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MAD COW DISEAS E
The Risks to Humans
J
. -PHILI
. DESLY `
ANDRÉ ICO T
MAD COW DISEAS E
The Risks to Human s
Translated froitn the French by David Marsh Ph D
DOM i hJ O S
Flammarion
JE A N-PHILIPPE D6SLY S
Jean-Philippe Deslys MD, PhD heads the Prion Research Group at the
CEA (French Atomic Energy Commission), where he is a senior
research scientist. He begaai his studies of prions in 19 8 5 by working
on iatrogenic Creutzfeldt-Jakob disease . Since 1996, Dr Deslys has
been an cxpert to the French Interdepartmental Committee on
Transmissible Subacute Spongiform Fncephalopathies (TSSEs) and an
outside expert to the WHO (World Health Organization) and the
American FDA (Food and Drug Administration) . He is the author of
numerous scientific publica .tions .
ANDRÉ PIC® T
André Picot PhD is an organic chemist and engineer and Director of
Research at the CNRS . He is a specialist in toxicochemistry, a new discipline he developed at the C1VRS, where he created and then directed
the Chemical Risk Prevention Unit(1989-2000) : He is also a French
member of a European Union group that sets the standards for chemical
products in the workplace . Dr Picot co-organizes and runs courses in
basic and applied toxicology at the CNAM (Conservatoire national des arts
et ntëtiers) in Paris and Lyon . He is the author of numerous articles and
scientific works, including the classic "Safety in Chemistry and
Biochemistry Laboratories" (Lavoisier, in French) which warned against
pathogenic prions in the early 1990s ,
Original title : La vache foll e
© Flammarion, 200
1 © Flammarion, 2002 for the Englislr translatio n
Printed in France by Hérissey, Evreux
CONTENT S
7
Foreword
The longstanding enigma of
prion diseases in humans
13 From cannibalism to kur u
13
1 7 The classic human forms : Creutzfeldt-Jakob disease
19 Contamination by growth hormon e
23
Spongiform encephaiopathies
are not ne w
23 Scrapie, an endemic disease of mysterious origin
27 Other natural animal diseases
30 What is the purpose of laboratory models ?
39
An infectious agent of the thir d
kind ; the prio n
39 Neither virus nor bacterium, a new form of infectious agent
42 The prion protein is present in all mammal s
44 A change in structure alters its properties
45 Does the prion resist all treatments ?
48 Genetic mutations in the prion diseases
50 Prion, virïno and other hypothese s
55 The search for analogous models : yeast prions
59
Mad cow disease and the
contaminatiohof human s
59 The emergence of mad cow disease
67 A new Creutzfeldt-Jakob disease
7 1 The risks to human s
71 Meat-and-bone meal fetlto our herbivores
75 Can we eat beef products ?
85 First the mad cow, now the mad sheep ?
86 The precautionary principle : shield or umbrella?
88 Contamination of the environment
90 Blood transfusio n
92 Protection of humans : risk management
and screening programme s
1 0 3 Cancluasiori
107 Appendices
109 Notes and references
113 Glossary
121 History of mad cow disease
126 Bibliograph y
The first time a speefafi.Zed term is used, it is signalled by an asteris&".
Its meaning is defined in the glarsa7y.
Foreword
MAD COW DISEAS E
mous . The British people discovered that these much
talked-about prions, which had potentially çontaininated the whole cattle population in Britain (over 180 000
cases to date), for which there was still no detection test,
and which resist almost all methods of decontamination,
may be found in health and energy-giving food . An official embargo was rapidly imposed on all beef by-products from the United Mngdom, and this cordon
sanitaire, coupled with various protective measures,
notably regarding meat-and-bone meal, partially restored consumer confidence elsewhere in Europe . But
doubts lingered . In Britain, restrictions were tightened,
notably through a ban on human consumption of all
cattle statistically of an age to develop the disease (over
30 months), while advertising campaigns, actively promoted the consumption of meat, whose price dropped,
all of which served to boost sales .
In late 2000 there was a new development . An increasing number of cattle were found to be contaminated
more than five years after the ban on the disease-transmitting meat-and-bone meal, whereas officially the risk
was supposed to have been controlled long before .
Investigative reports broadcast on television at peak viewing times revealed that contrary to what had frequently
been asserted, large quantities of contaminated meatand-bone meal had continued to circulate across Europe
with complete impunity. Moreover, screening for B S E
had been ineffective, and the protective measures implemented were manifestly inadequate in some countries
which considered themselves BSE-free . In Germany the
first cases of B SE were discovered at the end of 2000, following implementation of screening tests . The resulting
FOREWOR D
scandal surrounding the inadequacy of the precautions
taken by the authorities led to the resigna;tionof the
German Ministers of Health and of Agriculture . The
increasing numbers ofpeoplé affected by BSE (121 by
early 2002 in the United Kingdom), even though they
were contaminated before the introduction of precautionary measures in Britain, added to the confusion by
heightening the sense of unease and by considerably
altering the perceived risk to humans .
