
C
WD was first identified in the US in the late 1960s in captive mule deer in a
Colorado wildlife research facility, but it was not recognized as a spongiform
encephalopathy until 1978. Interestingly, its neuropathology included the
hallmark abnormality of vCJD (“daisy plaques”) before that disease appeared in the UK. The
known occurrence of CWD remained limited to captive mule deer until 1981, but within a decade
was discovered in free-ranging mule deer, white tail deer, and elk in Colorado and Wyoming.
By 2000, CWD had been identified in both farmed and free-ranging animals in several states near
the original endemic area, as well as in contiguous regions of Canada. Intensified surveillance in
recent years has identified what appears to be an ever-expanding geographic range, thought to
result more from commercial interstate transport of animals than from natural movement: cases
have turned up west of the Continental Divide in Colorado, in Wisconsin, in New Mexico, and
even in South Korea (from elk exported from Canada that have now caused several endemic
cases).
The pathogenesis of CWD has not yet received the same attention as scrapie or BSE, but what is
known is consistent with the general outlines of TSE: early involvement of the lymphoreticular
system, including gut-associated lymphoid tissue, and incubation periods ranging from 15 to 36
months, depending on the species and conditions of infection. Significant differences in the
amount and distribution of abnormal protein in different body tissues have been observed in deer
and elk, and although the protein has not been detected in either muscle or “antler velvet” – two
products consumed by humans – bioassays of these organs have not been undertaken.
Comparatively inefficient misfolding of the normal human protein can be initiated by in vitro
incubation with a misfolded cervid protein “seed”, and under experimental conditions, CWD can
be transmitted to a variety of species, including at least one primate species. Several years ago, in
a still unpublished study, two squirrel monkeys were inoculated intracerebrally with brain
homogenate prepared from a mule deer dying of CWD, and the disease transmitted to both
animals after incubation periods of 31 and 32 months. The clinical features and duration of illness
were not recorded, but neuropathological examinations revealed spongiform change without any
plaque formation. Immunostaining was not performed.
CJD was recently diagnosed in three unusually young patients who had consumed venison, and
although epidemiological and laboratory investigation failed to show a convincing link between
exposure and disease, the conclusion that these patients most likely had sporadic CJD must be
weighed against the fact that we do not know what CWD in humans would look like – it might
look like sporadic CJD, or vCJD, or it might have distinguishing characteristics unlike either form
of disease. The same may of course be said about human infections with scrapie or mink
encephalopathy, but we already know from epidemiological surveillance studies that scrapie does
not cause CJD (its incidence is similar in countries with and without scrapie), and mink tissues do
not enter the human food chain.
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Another potentially dangerous situation would arise if CWD were to find its way into non-cervid
animal species. In particular, if CWD were to be introduced and become endemic in livestock
species such as sheep and cattle, the animal and human food chains could be put at the same kind
of risk as happened with BSE. We know that sheep and cattle can be experimentally infected with
CWD by intracerebral inoculation, and tests are under way to determine whether oral dosing with
CWD brain tissue, or close contact with CWD-infected deer, can transmit disease to cattle.
Although food chain infection would require a series of breakdowns in the system of
precautionary measures already taken to prevent a BSE outbreak, including the banning of most
mammalian protein for use in ruminant feed, the potential for human error is a real and
unpredictable quantity.
25
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Although CWD presents more of problem to individuals (hunters, for example) than to public
health, individual infections could have public health consequences similar to those of vCJD:
apparently healthy individuals harbouring the infection during its incubation period could
possibly transmit disease via cross-contamination of surgical instruments or blood donations, and
after death, if disease is unsuspected, their organs could be harvested for donations. Without the
ability to establish a diagnosis of human CWD infection, or knowledge of the presence or absence
of infectivity in peripheral body tissues and blood, our understanding of the potential for human
risk will continue to depend solely on epidemiological inference.
T RANSMISSIBLE
In brief, CWD is spreading – or is now being recognized in areas distant from its original focus –
and may have the potential to infect humans. It is not known whether CWD exists undetected
outside North America, but the spectre of a US “mad deer” epidemic has excited much political
and media attention, and has brought CWD to the forefront of TSE research. Its unique and
troubling feature is that unlike scrapie, TME, and BSE, it occurs in both captive and wild-ranging
animals, which poses enigmas both for understanding the means by which it is transmitted from
animal to animal, and for devising strategies to prevent its spread. When the disease is diagnosed
in captive animals, herds can be culled or entirely destroyed, but this strategy cannot be used for
animals in the wild.
T RANSMISSIBLE
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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EVALUATION OF RISK
ssessing the risk of transmitting BSE to animals or humans by products of
bovine origin is accomplished by a simple and straightforward set of criteria;
the difficulty lies in the fact that data to interpret the criteria are frequently
incomplete or non-existent.
A
The criteria can be summarized as follows:
1. Geographic source of bovines
2. Tissue source of the bovine products
3. Processing effects on infectivity
4. Route of product administration
The establishment of a “risk hierarchy” according to the geographic origin of source cattle is by
far the most difficult and disputed item in the whole assessment scheme: the criteria used by the
European Commission include: adequacy of surveillance to ensure detection of BSE cases; culling
schemes; importation of live cattle or meat and bone meal from BSE-affected countries; rendering
and other feed preparation practices; and types and implementation of feed bans (Annex 3). Table
4 shows the current placement of all countries evaluated to date; the list continues to be expanded
and updated as data become available. For economic reasons, all countries naturally wish to be
considered as having as low a risk as possible, and some have disagreed with the category in
which they have been placed by the Commission.
Moreover, assessment of risk at the national level does not take into account the possibility that
regions or even particular herds of cattle in a country that has identified BSE within its borders
may be at much lower risk than other regions or herds, or even be risk-free. The reason is not that
TABLE 4
Geographical BSE Risk (GBR): categories of evaluated countries as of 1 January, 2003
GBR level
Presence of one or more cattle clinically or pre-clinically infected with the BSE agent
I
Highly unlikely
Argentina, Australia, Botswana, Brazil, Chile, Costa Rica, El Salvador, Iceland,
Namibia, New Zealand, New Caledonia, Nicaragua, Norway, Panama, Paraguay,
Singapore, Swaziland, Uruguay, Vanuatu
II
Unlikely but not excluded
Canada, Columbia, India, Kenya, Mauritius, Nigeria, Pakistan, Sweden, USA
III
Likely but not confirmed, or confirmed at a lower level
Albania, Andorra, Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Israel, Italy,
Latvia, Lithuania, Luxemburg, Malta, the Netherlands, Poland, Romania, San Marino,
Slovakia, Slovenia, Spain, Switzerland, Turkey
IV
Confirmed, at a higher level
Portugal, UK
Notes: The GBR is a qualitative indicator of the likelihood of the presence of one or more cattle being
infected with BSE, pre-clinically as well as clinically, at a given point in time, in a country. Assessment of
the GBR is based on the assumption that BSE arose in the United Kingdom (UK) and was propagated
through the recycling of bovine tissues into animal feed. Later, the export of infected animals and infected
feed provided the means for the spread of the BSE-agent to other countries where it was again recycled
and propagated via the feed chain. For all countries other than the UK, import of contaminated feed or
infected animals is the only possible initial source of BSE that is taken into account. These GBR national
categories are current as of December 2002; regular updates are available on the EC Food Safety web site:
http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html
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It is fortunate that the two most heavily consumed bovine products – meat and milk – do not
contain any demonstrable infectivity. Homogenates of bovine muscle have not transmitted
disease to intracerebrally inoculated mice and cattle, and milk has not transmitted disease to
intracerebrally inoculated mice, or (even more convincingly) to nursing calves of BSE-affected
mothers. The absence of detectable infectivity in blood means that organs that are not inherently
TABLE 5
The distribution of infectivity in tissues of cattle experimentally infected with BSE
Bioassay in mice
Months after oral exposure
Frontal cortex
Caudal medulla
Spinal cord
Dorsal root ganglia
Trigeminal ganglia
Distal ileum
Bone marrow
2
6
10
14
18
22
26
≥32
-
+
-
+
-
+
-
+
-
nt
-
nt
-
+
+
+
+
+
(+)
nt = not tested. Parentheses indicate a single positive result at 32 months, but negative results at 36 and 40
months after oral exposure. Negative tissues (assayed by intracerebral and intraperitoneal inoculation of
mice): pituitary, dura mater, CSF, peripheral nerves, skeletal muscles and tendon; non-ileum GI tract, liver,
pancreas, salivary glands; spleen, tonsil, lymph nodes, blood clot, buffy coat, serum; kidney, lung, trachea,
heart muscle/valve/ pericardium; testis, prostate, ovary, placenta; skin, fat, bone; milk, urine, feces.
Bioassay in cattle
Months after oral exposure
Caudal medulla/spinal cord
Caudal medulla
Spinal cord
Distal ileum
Tonsil
6
10
18
22
26
32
nt
ot
ot
+
ot
nt
ot
ot
+
+
nt
ot
ot
+
ot
ot
nt
nt
nt
nt
ot
ot
ot
nt
ot
+
nt
nt
ot
nt
nt = not tested; ot = on test (assay in progress and negative to date: survival range as of 1 January, 2003 is
46-59 months after intracerebral inoculation). Tonsil has transmitted to only 1 of 5 assay animals.
Other tissues for which assays are in progress and negative to date (7-33 months after oral exposure): skin,
skeletal muscle, and peripheral nerve; mesenteric, pre-scapular, and popliteal lymph nodes; liver, kidney,
and spleen; tonsil, thymus, bone marrow, and buffy coat.
27
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Evaluation of risk according to the bovine tissue source of the product is on more solid ground,
because experiments have been conducted that define which tissues are infectious and which are
not. Early infectivity bioassays employed strains of mice for tissue inoculations, but the partial
species barrier effect was understood to reduce the assay sensitivity. Shortly thereafter, parallel
bioassays were initiated in cattle, and still preliminary results confirm the earlier results in mice:
as shown in Table 5, they indicate a distribution of infectivity largely limited to nervous system
tissues – and much less widespread than occurs in other TSEs, such as scrapie in sheep and CJD
in humans.
T RANSMISSIBLE
such variability is unappreciated, but rather that it is a practical impossibility to collect reliable
data from which to assess risk in smaller geographic units than entire countries. In special cases,
commercial firms have requested individual risk assessments in order to be permitted to import
and sell bovine products from isolated source herds in countries that would not qualify under the
nation-by-nation risk assessment scheme.
T RANSMISSIBLE
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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TABLE 6
Bovine materials used by humans (not exhaustive)
CONSUMABLES1
NON-CONSUMABLES1
Used directly (or after minimal processing)
Meat on the bone2 (T-bone, ox tail)
Deboned meat
Offals3 (e.g., liver, heart, kidney,
thymus, and brain)
Fat4 (suet)
Bone (soup and broth)
Brain and endocrine powders in some
unregulated “health food” supplements
Bone, heart valves, pericardium,
and trachea used in biologicals
and medical devices
Used after processing
Milk and milk products (e.g., butter,
lactose and casein)
Rennet (chymosin and pepsin derived
from abomasum) used in the
production of cheese, whey and
whey products such as lactose
Bone2 and skin to make gelatin,
gelatin derivatives, and collagen
Meat, including tongue, meat
extracts used as flavouring, and
“mechanically recovered meat”5
Tripe (fore-stomachs)
Tallow6 (used for frying and in
food as shortening) and to
make tallow derivatives7
Fat4 (suet), beef drippings
Blood and blood products
(e.g., hemoglobin to clarify wine)
Intestines (duodenum to rectum)
for natural sausage casings
Milk and milk products (e.g., skim milk, lactose
from whey – used as excipient and stabilizer)
Bone2 and skin to make gelatin
(injectables) and gelatin derivatives (used for
amino acids and blood expanders)
Meat and meat products (used in media to grow
bacteria for vaccines)
Blood products: (e.g., fetal calf serum used in
cell culture growth media for viral vaccines,
stem cells, and recombinant products;
thrombin used as a hemostatic agent)
Tallow6 (e.g., soaps)
Tallow derivatives7 (e.g., buffers, cosmetics, and
suppositories)
Other tissues
Lungs (e.g., surfactant injections)
Intestines (e.g., catgut)
Endocrines (e.g., pancreatic insulin)
Heart, arteries, trachea, tendon, joints used to
prepare collagen, elastin, and various chemicals8
Brain, marrow, and viscera9 used in cosmetics
1 With the advent of BSE, safe sourcing has been practiced by avoiding tissues that are known to harbour
or replicate TSE agents, eliminating ruminant materials, or where there is no satisfactory alternative,
sourcing them from countries, herds and animals with an acceptably low geographical BSE risk
assessment. This is therefore not a table of risk, but rather a reminder of the extent to which humans are
exposed to products of bovine origin.