This set the stage for a growing feeling of defiance and
massive rejection of all beef products, regardless of their
quality. With this continuing uncertainty, a lasting crisis
in cqnfidence has grown and efforts to reassure have failed to convince consumers, who above all wish to be
informed and protected . Gonsuzners wonder just what
they can still eat .
Our goal in this book, whose paperback format 9
makes it accessible to a wider audience, is to provide
readers with information as objective as possible together with the relevant scientific background, so tha t
they can take stock of the situation and understand this
ever-changing subject which troubles, even frightens,
the general public .
We shall first describe current understanding of prion
diseases and of these curious prions, and then focus on
mad cow disease and its consequences for humans .
TSSÉs* (transmissible subacute spongïform encephalopathies) principally include Creutzfeldt Jakob disease in
humans and scrapie* in sheep and goats, and since 1985
bovine spongiform encephalopathy (BS E), the scientific
name for mad cow disease . These are degenerative
diseases of the central nervous system* . Under the
MAD COW DISEAS E
microscope, the brain appears to be riddled with holes,
like a sponge, whence the term spongiform . These holes
result in dementia and various neurological disorders,
notably loss of balance . Such signs only appear after a
long and silent period of incubation which ranges from
four to over forty years in humans .
Mad cow disease, and prion diseases in general, constitute a biological enigma and the sale example of a transmissible disease whose origin is still unknown . Two
studies have enhanced our understanding of prion
diseases . First, two French veterinarians, Paul-Louis
Chelle and Jean Cuillé, demonstrated in 1936 that scrapie is transmissible to other sheep . D . Carleton Gajdusek
(Nobel prize in 1976) later brilliantly demonstrated, notably in studies of kuru*, that human-to-human transmission also occurs . Second, in the early 1980s, Stanley B .
10 Prusiner (Nobel Prize in 1997) and his collaborators
revealed the key role of a protein* of the host (affected
individual), the pricrn protein, or PrP, which accumulates
in the brain in an abnormal form (p`rPc or prPrP$) . In
France, the pioneers were a neurologist, Françoise
Cathala, and a military doctor, Louis Court, who for
almost two decades from the early 1970s onwards
through enthusiasm and perseverance succeeded in
continuing his research into a subject which was of scant
interest to officialdom .
The causal agents of TSSEs are called unconventional
transmissible agents* (UTAs), unconventional slow
viruses*, more speciîically, virinos* or prions . These
agents multiply in the lymph tissue (lymph nodes, spleen,
tonsils, appendix . . . ) and the central nervous system
(brain and spinal céard) .lhe degree of infection depends
FOREWOR D
on the causal agent, the route of inoculation, and the
genetic* makeup of the host, i .e. the infected individual.
TS SE does notresuilt in inflammatory signs in the brain whence the ierrn encephalopathy rather than encephalitis
- or in the rest of the body . The lack of a response by the
host's immune defence system greatly hampers the development of a serological diagnostic test (by detection of
antibodies*) . As yet, there is no simple test for direct
detection of these infectious agerats . In practice, diagnosis
is only possible once the clinical signs become apparent,
and is then confirmed or rejected following histological*
examination of the brain. No effective treatment has been
developed, and the clinical course is unremittingly fatal .
11
Spongio °
This »ticrdgraph ofsecta'oned brain illustrates the characterrstic
féatures of'sfiongiosis in a man who died of Creut7yreItlt-jakab disease
(whife, unstained areas corresondto holes).
MAD COW DISEAS E
Never a day goes by without reference to mad cow
disease, and in the absence of clear scientific answers, the
airing of wholly contradictory opinions in the media
creates a general climate of suspicion . A huge divide
separates those who consider the danger to humans to be
negligible and others who predict hundreds of thousands of deaths . Like the cows before them, humans
have become disoriented, and lacking definitive answers
are voicing a pressing desire for reliable information,
which we seek to provide in this book .
The ~~~~~~ an ~.~~ ng en i gma
of prxÎ on diseases in ~ uman ~.~
From 1 ~ism t kuru 1 3
In 1957, Vincent Zigas, an Australian mili.tary doctor,
and Daniel Carleton Gajdusek, an American paediatrician, first described kuru, a strange disease which affected the Fore tribewhich was still living in the Stone Age
in the highlands of Papua New Guinea . In the Fore language, kuru means `shivering or trembling', and was characterized by a loss of balance and dementia . Kuru was
responsible for over half of the recorded deaths in the
most severely affected villagés . This epidemic seems to
have started in the early 20th century and peaked in the
1950s. Adult women (about eight women for oneman)
and the children of both sexes (one third of cases) were
its principal victims .