2 Some bone material, such as skull and vertebral column (excluding the tail) from cattle over certain
ages are classified as specified risk materials (SRM) in the EU.
3 SRM include skull, brain, ganglia, eyes, tonsils, intestines and spinal cord, and in the EU and some
other countries these are compulsorily removed for destruction.
4 Some fat (e.g. mesenteric fat) is classified as SRM in the EU.
5 Production of mechanically recovered meat from ruminant animals in the EU is banned. Elsewhere, its
inclusion in meat products usually does not exceed 5–10% by weight.
6 Tallow (bovine rendered fat) is one of the two major end products from rendering bovine carcass waste.
Tallow is not used in regulated medicines, but can be used in soaps, topical creams, cosmetics, and
suppositories. Tallow from SRM is not permitted for use except under license as a fuel.
7 Tallow derivatives include glycerol, fatty acids and their esters, stearates, polysorbates, and sorbitan
esters. Tallow derivatives are produced from tallow by hydrolysis at temperatures and pressures that have
been shown to sterilize TSE infectivity. They are used in the manufacture of some medicines and
cosmetics and may also be used in plastics (food wrappers).
8 Glucagons (replaced by recombinant technology in some countries), corticosterol,chondroitin sulfate,
vitamin D3, cholesterol, and cholic acid.
9 Liver, thymus, heart, mammary glands, placenta, ovary, and spleen.
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Although the minimum infective dose for zoonotic forms of TSE has not been established, the
route of administration of products for human use certainly plays a role in the determination of
risk. TSE is not easily transmitted; it usually requires some form of tissue penetrating event. Direct
inoculation into the brain is by far the most efficient method of transmitting disease, but apart
from therapy for certain types of neurological disorders (for example, CNS mycoses), this route is
irrelevant to human exposure. Peripheral routes of inoculation are much less efficient: five to ten
times more infectious material is needed to transmit disease by the intravenous than by the
intracerebral route, and other peripheral routes (intramuscular, intraperitoneal, and
subcutaneous) are even less efficient. Experimental transmission has also been accomplished by
contaminating dermal, ocular, and gingival abrasions, although application of infectious material
to unbroken skin does not transmit disease. Although the oral route of infection is at least 100 to
1000 times less efficient than intracerebral inoculation, ingestion is certainly the means by which
non-bovine species became infected with BSE, and it may well be the natural route of contagion
for scrapie and CWD.
How does all of this information bear on the question of risk of individual products? For example,
would ingestion of a frankfurter made with MRM from cattle in Japan be more or less risky than
the application of an anti-aging skin cream made with tallow from cattle in Belgium? How to
compare the risk from a vaccine containing fetal calf serum from Italy with that of a capsule made
from gelatin obtained from cattle in the Ukraine? Has BSE silently crossed back into sheep,
carrying its newly acquired pathogenicity for humans, and should we be concerned about ovinederived consumables? (In contrast to BSE-infected cattle, experimentally infected sheep show the
pathological form of prion protein in lymph nodes and in both the large and small intestines, and
blood transfusions can transmit disease to other sheep).
The answers to these teasing kinds of questions are of course unknown, and merely reinforce the
common sense conclusion that the risk inherent in any foods or other products should be
minimized as much as possible by appropriate selection of animals, avoidance of tissues known
to be infectious, and use of production steps known to eliminate (or at least reduce to an
untransmissible level) any infectivity that might be present in the unprocessed tissue.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
The third issue in assessing risk – the degree to which processing may reduce any infectivity that
might be present in the tissue – requires separate data for each product in question (i.e. validation
testing of infectivity clearance), which are available for only a fraction of the hundreds if not
thousands of products containing, or exposed to, bovine tissues (Table 6). As it happens, the
processing of gelatin (from hides and/or bones) and tallow (from fat), which are the starting
materials for a multitude of products, ranging from pharmaceuticals to foodstuffs to cosmetics,
have been found to reduce infectivity by a much greater level than could ever possibly be present
in source material. The processes by which other tissues, such as pancreas (for insulin), lung (for
surfactant) and stomach (for rennet), are prepared have not as yet been individually validated for
infectivity reduction, but none of these source tissues appears to be infectious. Excellent up-todate risk assessments of various products of bovine origin are available from the Scientific
Steering Committee of the European Commission, an independent body that advises the
Commission on matters related to consumer health and protection (http://europa.eu.
int/comm/food/fs/sc/ssc/outcome_en.html).
T RANSMISSIBLE
infectious are unlikely to be contaminated by “passenger infectivity” in circulating blood. Thus,
the only tissues outside the central nervous system (brain and spinal cord) that pose a risk of
infectivity are the trigeminal and paraspinal ganglia, the distal ileum, and possibly bone marrow
and tonsil. The anatomic isolation of ganglia and of lymphatic elements within the distal ileum
minimizes the risk of cross-contamination of neighbouring tissues, and careful removal of the
tongue similarly minimizes its cross-contamination by tonsillar tissue.
T RANSMISSIBLE
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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GOVERNMENT RESPONSES AND THE
CURRENT SCENE
G
overnments worry when the power of public concern is brought to bear on
issues with which they may be involved in a regulatory capacity, and many
governments have acted to minimize risk from BSE both to animals and to
humans, and to defuse what has at times been a media-induced panic. Government responses
have varied from urgent to desultory, as outlined in Table 7.
The single most important measure taken by the UK was decreed as soon as contaminated meat
and bone meal were proposed as the most likely vehicle of infection: their use in ruminant feed
was prohibited in July 1988. The same prohibition was not instituted by the European Union
until six years later, when it had became painfully obvious that exported BSE was causing
indigenous outbreaks via the same carcass recycling mechanism that had spread the original
epidemic in the UK.
During this same period, numerous import-export regulations were also initiated that had the
effect of preventing further international transfer of infection, and the prohibition of high-risk
tissues and tissue products from entering either the animal or human food chain essentially “finetuned” the earlier, more limited restrictions. Some of these have since been deemed unnecessary
(for example, the short-lived ban on the exportation of bone-in meat) as experimental data have
accumulated to indemnify many products as having a negligible potential for transmitting
disease.
Responses varied from country to country: the US, which had the good fortune to be nearly selfsufficient with respect to breeding animals and producing dietary supplements, imported very
few animals and only one small shipment of feed during the 1980s; it prohibited importation of
bovines and bovine products in 1989. Japan, which imported hundreds of tons of MBM from the
UK and continental Europe during the same period, did not institute any bans or surveillance
programs and learned to its chagrin in 2001 that it was host to some BSE-infected cattle. It seems
self-evident today that any country that imported significant quantities of cattle or MBM from the
UK during the period from 1980 through 1991, when all exports were banned, or from countries
of continental Europe through the end of the century, must consider itself at some risk of having
cattle with BSE.
Despite the issuance by many governments of a package of prohibitions that could reasonably be
expected to eliminate the veterinary and public health risks of BSE, loopholes often exist that
could allow the spread of disease. The situation in the US is illustrative:
•
No rules exist that require the rendering process to be capable of sterilizing BSE infectivity.
Most feed mills render carcasses under conditions that cannot be guaranteed to sterilize.
•
Feed for ruminants and non-ruminants can be processed in the same feed mills, creating
the potential for cross-contamination. Thus, rendered carcasses of deer and elk with
chronic wasting disease could conceivably be fed to cattle.
•
If farmers or ranchers mistakenly or deliberately use non-ruminant feed for ruminants, the
mammalian to ruminant feed ban would be bypassed. Spontaneous TSE occurring in nonmammalian species, if it occurs, would also escape the mammalian to ruminant feed ban.
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Great
Britain2
Ban on ruminant protein in ruminant feed
Ban on export of UK cattle born before July 1988 feed ban
Ban on export of UK cattle >6 months of age
Ban on SBO3 in human (1989) and animal (1990) nutrition
July 1988
Ban on export of SBO and feed containing SBO to EU countries
High-risk waste to be rendered at 133°C/3 bar/20 min
Ban on export from UK of SBO and feed containing SBO
to non-EU countries
Ban on mammalian MBM4 in ruminant feed
Ban on mammalian protein in ruminant feed5
Rendering methods must sterilize BSE
SBO ban broadened to include the bony skull
MRM6 from bovine vertebral column banned and export prohibited
Removal of lymph nodes and visible nervous tissue from UK
bovine meat >30 months exported to EU
Ban on export of all UK cattle and cattle products except milk
Slaughtered/dead cattle >30 months (except certain beef cattle
>42 months) ruled unfit for any use (hides for leather excluded)
Mammalian MBM prohibited from all animal feed/fertilizer
Mammalian MBM and MBM-containing feed recalled
Mammalian waste (except fat) to be rendered at 133°C/3 bar/20 min
BSE cohort cattle in UK ordered slaughtered and destroyed
Replace human plasma and plasma products for use in
the UK with imported sources
Slaughter and destruction of offspring born to BSE-affected
UK cattle after July 1996
Leukodepletion of whole blood donations from UK residents
Ban on cattle and sheep SRM7 throughout the EU
Ban on MRM production from any part of cattle, sheep and goats
Ban on mammalian protein in all livestock feed
Ban on slaughter techniques that could contaminate cattle
carcasses with brain emboli (e.g., pithing or pneumatic stun guns)
Immunologic brain examination on all slaughtered cattle
>30 months of age
1
2
3
4
5
6
7
European
Union2
July 1989
Mar 1990
Nov 1989/
Sept 1990
Sept 1990
Nov 1990
July 1991
June 1994
Nov 1994
Jan 1995
Aug 1995
Dec 1995
Jan 1996
Mar 1996
Mar 1996
Mar/Apr 1996
June 1996
July 1996
Jan 1997
Aug 1998
Jan 1999
July/Nov 1999
Jul 2000
Jan 2001
Jan 2001
Jan 2001
Jan 2001
The most important measures are shown in boldface type.
In Northern Ireland and Scotland, dates of implementation were sometimes different than those shown
for England and Wales; also, individual EC countries often adopted different measures on different
dates.
SBO = Specified bovine offals (brain, spinal cord, thymus, tonsil, spleen, and intestines from cattle
>6 months).
MBM = meat-and-bone meal (high-protein residue produced by rendering).
Some exemptions, e.g., milk, blood, and gelatin.
MRM = mechanically recovered meat (residual meat derived from bones, including vertebral column
with dorsal root ganglia and possibly spinal cord in situ).
SRM = specified risk materials (all tissues shown to be infectious in cattle, sheep, or goats; where
infectivity is limited to animals over a certain age, the ban applies to animals over that age. The
definition of SRM changes as new information is acquired).
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Precautions1
T RANSMISSIBLE
TABLE 7
Principal governmental measures taken to protect human and animal health
T RANSMISSIBLE
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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•
Organs known to be infectious in cattle with BSE (including brain) are not prohibited from
human consumption.
•
Plate waste from restaurants, which could contain bovine brain or paraspinal ganglia in
the uneaten remains of some cuts of meat, could be recycled to cattle in feed produced by
rendering plants.