Despite the remoteness of these highlands, which atthe
THE LONGSTANDING ENIGMA OF PRI O N DISEASES IN HUMAN S
time were virtually inaccessible, some doctors were fascinated by the epidemic, which was highly localized and
seemed not to extend to nearby tribes or to Whites in
contact with the Fore people . Food seemed to be a determinant factor in the appearance of the disease, even if
plants and insects (standard items in ïhe Papuans' diet) seemed not to be implicated. A whole variety of explanations
were proposed, including endocrine disorders (hormonerelated diseases) and female sex-linked genetic anomalies,
but also mycotoxins (the toxic principles of mushrooms),
and even the effect of volcanic ash . In fact, kuru was transmitted through ritual cannibalism which was believed to
confer vitality during secret funeral rites . In these primitive
societies, warriors and hunters were deemed superior and
ate the deceased's muscles, the seat of strength, leaving the
less choice cuts, including the (highly infectious) brain, t o
14 the women and children. A massive ethnological study,
supervised by Daniel Carleton Gajdusek, in which contaminating `feasts' were reconstituted, revealed the singular
finding that all the cases of kuru were linked through a
human chain of infection ,
Gajdusek precisely described the clinical signs of
kuru, distinguishing three stages . In the first stage, there
was a loss of balance, indicative of early involvement of
the cerebellum (part of the metencephalon situated
behind the brain stem) . The cerebellum controls the
regulation of muscle ton.eand allows harmonious interplay between the various muscles involved in maintaining balance, whence the first signs observed in the
afflicted . Abnormal eye movements and trembling
were the other symptoms, which combined to render
this disease highly disabling .
FROM OANNIBALISM TO KUR U
At the second, so-called sedentary stage, the victim
could only move around with the aid of awalkïng stick,
as the muscles progressively became paralyzed . The victim's spirits varied : some laughed constantly, whence
kuru's nickname of 'laughing death' ; others were plunged into a melancholic depression . At the endstage, the
patient was totally disabled, incontinent, and unable to
speak or to eat unaided . Death generally occurred within
a year of the appearance of the first symptoms . Patients
survived longer with hospital care, but this just prolonged the suffering, so their families would soon take them
home to their village to die in peace .
Early attempts to inoculate various animal species
(mice, rats, rabbits, guinea pigs) failed. However, in 1959
William Hadlow, an American veterinarian, noted similarities between kuru and scrapie, and this observation
convinced Gajdusek that the experimental inoculations 1 5
should be continued, above all in primates, while extending the period of observation. The first successful transmission was only achieved seven years later in the
chimpanzee, following an injection into the brain . In
addition to the lesions of spongiosis (holes giving the
brain a spongelike appearance) indicative of neuronal
degeneration (see photo p . 11), histological examination
of the chimpanzee brain revealed the amyloid* plaques
characteristic of kuru. These amorphous protein deposits
comprise the abnormal form of the prion protein, l'rP1e19
and are identical to thé lesions found in humans .
Experimental transmissions of kuru were finally achieved in more than one dozen species of Old and New
World monkeys . Of more than one million infectious
units per gram of human brain, a single unit was enough
THE LONGSTANDINGÉNIGMA OF PRION DISEASESW HUMAN S
to kill one monkey. Transmission of kuru following ingestion of brain tissue from victims of the disease proved
feasible in certain monkeys, but the infectious agent
could not be detected in muscle samples . Transmission in
primates is easier by scarification*, suggesting that containination among the Fore people may have occurred
through the skin, during preparation of the feast . The
women used rather primitive and coarse methods to cut
up the corpse and remove the brain .
Once these anthropophagic practices were outlawed in
1957, the number of cases of kuru dropped dramatically,
and the increasingly rare victims hadin fact been contaminated 35 or even 40 years before . The last deaths were
recordeçlin 1998 .Theyoungestvictims wereat least four
and a half years old, which can be taken as the minimunt
period of incubation of kuru under `natural' conditions .
16 The disease is not transmissible by contact with sick
patients . No case of kûru has been reported in Papuans
who have not participated in ritual cannibalism, or in
Europeans living among the Fore tribe . Since cannibalism ceased, no child born to, and generally breastfed by,
an infected mother (incubating the disease or with fullblown kuru) has developed kuru .
Kura killed over 3000 people in a population of
approximately 30000 . The epidemic may have started
with a sporadic* case of CreutzfeldtJakob disease . Our
understanding of the classical forms of prion diseases in
humans has benefited immeasurably from these pioneering studies of kurnz,
THE CLASSICAL HUMAN FORM S
The lassical human for æ
Creutzfeldt-Jakob diseas e
Creutzfeldt Jakob disease (CJD) is the most frequent
human form of the prion diseases . First described by the
German doctors Hans Creutzfeldt and Alfons Jakob in
1920 and1921 ; respectively, it was given more than 80
different names over the years, before being clearly identified as one of the TSSEs . The similarity of its histological brain lesions to those observed in kuru led Gajdusek's
team to study and demonstrate the transmissibility of
Creutzfeldt Jakob disease 2.
In its usual form, which accounts forover 80% cases of
TSSE in humans, CJI) appears sporadically, striking isolated individuals rather than as an epidemic . Its annual
frequency is 1 .4 cases per million inhabitants, representing approximately one in 7000 deaths in France, with ï
an average of 80 cases a year. CJ D usually becomes evident as dementia (defined as a deeply diminished intellect; with impaired memory, altered intelligence,
temporospatial disorientation and perturbed social behaviour), which begins progressively, in patients aged 6065 years, and is gradually combined withneurolog7cal
disorders (loss of balance and visual disorders) and
muscle contractions (myoclonia.*) .
The first signs are variable . In 40% of cases, CJD
begins insidiously as progressive mental deterioration . In
35% of cases,the early signs are solely neurological and
when isolated may lead to diagnostic errors . The other
forms combine mental impairment and neurological
signs from the outset. The course of the disease is unremitting, and is apparent in dementia with confusion and
THE LONGSTANDING ENIGMAOFPRION DISEASES IN HUMAN S
mutism, often associated with cortical blindness (the
patient becomes blind because of involvement of the
brain and not the eyes), hallucinations accompanied by
loss of motor coordination, and abnormal movements .
Death ensues within a few months, and the clinical diag-
18
nosis is confirmed by histological examination of the
brain and screening for RrpeS, tlie abnormal form of the
prion protein (see p . 44) .
In the absence of a determined infectious cause, Stanley
Prusiner proposed an origin solely linked to the individual who harbours the prion protein that has become
pathogenic : a somatic mutation* of the PrP occurs, by
analogy with the mutations of genes* implicated in most
cancers . However, this hypothesis suggests that there is an
age-related rise in the number of cases and not a peak at
60-65 years followed by a decrease . Cases of sporadi c
CJD do not seem to be linked to scrapie, since scrapiefree countries like Australia and New Zealand have the
same frequency of CJD as the rest of the world, but the
existence of an unidentified animal reservoir cannot be
excluded. Viruses responsible for African haemorrhagic
fevers, such as the Ebola virus, which are the most dangerous in the world and which occur in outbreaks and
then disappear for ten years, persist in an animal reservoir
during the silent periods, since all contaminated humans
die within a few days (the parasite needs a host to survive) . Despite strenuous efforts, notably by the WHO, the
Ebola virus reservoir has still not been identified . One of
the current hypotheses is the endemic presence of the
virus in a non-pathogenic form in rodents . Only occasionally then would a mutant virus pathogenic for humans
appear and cause a new outbreak of the disease .
CONTAMINATION BY GROWTH HORMON E
Natural transmission of CJD between humans (in a
couple, notably) does not seem to occur, but the disease
can be transmitted experimentally to primates by injection of brain tissue of an affected individual, Inadvertent
transmission may also occur via medical treatments .
Such transmission of CJD is called iatrogenic*, in which
a healthy patient is contaminated by material from an
undiagnosed but infected individual (during the clinically silent phase or when the first signs are atypical) .
Such transmission may be due to the use of contaminated surgical instruments, dura mater implantation, corneal grafts, or to injection of growth hormone derived
from human pituitaries .
Certain forms of CJD can affect several members of
the same family and are linked to a genetic mutation.
They are rare (representing 10 to 15% of cases of CJD) ,
and include familial CreutzfeldtJakob disease, Gerstmann- 19
Straussler-Scheinker syndrome, and fatal familial insomnia. These forms are sometimes qualified as purely
genetic as there is no identified infectious cause . Yet they
can be transmitted experimentally to normal animals,
albeit with more diffcultythan the classical forms. Hostrelated susceptibility, principally linked to the gene
coding for PrP, should therefore not be confused with the
presence of an infectious agent, to wit an unconventional
transmissible agent, or prion .
i ti growth hormon e
France unfortunately has the greatest number of cases
to date of iatrogenic Creutzfeldt Jakob disease linked to
THE LONGSTANDING ENIGMA OF PRION DISEASES IN HUMAN S
growth hormone (over half of the 140 cases in the world) .
The first cases appeared in 1989 in unusually young
children (10 to 11 years old) . Of nearly 980 children
treated between January 1984 and June 1985 by human
growth hormone extracted from the pituitaries of human
cadavers, 76 (i .e . nearly 811/6) thus far have developed this
deadly disease and new cases appear regularly. In the
mid-1980s, genetic engineering* was not yet capable of
producing these growth hormones from bacteria . As
hormones of animal origin were ineffective (because of a
difference in receptor - target of the hormone - between
humans and pigs or cattle), it was necessary to use
growth hormone collected from human cadavers.
The same iatrogenic CJD was observed in the United
States, where the first cases were described in 1985, and
in the United Kingdorn, where national programmes fo r
20 production of growth hormone were also implicated,
unlike the pharmaceutical industry whose stricter
manufacturing rulesand application of Good Laboratory
Practice meant that it could not be incriminated3 .Thes
ena#iolprgameswrimplentdoslveh
shortage of growth hormone, or rather of human
pituitaries, thereby ensuring access to treatment
regardless of the patient's financial wherewithal . This
contamination is a reminder of the remarkable capacity
of prions to resist most purification or decontamination
processes . It also illustrates how difficult it is to predict
the outcome of the contamination of a human
population . We now have over fifteen years of experience
of transmission between humans (no species barrier,
which diminishes the efficacy of the transmission and
increases the incubation period) by a, relatively effective
CONTAMINATION BY GROWTH
HORMON E
contamination route (injections more so than the oral
route), and yet cases still arise at a steady rate (on average
four to five a year) .
Individual susceptibility also plays an important part .
The gene coding for PrP (the prion protein) is an essential
factor in the contamination of patients who developed
CJD after receiving human growth hormone in France .