•
Mechanically separated meat expressed from crushed carcasses can be added to cooked
and uncooked meat products, up to a concentration of 30% by weight. Since 1997, spinal
cord has been removed, but the vertebral column, including the paraspinal ganglia, can
still be processed and used in many products – hot dogs, sausages, canned beef, luncheon
meats, and soups and stews. A recently introduced and much safer process, “advanced
meat recovery”, has not yet completely replaced the crushing method.
•
Glandular dietary supplements containing various animal organ powders, including cattle
brain, were often imported from the UK or countries in continental Europe until the US
Department of Agriculture import ban in 1989. The ban relies on proper labelling of the
shipment and can be abused.
Government regulatory agencies have also addressed the problem of possible “secondary”
disease transmission, that is, the transmission of vCJD from an individual during the unsuspected
pre-clinical phase of disease to other humans via blood and organ donations, or from crosscontamination of instruments used in surgical or other invasive procedures. In particular, blood
and blood products have received ongoing attention in many countries, and donor deferral
policies have been implemented that have become almost arcanely complicated, as more and
more countries report BSE (or vCJD). In the US, for example, deferrals differ for prospective
donors with different length of residence times in the UK and continental Europe, and for military
personnel stationed either north or south of the Alps (“Hannibal’s line”). Consider the plight of a
soldier stationed in Turkey who en route had stayed three weeks at a base in the UK, and also had
vacationed in Switzerland.
The situation with respect to deferral of organ donors is less well defined, depending as it does on
rules adopted by individual organ bank associations. In general, it can be said that these associations
are aware of the potential risk posed by patients with vCJD, and of policies applied to blood donors,
and that they tend to issue rules that are in line with these policies. The situation with respect to
instrument cross-contamination is entirely unregulated, and it is likely that many if not most
hospitals continue to employ standard decontamination procedures, which are insufficient to
guarantee the sterility of instruments used on patients with vCJD (or even sporadic CJD).
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S
It stands to reason, therefore, that the elimination of scrapie could be the single most effective
means by which further outbreaks of TSE could be avoided. As used by epidemiologists, the word
“elimination” implies repeated successes in reducing infection to undetectable levels in various
regions of the world – like putting out brush fires – and is sharply distinguished from the term
“eradication”, which is defined as the total global disappearance of a disease. Examples of
elimination are measles and polio; the only example of eradication is smallpox. While it may
never be possible to eradicate scrapie, its elimination is now a practical goal. This idea is neither
new nor particularly original, but it is sometimes lost from view in the imbroglio of dealing with
the secondary diseases. Nor has it escaped the attention of governmental agencies in some
countries, which have made sporadic efforts to eliminate or at least reduce the burden of disease
among their sheep populations. Alas, the disease has proved more durable than the will of its
antagonists, and all past efforts have eventually languished and disappeared.
Today, however, we have tools that did not exist even 15 years ago, and it would seem more than
timely, in view of recent events, once again to take up the campaign. Immuno-detection of
pathological protein in the third eyelid of sheep has proved to be a very useful method for the
random screening of flocks for the presence of scrapie, and biopsy of the olfactory epithetium may
be an even better index of infection. Equally important is the association of specific prion
genotype with post-exposure risk of developing clinical scrapie, which makes possible the
selective breeding of sheep, so that flocks are eventually composed of the most scrapie-resistant
genotypes. National control and eradication schemes have been developed or are under
development in several countries (the Netherlands, Great Britain, France and the US). In Great
Britain a national voluntary scheme has started with pedigree and pure-bred flocks: breeding
eligibility is limited to rams with the more resistant genotypes, and rams with the most
susceptible genotypes are slaughtered or castrated.
With respect to BSE and vCJD, it is evident that strict compliance with the following three
principles should eventually make these diseases a matter of history:
1.
Identification and destruction of all clinically affected cattle
2.
Prohibition of all mammalian protein for use in ruminant livestock
3.
Prohibition of all high-risk tissues for use by humans
Plugging loopholes to these rules (when they exist or are identified) will accomplish the task more
quickly, but compliance with the basic rules is the more important issue, and experience shows
that compliance is never perfect. Also, a continued awareness of the unlikely but still possible
vertical or horizontal BSE transmission within herds, and of the backcrossing of BSE to sheep (or
its spread to other species) is needed, but it is made difficult by the absence of any practical test
to distinguish BSE from scrapie. Efforts to develop such a test should receive high priority.
33
SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
crapie, recognized since at least the 18th century to be a contagious disease of
sheep, may lie at the root of all other forms of animal TSEs. It has certainly been
the cause of at least some outbreaks of TME in mink, it was probably the cause
of BSE in cattle (in turn spreading TSE to other species by exposure to contaminated recycled
livestock feed), and it may be the cause of CWD in deer and elk.
T RANSMISSIBLE
CONCLUSIONS AND RECOMMENDATIONS
T RANSMISSIBLE
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Continuing attention also needs to be given to the possibility of secondary vCJD infections from
blood or tissue donations and surgical procedures. Donor deferral policies should be tailored to
estimated national risks of vCJD infection or, where vCJD has not occurred, to estimated national
risks of BSE infection, and should be regularly reviewed and revised as new data accumulate. In
particular, as risk diminishes, deferral criteria should be relaxed in a timely manner. Prevention of
secondary cases from surgical cross-contamination is easier in principle and more difficult in
practice. Effective sterilization procedures are well defined (exposure to NaOH and autoclaving
at 134°C) but need to be advertised and possibly mandated by national infection control
organizations.