For nearly five years, in all the observed cases (i .e . 30
children or young adults) there was a single type of prion
protein, a feature seen in only 50 % of the normal
popula.tion_ The other half of the population, including
the children treated by growth hormone, has two types of
PrP which differ by a single amino acid4 (heterozygosity*
at codon* 129) : one from the father's genetic material, the
other from the mother's . This difference was therefore
apparently enough to make the carriers resistant to the
disease .
2
Yet the interpretation seems less optimistic today, as the
first cases of affected people carrying the two types of PrP
(heterozygotes*) were recorded in 1994, and their
number continues to rise . Moreover, in kuru it was
observed that this lzeterozygosity at codon 129 only
delayed onset of the disease and did not afford genuine
protection . In experimental models, the disease is
seemingly slowed whentwo different PrPs are present in
the same animal . Overall, individual genetic susceptibility
allows the onset of the disease to be delayed, but not
necessarily avoided .
1
Spongiform encephalopathies
are not new
Scraple, an e ic disease 23
of mysterious origin
SPONGIFORM ENCEPHALOPATHIES ARE NOT NE W
Transmissid6e subacute spongitorm
encephaBopathies in humans and animals
~~
Sheep
Goat
~~~~~
a .;:^
I
~~
-
RI
1
~~TRj ~
E77
Scrapie
around 1730
Scrapie
?
Kuru
around 1900
1920
1926
1992
Man
Creutzfeldt-Jakob disease
Mink
Transmissible mink encephalopathy
1947
Wapiti and mble
deer of the Rocky
Mountain s
Chronic wasting disease
1967
Cattle
Bovine spongiform encephalopathÿ
1985
Gerstmann-Straussler-Scheinker syndrome
Fatal familial insomnia
Nyala
Gemsbok,
Spangiform encephalopathy
Cape élk; I~rabian ~~ ~ ~
1986
1987
1989
oryx, Great kud u
Caf
Feline spongiform encephâlopathy
1990
Cheetah, puma
Spongiform ençephalopathy
1992
Mouflon
(wild sheep)
Spongiform encep halo p athy
1992
affects oth er small ruminants, notably goats, and is foun d
in most countries of the world, except for Australia and
New Zealand . Principally studied in Suffolk sheep, natural scrapie is an endemic disease and affects animals aged
two to five years (older sheep are rarely kept, for commercial reasons) . It is present throughout Europe, and in
some flocks affects up to 30% of animals - a frequency of
about 0 .5 to 2% per year being considered as the c mmonest,Iéike numerous animal diseases, scrapie posed no
particular danger to human health, but was deemed shameful and therefore occulted: Today scrapie is a notifiable
disease only in some countries, including France .
SCRAPIE, AN ENDEMIC DISEASE OF MYSTERIOUS ORIGI N
Scrapie in sheep and goats has a period of incubation
which may reach two to three years . Classically there are
two forms . The clinical form is characterized by hyperexcitability and trembling, initially of the head and later
of the whole body. The pruriginous form is manifested
by pruritus* which causes the sheep to scrape (whence
scrapie) or nibble themselves in an attempt to relieve the
irritation, causing loss of wool and extensive lesions .
Infected animals are no longer able to coordinate their
movements, lose the ability to stand, and die of exhaustion (cachexia) after a few months .
The transmissibility of scrapie within flocks was
demonstrated in 1936 by French veterinarians, and
confirmed on a large scale by British vets . In 1939,
nearly 7% of 18000 sheep vaccinated four years before
against louping-ill (a nervous disease caused by a virus
transmitted by ticks) developed scrapie . The vaccine had ~5
been produced from the brains of sheep contaminated
by a strain* of scrapie .
There is natural horizontal translnission* between animals within a flock which could be linked to placentophagia (when an infected ewe litters, the infectious .
placenta is eaten by her or by other ewes) or to contamination of pastures by the placenta, or even by faeces.
Pastures grazed by infected animals could contaminat
. It was shown experimen-enwflocksrvtyea
tally (see p . 89) that infectiousness persists in the soil 7
for several years, whence the danger to health posed by
the burial of the carcasses of scrapie-infected animals .
This persistent infectiousness has led to a ban on the use,
in fertilispr, of proteins from animal offal . The scrapie
agent was also identified in hay mites 8 (miniscule spi-
SPONGIFORM ENCEPHALOPATHIES ARE NOT NE W
ders), suggesting the possibility of a hitherto unidentified
animal reservoir.
Vertical transmission also seems possible, since some
embryos collected from an infected ewe and transferred
into a healthy surrogate ewe subsequently develop scrapie. The sheep model is nonetheless particular, because
high levels of infectious agents are found in several peripheral organs (tonsils, spleen, lymph nodes, intestine,
placenta), unlike what is observed in cattle and in
humans, where infectious agents are limited to the central nervous system (brain, spinal cord) . This particularity
could explain the putative contamination of the environment (scrapie-infected pasture) by the placenta or even
faeces, and the possibility of horizontal transmission (between animals of a given flock), thereby explaining the
endemic character of the disease in sheep and goats.
26 The infectious agent of natural scrapie is present very
early, from 3-4 months, in the lymphoid nodules (called
Peyer's patches) of the intestine, and then progressively
in other lymph organs (lymph nodes, spleen) ending up
in the central nervous system, which indicates early
contamination by the oral route .
Scrapie eradication programmes have generally failed,
such as in the United States, and in Iceland despite carefully organized and extensive culling and repopulation .
On the other hand, scrapie seems to have disappeared
naturally in Australia and New Zealand, countries that
are officially scrapie-free but which at the time of King
George fTÎ. (who reigned from 1760 to 1820) introduced
British sheep which were certainly contaminated . At that
time scrapie posed major economic problems, and
Claridge writing in 1795 noted that it was considered the
OTHER NATURAL ANIMAL DISEASE S
worst calamity to befall His Majesty's sheep breeders .
The explanation could relate to the type of breeding,
which favours horizontal transmission in Iceland (an
island in thé North Atlantic), where sheep are stabled
dûring the long winter months and fed potentially soiled
hay, whereas extensive land is available in Australia and
New Zealand, thus theoretically limiting the risk of pasture-borne contamination .
Other natur 1 animal disease s
Spongiform encephalopathy of the min k
This rare disease (transrrtïssible mink enceiiuzlopathy) has
ravaged mink farms following foodborne contaminatîon .
The first outbreak occurred in 1947 in the United States,
in a farm in Wisconsin . All the adult minks showed signs 27
of lack of motor coordination, drowsiness and major
autonomic disorders (the autonomic nervous system
controls the functioning of the viscera, respiration, blood
pressure and thermoregulation), and then died. The
disease also developed in 125 minks sold seven months
before to a farm in Minnesota . In 1961, the disease appeared in five farms in Wisconsin all of which used the same
supplier of animal feed, but mortality was lower (between
10 and 30%) . In 1963, two outbreaks were reported in
Canada, and another occurred in Wisconsin . Here too,
there was a common feed supplier. Outbreaks also occurred in Finland, and in what were then East Germany and
the USSR . The most recent case was reported in 1985 in
Stetsonville, in the United States, twenty-two years after
the last reported American ocatbreak .9
SPONGIFORMENCEPHACOPATHIES ARE NOT NE W
The silent incubation period is estimated to last from
seven to twelve months . The first signs are hyperexcitability and marked aggressiveness, during which the animal may injure itself, Drowsiness then predominates,
often accompanied by blindness, and is followed by
paralysis of the hind legs and death, which occurs after a
few weeks . There is no natural transmission between
minks, which therefore constitute an epiderniological*
`dead end' : prevention therefore only involves monitoring of food sources .
Although the epidemiological data clearly incriminate
the animal feed, the actual cause of the disease is still unknown . Minks are carnivores and may have been fed the
carcasses of sheep that had succumbed to scrapie .
Laboratory experiments have shown that certain strains
of scrapie are transmissible to mink by the intracerebra l
28 route . However, no mink has developed the disease by
eating the brains of sheep contaminated by the scrapie
agent . In contrast, contamination by the oral route was
easily achieved using the brain of cattle with bovine spongiform encephalopathy . On at least two occasions, and
notably for the most recent outbreak, at Stetsonville, it
seems that the minks were never given feed containing
sheep offal, but were fed the carcasses of cows found dead
or culled because of downer cow syndrome (in which no
T S SE screening seems to have been done) . This suggests
that the minks were contaminated by material from cattle
that had developed an undetected sporadic prion disease.
Chronic wasting disease of wsld ruminant s
Chronic wasting disease (G"); which is similar to
scrapie, affects free-ranging and farmed ruminants in the
OTHER NATURAL ANIMAL DISEASE S
United States, principally the mule deer of the Rocky
Mountains, as well as the white-tailed deer and the ellc .
This disease was observed for the first time in the early
1960s in a herd of mule deer raised in captivity in a wildlife research facility at Fort Collins (Colorado) . It was
subsequently observed near Kremmling (Colorado) and
Wheatland (Wyoming) in mule deer and in farmed elks .
Marked horizontal transmission occurs in the adult animals, above all in captivity, because of overcrowding .
After three years the disease generally affects all mule
deer in the herd and approximately 30% of the herd in
the case of elks, which appear less sensitive . It is highly
recommended that the whole herd be culled as soon as
the first sick animal is observed, to avoid both contamiriating the environment and pointless replacement over
years of the herd by healthy animals, which may succumb in turn .
The systematic search for signs of the disease by microscopy 1a has shown that the disease was not clinically diagnosed in 97% of the cases . In Wyoming, only four of 133
animals found to be positive manifested clinical signs :It
was only in 1981 that the disease was described in wild
animals, but it is probable that undetected cases occurred
much earlier. The frequency (or prevalence) of CVV D
was estimated as approximately 1% in 1984 and rose
gradually to 3 .5% in 1998 in North-West Colorado .