Research priorities should include efforts to develop a rapid and reliable laboratory method to
distinguish different strains of TSE irrespective of the affected species, to develop an
immunological screening test to detect preclinical TSE infection, and to explore new technologies
for reducing infectivity in processed meat products (for example, heating under high pressure has
recently been shown to conserve the quality of processed meat while substantially reducing TSE
infectivity). The whole question of a spreading danger from CWD needs the same kind of urgent
attention that was given to BSE when it first appeared, including knowledge of its host range, its
possible occurrence outside North America, and its characteristics in experimentally infected
primates (assuming human infection remains unidentified). It could be argued that unrecognized
TSE in other animal species should also be systematically investigated, although the huge
investment of resources needed for such studies might be better spent on more immediate
concerns. On a more fundamental note, molecular genetic studies might be profitably directed at
demystifying the unique association of human susceptibility to BSE with the homozygous
methionine codon 129 genotype, and renewed attention paid to the carbohydrate moieties of the
prion protein, which appear to constitute the principle biochemical distinction between the
BSE/vCJD and other strains of TSE.
An important practical concern is the potential risk to travellers and temporary residents of
countries in which BSE has already occurred or is thought likely to occur but be unrecognized. In
view of the delays of many nations to implement precautionary measures and surveillance
programs to detect disease, it would probably be prudent for travellers to countries that imported
significant quantities of cattle or feed from Europe to avoid products that contain processed meat
for at least the next few years.
Finally, a difficult issue must be addressed. Does the risk of BSE (or other as yet undetected types
of animal TSE) warrant the permanent elimination of animal protein from livestock feed? The
rendering industry as presently constituted would disappear, and the incineration and landfill
industries would expand in conjunction with the production of nutrition crops such as soybeans.
Would the increased incineration and landfill disposal have adverse environmental effects?
Would plant protein be as effective as animal protein, and would its increased production result
in a shortage of arable land for other purposes? Is the public prepared to pay more for meat or eat
only as much as can be produced from range-fed animals? And would such a policy compromise
the already marginal status of animal and human nutrition in developing countries? These are
questions that must be decided, not by scientists and government regulators, but in national
debates by society as a whole.
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Ferguson, N.M., Ghani, A.C., Donnelly, C.A., Hagenaars, T.J., and Anderson, R.M. (2002).
Estimating the human health risk from possible BSE infection of the British sheep flock. Nature,
415:420-424.
Foster, J.D., Hope, J., McConnell, I., Bruce, M., and Fraser, H. (1994). Transmission of bovine
spongiform encephalopathy to sheep, goats, and mice. Annals of the New York Academy of Science,
724:300-303.
Foster, J.D., Parnham, D.W., Hunter, N., and Bruce, M. (2001). Distribution of the prion protein in
sheep terminally affected with BSE following experimental oral transmission. Journal of General
Virology, 82:2319-2326.
Ghani, A.C., Ferguson, N.M., Donnelly, C.A., and Ferguson, R.M. (2000). Predicted vCJD mortality
in Great Britain. Nature, 406:583-584.
Hilton, D.A., Fathers, E., Edwards, P., Ironside, J.W., and Zajicek, J. (1998). Prion immunoreactivity
in appendix before clinical onset of variant Creutzfeldt-Jakob disease. Lancet, 352:703-704.
Hunter, N., Foster, J., Chong, A., et al. (2002). Transmission of prion diseases by blood transfusion.
Journal of General Virology, 83:2897-2905.
Hunter, N., Foster, J., Chong, A., et al. (2002). Transmission of prion diseases by blood transfusion.
Journal of General Virology, 83:2897-2905.
Kao, R.R., Gravenor, M.B., Baylis, M., et al. (2002). The potential size and duration of an epidemic
of bovine spongiform encephalopathy in British sheep. Science, 295:332-335.
Kimberlin, R.H. (1996). Bovine spongiform encephalopathy (BSE) and public health: some
problems and solutions in assessing the risks. In: Transmissible Subacute Spongiform
Encephalopathies: Prion diseases, Court, L. and Dodet, B., editors. Elsevier, Amsterdam, 487-502.
Lasmézas, C.I., Fournier, J.-G., Nouvel, V., et al. (2001). Adaptation of the bovine spongiform
encephalopathy agent to primates and comparison with Creutzfeldt-Jakob disease: implications
for human health. Proceedings of the National Academy of Science (USA), 98:4142-4147.
Race, R., Raines, A., Raymond, G.J., Caughey, B., and Chesebro, B. (2001). Long-term subclinical
carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine
spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans. Journal of Virology,
75:10106-10112.
Scientific Steering Committee of the European Commission for Food Safety.
http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html
Sources of BSE infectivity. Project MO3108 for the Food Standards Agency, London, October 2002.
www.foodstandards.gov.uk
Valleron, A.-J., Boelle, P.-Y., Will, R., and Cesbron, J.-Y. (2001). Estimation of epidemic size and
incubation time based on age characteristics of vCJD in the United Kingdom. Science, 294:17261728.
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Brown, P., Will, R.G., Bradley, R., Asher, D.L., and Detwiler, L. (2001). Bovine spongiform
encephalopathy and variant Creutzfeldt-Jakob Disease: background, evolution, and current
concerns. Emerging Infectious Diseases, 7:6-16.
WHO Health Topics.
http://www.who.int/health_topics/en
WHO Regional Office for Europe. Information on Transmissible Spongiform Encephalopathy (TSE).
http://www.euro.who.int/eprise/main/WHO/Progs/FOS/MainActs/20010913_1
The European and Allied Countries Collaborative Study Group of CJD (Euro CJD) plus the
Extended Collaborative Study Group of CJD (NEUROCJD).
http://www.eurocjd.ed.ac.uk
The UK Creutzfeldt-Jakob disease surveillance unit.
http://www.cjd.ed.ac.uk
OIE (World Organization for Animal Health).
http://www.oie.int
Department for Environment, Food, and Rural Affairs (DEFRA). BSE.
http://www.defra.gov.uk/animalh/bse/index.html
The European Commission (EC): Food safety: Bovine Spongiform Encephalopathy (BSE).
http://europa.eu.int/comm/dgs/health_consumer/index_en.htm
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Scientific Steering Committee of the European Commission for Food Safety.
http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html
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Conclusions and Recommendations
Brown, P., Cervenáková, L., and Diringer, H. (2001). Blood infectivity and the prospects for a
diagnostic screening test in Creutzfeldt-Jakob disease. Journal of Laboratory and Clinical Medicine,
137:5-13.
Brown, P., Meyer, R., Cardone, F., and Pocchiari, M. (2003). Ultra-high pressure inactivation of TSE
(prion) infectivity in processed meat: a practical method to prevent human infection. Proc. Natl.
Acad. Sci (USA), in press.
Detwiler, L., and Baylis, M. (2003). The epidemiology of scrapie. In: Risk Analysis of BSE and
TSEs: Update on BSE and Use of Alternatives to MBM as Protein Supplements. Revue Scientifique
et Technique. Office International des Epizooties (OIE), Paris, 2003, in press.
O'Rourke, K.I. (2001). Ovine scrapie. New tools for control of an old disease. Vet Clin North Am
Food Anim Pract, 17:283-300.
O'Rourke, K.I., Duncan, J.V., Logan, J.R., et al. (2002). Active surveillance for scrapie by third eyelid
biopsy and genetic susceptibility testing of flocks of sheep in Wyoming. Clinical and Diagnostic
Laboratory Immunology, 9:966-971.
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Zanusso, G., Ferrari, S., Cardone, F., et al. (2003) Detection of pathologic prion protein in the
olfactory epithelium in sporadic Creutzfeldt-Jakob disease. New England Journal of Medicine, 348:
711-719.
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The titer of infectivity in sheep brain/spinal cord (CNS) is ~ 5 log10LD50/g, as bioassayed by
intracerebral inoculation of mice (mouse intracerebral LD50).
The weight ratio of CNS to carcass ranges from 1:100 to 1:400; if we use the lower ratio (10-2) in
order to approximate the additional small infectivity contribution from peripheral tissues, the
total carcass infectivity is ~ 3 log10 LD50/g.
A typical proportion of ovine to total tissue from all animal species in a batch of carcasses (in
the UK) is ~ 10-1, reducing the input infectivity to ~ 2 log10 LD50/g. Exposure to steam heat
during processing would reduce infectivity by at least another 1 log10LD50/g, such that any
remaining infectivity in the heated carcass mass would not have exceeded 1 log10LD50/g.
The weight ratio of carcass to meat and bone meal (MBM) is ~ 5:1, and if all infectivity
segregates with MBM, the amount of infectivity in 5 g of heated carcass tissue
(≤1 log10LD50) would be present in 1 g of MBM.
A final tallow extraction step would further reduce infectivity by about 1 log10LD50/g, yielding
an MBM infectivity level hovering close to zero, which because of the comparative inefficiency
of oral infection would have very little chance of transmitting disease.
Elimination of the tallow extraction step would have preserved the ≤1 log10LD5/g level of
infectivity in MBM. A calf consumes ~ 2 kg of feed per day, of which ~ 4.5% (90g) consists of
MBM. It follows that 90g x ≤1 log10LD50 ≤ 900 (mouse intracerebral) LD50, = infective dose that
might have been consumed by a calf on any given day, assuming the MBM had come from a
batch of carcasses containing an infected sheep (the estimated overall incidence of scrapieinfected sheep in the UK is about 1%).
The point of this arithmetical exercise is not so much to establish exact limits of infectivity before
and after rendering as to illustrate the fact that at infectivity levels near the threshold of
transmissibility, small reduction effects – usually dismissed as insignificant – may have large
consequences.
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Calculation of the amount of TSE infectivity that might have been present in meat and bone
meal before and after elimination of a final tallow extraction step during the rendering process
(ca. 1980)
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ANNEX 1
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
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ANNEX 2
Calculation of the possible amount of BSE infectivity in a product containing mechanically
recovered meat (MRM)
MRM is produced from carcasses from which heads, limbs, and offals have been removed,
consisting of shoulder girdle/ribs/vertebral column/pelvic girdle skeletons with associated
untrimmed muscle, fat, and connective tissue. The only infectious tissue that would invariably
have been present was paraspinal ganglia embedded within the vertebral column.
The titer of infectivity in BSE tissues has been measured only in brain, which has a titer of
6 log10LD50/g, as assayed by intracerebral inoculation of cattle. Thus, ganglia infectivity must
be estimated by analogy with scrapie. In scrapie-infected sheep, spinal cord infectivity is ~ 1 log
lower, and sciatic nerve ~ 3 logs lower, than brain. Assume that the titer of ganglia is
intermediate between these two neural tissues, or ~ 2 logs lower than brain, giving a BSE
ganglia titer of ~ 4 log10LD50/g.
The total weight of paraspinal ganglia in adult cattle is ~ 30 g, and thus the total amount of
infectivity in the ganglia is 30 x 4 log10LD50 = 3 x 105 LD50.
MRM is produced in batches of ~ 5 tons (= 45 x 105 g). Assuming one BSE carcass
is included in the carcasses used to produce a batch of MRM, the concentration of infectivity in
the batch would be 3 x 105 LD50/45 x 105 = 3/45 = 0.07 LD50/g.
An average hot dog weighs 2 oz (60 g) and may contain up to 10% MRM. The amount of
infectivity in such a hot dog would be 0.07 LD50/g x 60 g x 10% = 0.42 (intracerebral) LD50, an
amount unlikely to transmit disease by the oral route, but consumption of several hot dogs over
a day or two could increase the chances of transmission.
However entertaining it may be to play with numbers in this way, the fact is that the conclusions
to be drawn from this exercise are even more fragile than those from estimating infectivity in meat
and bone meal, because:
1. The estimate of infectivity in paraspinal ganglia from BSE cattle is a hybrid of incomplete data
from BSE–infected cattle and scrapie-infected sheep and, although reasonable, could be wrong
by at least a factor of 10 in either direction.
2. If spinal cord had not been removed from the vertebral column, the amount of infectivity in the
resulting batch of MRM would increase at least 10-fold (spinal cord weight being about 300 g,
and possibly having a higher titer than ganglia).
3. The magnitude of a bovine-human species barrier (if any) is entirely unknown.
4. The efficiencies of oral versus intracerebral infection in humans is also unknown, but by
analogy with scrapie would probably be at least 1:100, and still incomplete oral dosing studies
in cattle suggest a ratio as high as 1:100,000.