Researchers estimate that over the same period, the frequency of CWD in deer rose from 3% to 13% in SouthEast Wyoming . Uncontrolled, CWD could decimate
deer herds over the next thirty to fifty years and invade
neighbouring states, particularly as it is transmitted to
other species, notably white-tailed deer, which are much
29
SPONGIFORMENCEPHALOPATHIES ARE NOT NE W
more mobile, and elks, which are transported around the
country for breeding purposes it .
In Canada, contaminated herds of over one thousand
wapitis were recently culled to halt propagation of
CWD . The recent description of three cases of CJD in
hunters under forty years of age, who may have had
contact with the animals of these regions, alerted the
Center for Disease Control in Atlanta, which monitors
emergence of diseases worldwide, to the risk of transmission to humans . For the moment, no causal link has
been established, but as a precautionary measure for
blood transfusion, hunters of this area could be prohibited from donating blood 12 0
What the purpose
of laboratory mod 1 ?
The mouse, the mQdél par excellenc e
The mouse is the animal the most frequently sensitive
to natural diseases and constitutes the reference model
for the detection of infectious agents, their quantification
and the study of their properties, and for studying disease
progression. Laboratories work with syngeneic* strains
of animals (mice) with the same genetic makeup from
lines obtained by successive controlled crosses, so as to
minimize variability betweén individuals.
Disease control. The mouse constitutes an easily reproducible in vivo niodel which for years has been the most
reliable research tool in studies of prions, whose exact
nature is still unknown, which are invisible under the
microscope, and which do not multiply in the test tube .
WHAT IS THE PURPOSE OF LABORATORY MODELS ?
As an example, the classical response 13 of a mouse following direct injection into the brain (the most effective
route of contamination) is as follows . For five months,
nothing seems to happen, and then within a few days all
the animals start to present clinical signs (balance disorders, abnormal gait) which worsen and lead inexorably
to death in one month . This is therefore a biological
clock of astonishing precision since it is possible, at the
time of contamination, to predict to within a few days the
death of the animal six months later.
On contamination by the oral route, these infectious
agents are first found in the Peyer's patches of the small
intestine (lymphoid nodules which constitute the first line
of defence of the immune system) at the time of passage
through the intestinal barrier: l They then diffuse through
the lymph and blood systems to all the lymph tissues
(lymph nodes, tonsils, spleen), where they multiply 31
before invading the nervous system from different nerve
endings in contact with the contaminated tissues . The
propagation along the nerves occurs in both directions ,
and so at the endstage of the disease the central and peripheral nervous systems are completely invaded, regardless of the initial route of inoculation . The infectious
agents are always most abundant in the central nervous
system, which, because of the weight of the brain and spinal cord, accounts in absolute terms for most (over 95%)
of the infectivity found in the animal .
~ ,)uantiirtg infectious tagents. In practice, the mouse
model allows determination of the infectious titres, i.e .
measurement of the quantity of infectious agent present .
The lower the infectious dose, the longer the incubation
of the disease . In the mouse model, animals contamina-
SPONGIFORM ENOEPHALOPATHIE5 ARE NOT NE W
ted with a homogenate* ofinfeetioûa brain diluted to 1%
(0 .2 mg for 201zlinjectéd,i.e. 20 millionths of a litre) all
die within a few days after 180 days, whereas the mice
that received a dose one million times lower die between
320 and 400 days after the injection, and sometimes
even survive .
There is enough infectious agent in one gram of brain
to kill between one million and one thousand million
mice . Yet our state-of-the-arttechniques have still not succeeded in detecting any infectious microorganism .
A classification of tissues and organs was proposed following quantification of the infectious agents in scrapieaffected sheep, and underpins precautionarji measures
for the exclusion of high-risk organs . Essentially, the
32
most infectious tissue in the sheep is the central nervous
system (brain and spinal cord), followed by the lymph
organs and tissues (lymphnodes,spleen ; tonsils, Peyer's
patches, appendix and, more generally, intestine, placenta) with 30 to 100 times less . Muscle, milk, urine, skin
and fur are considered to be free of detectable infectivity .
The limits of the mouse model should be borne in
mind : the volume injected into the mouse by the intracerebral route cannot exceed 20 microlitres, i.e . 2 mg of
tissue if it is homogenized at10% . This implies that the
same quantity of agent that would give a mouse the
disease if concentrated in 2 mgof tissue would be undetectable if diluted in 2 grazns . However, the inability to
detect the infectious agent does not mean that there is
none in the sample, but rather that its concentration is
insufficient in the 2 mg tested .
These possibilities of assessing the presence of prions
also allow determination of the relative efficacy ofdiffe-
WHAT IS THE PURPOSE OF LABORATORY MODELS ?
rent routes of infection . As expected, the most effective
route is direct contamination of the brain, followed by
the intravenous route, which is ten times less effective,
and then the intraperitoneal route (injection through the
abdominal wall), which is approximately 400 times less
effective . For the intravenous and intraperitoneal routes,
respectively 90 and 99 . % % of the prions injected are eliminated or destroyed, notably by the macrophages*,
before having reached theirtarget, i .e . the cells in which
they can multiply before invading the central nervous
system. This is still more marked for the oral route,
which is considered to be 100000 times less effective
than the intracerebral route, since the vast majority of
the prions ingested do not even cross the intestinal barrier. Transmission does not occur through healthy skin,
but is relatively effective through damaged skin (scarification) and subcutaneously, albeit 50 times less effecti- 3 3
vely than by the intracerebral route . Passage across the
conjunctival mucosa is only five times less effective than
the intracerebral route . These orders of magnitude are,
however, observed in a particular experimental
model14 . The data change depending on the type of
mouse and the prion strain .