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Factors considered in assessing the geographic BSE risk (GBR)
Results of BSE surveillance:
- Number of cattle, by origin (domestic/imported), type (beef/dairy), age, method used to
confirm the diagnosis, and reason the animal was examined (central nervous system
symptoms, BSE suspect, BSE-related culling, other)
- Incidence of reported BSE cases by year and birth cohort
BSE-related culling
- Culling schemes, date of introduction, and criteria used to cull
- Information on animals already culled in the context of BSE
Import of Cattle and Meat and Bone Meal (MBM)
- Imports of live cattle and/or MBM from UK and other BSE-affected countries
- Information that could influence the risk of imports to carry the BSE agent (BSE status of
the herds of origin of imported cattle, precise definition of the imported animal protein,
etc.)
- Main imports of live cattle and/or MBM from other countries
- Use made of the imported cattle or MBM
Feeding
- Domestic production of MBM and use of MBM (domestic and imported)
- Domestic production of composite animal feed and its use
- Potential for cross-contamination of feed; measures to reduce and control it, results of the
controls
MBM bans
- Dates of introduction and scope (type of animal protein banned for use in feed in different
species; exceptions, etc.)
- Measures taken to ensure compliance
- Methods and results of compliance control
Specified Risk Material (SRM) bans
- Dates of introduction and scope
- Measures taken to ensure compliance
- Methods and results of compliance control
Rendering
- Raw material used (type; annual amounts by type)
- Process conditions and their share of the annual total domestic production
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SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE
Surveillance of BSE
Measures in place to ensure detection of BSE cases:
- Identification system and tracing capacity
- Date when BSE became notifiable and criteria for a BSE suspect
- Awareness training (when, how, who was trained)
- Compensation (since when, and how much in relation to market value)
- Other measures taken to ensure notification of BSE suspects
- Specific BSE-surveillance programs and actions
- Methods and procedures (sampling and laboratory procedures) used for the confirmation
of BSE cases
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ANNEX 3
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Acknowledgments
ILSI Europe and the Emerging Pathogen Task Force would like to thank the main author of this report, Dr. Paul Brown,
National Institutes of Health, Bethesda (USA) as well as the other authors who helped the members of the Emerging
Pathogen Expert Group on TSE:
Dr. Ray Bradley (UK),
Dr. Linda Detwiler, USDA/APHIS Veterinary Service (USA),
Dr. Dominique Dormont, Commissariat à l'Energie Atomique (F) and
Prof. Robert Will, National Creutzfeldt-Jakob Disease Surveillance Unit (UK).
ILSI Europe and the Emerging Pathogen Task Force would also like to thank the International Forum for TSE and Food
Safety (TAFS) for their scientific review and endorsement of the report.
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Other ILSI Europe Reports
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Addition of Nutrients to Food: Nutritional and Safety Considerations
An Evaluation of the Budget Method for Screening Food Additive Intake
Antioxidants: Scientific Basis, Regulatory Aspects and Industry Perspectives
Applicability of the ADI to Infants and Children
Approach to the Control of Entero-haemorrhagic Escherichia coli (EHEC)
Assessing and Controlling Industrial Impacts on the Aquatic Environment with Reference to
Food Processing
Assessing Health Risks from Environmental Exposure to Chemicals: the Example of Drinking Water
ß-Carotene, Vitamin E, Vitamin C and Quercetin in the Prevention of Generative Diseases –
The role of foods
Detection Methods for Novel Foods Derived from Genetically Modified Organisms
Exposure from Food Contact Materials
Food Additive Intake – Scientific Assessment of the Regulatory Requirements in Europe
Foodborne Viruses: An Emerging Problem
Food Consumption and Packaging Usage Factors
Food Safety Management Tools
Functional Foods – Scientific and Global Perspectives
Markers of Oxidative Damage and Antioxidant Protection: Current status and relevance to disease
Method Development in Relation to Regulatory Requirements for the Dectection of GMOs in the
Food Chain
Overview of Health Issues Related to Alcohol Consumption
Overweight and Obesity in European Children and Adolescents: Causes and consequences –
prevention and treatment
Packaging Materials: 1. Polyethylene Terephthalate (PET) for Food Packaging Applications
Packaging Materials: 2. Polystyrene for Food Packaging Applications
Packaging Materials: 3. Polypropylene as a Packaging Material for Foods and Beverages
Recycling of Plastics for Food Contact Use
Safety Assessment of Viable Genetically Modified Micro-organisms Used in Food
Safety Considerations of DNA in Foods
Salmonella Typhimurium definitive type (DT) 104: A multi-resistant Salmonella
Significance of Excursions of Intake above the Acceptable Daily Intake (ADI)
The Safety Assessment of Novel Foods
Threshold of Toxicological Concern for Chemical Substances Present in the Diet
Validation and Verification of HACCP
To order
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Belgium
Phone: (+32) 2 771 00 14
Fax: (+32) 2 762 00 44
E-mail: [email protected]
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The International Life Sciences Institute (ILSI) is a nonprofit, worldwide
foundation established in 1978 to advance the understanding of scientific
issues relating to nutrition, food safety, toxicology, and the environment. By
bringing together scientists from academia, government, industry, and the
public sector, ILSI seeks a balanced approach to solving problems of
common concern for the well-being of the general public. Head-quartered
in Washington, DC, USA, ILSI has branches in Argentina, Brazil, Europe,
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as a Focal Point in China. ILSI’s global branch, the ILSI Health and
Environmental Sciences Institute, focuses on global issues of human
health, toxicology, risk assessment, and the environment. ILSI is affiliated
with the World Health Organization as a non-governmental organization
(NGO) and has specialized consultative status with the Food and
Agriculture Organization of the United Nations.
ILSI Europe was established in 1986 to identify and evaluate scientific
issues related to the above topics through symposia, workshops, expert
groups, and resulting publications. The aim is to advance the understanding and resolution of scientific issues in these areas. ILSI Europe is
funded primarily by its industry members.
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