Detecting cd6~rerent strains offirâons . In biology a strain is all
the individuals produced by successive subcultures of a
microbial colony . A prion strain results from the transmission of a prion disease to several successive hosts . In
practice, the mouse is infected with the prion strain, and
when it develops the disease its brain is used to inoculate
another mouse (in what is calleda `passage' between animals), thereby `stabilizing' the strain, whose properties are
constant through later passages . These properties charac-
SPONGIFORM ENCEPHALOPATHIES ARE NOT NE W
teristic of a strain are notably the period ofincubation for
a given type of mouse (syngeneic animals) and the pattern
of histological lesions (tissular lesions visible under the
microscope) in the different regions of the brain .
Over 20 different strains have been isolated in the
experimental mouse model, principally strains of scrapie, but also of other animal and human TSSEs . It is
noteworthy that it has never proved possible to transmit
certain scrapie, and above all CJ D , strains to rodents .
The stability of the strains varies . For the most stable
strains, these properties can be kept even when changing
species (mouse-hamster-mouse passage, for example),
whereas for other strains this leads to the selection of
variants with different properties . It is also possible to
mix strains, to perform several successive passages in the
animal (over several years) during which only the domi34 nant strain is visible, before again isolating each of the
starting strains .
A) Thé species barrier is determined by the host's PrP (prion protein),
which accumulates in the form ofRP°`S in cases of t ransmission .
A strain of mouse FITa4 which, is not transmitted to the hamster
can be transmitted to a transgenic* mouse w hich has both hamster
and mouse .l'rP, but then will only be transmissible to the mouse
(a- 1): In contrast, a hamster strain cannot be transmitted to
the mouse, even ajter a passage in a transgenic mouse
(a-2). B) Some strainscan be transmitted to other species but
variants with derent properties are then selected (b: strain 22C
of the mouse becomes strain 22H after passage in the hamster) .
Q Certain particularly stable strains conserve all8heir characteristics
after their transmission to other species (e: the strain of bovine
spongiform encephalopathy) .
SPONGIFORMENCEPHAL©PATHIES ARE NOT NE W
In 1975, the experiments of Alan Dickinson demonstrated the existence of healthy can-iers . Certain strains
multiply in the lymph organs without ever generating the
disease (absence of neuroinvasion) during the animal's
lifetime . This notion of the healthy carrier was recently
demonstrated again in the mouse and hamster by the
team of Bruce Chesebro in Montana (United States), and
then by John Collinge and colleagues in London . A
strain of hamster prion can multiply weakly in the mouse
without inducing the least pathological sign and then
recontaminate the ha:mster 15 .
This could pose majorpublichealth problems in cases
where a pathogenic agent persists in an animal or human
population. These strains also exhibit variable resistance
to decontamination and some are par ticularly resistant to
3G
heat treatments .
Controlling the genetic makeup . The power of the inurine
model (mouse) is largely due to the fact that its genetic
makeup is under our control . Researchers can work
either with syngeneic animals, when they wish to study
just the variations related to the different strains of prion,
or with animals that only differ by a limited number of
genes, or even by a single gene, so as to assess the
influence of the genetic heritage on disease onset and
outcome . The mouse model has been used to reveal the
principal gene responsible for susceptibility to TSS]Es .
Initially baptised SI N C (for Scrapie INCubation period)
e,itwasubeqnlyhot gecdinforth
prion protein (PrP), a normal protein which is neither
viral nor bacterial but is a part of the host since it is present in all mammals (see p . 42) . Understanding of the
role of the prion protein has been greatly enhanced by
WHAT IS THE PURPOSE OF LABORATORY MODELS ?
genetic manipulations producing transgenic animals
expressing the prion protein of mice (or of other species)
at very high levels, or not at all .
The hamster
The golden hamster has the advantage of developing
spongiform encephalopathy twice as fast as the mouse
(in under three months), following inoculation with a
particular experimental strain of scrapie 1 6 which accumulates ten times more infectious agent than in the
mouse. The hamster model has been used to assess the
different physicochémical properties of prions and was
used by,Stanley Prusiner in 19 8 2 in the first purification
of the prion protein .
The cat
The CJD agent istransmissible to the cat by the intracerébral route (transmission in approximately one third
of cases), unlike the agents of kuru and scrapie . Its period
of incubation ranges from two to five years . The disease
lasts from one to five months and is apparent in an
absence of grooming, nervousness, staring, and often
trembling . Marked sleep dysfunction, with loss of REM
(rapid eye movement) sleep and onset of apparent death
states (catalepsy), are observed in over half of the animals . These anomalies are similar to those described in
fatal familial insomnia in humans .
The monkey
The first experimental transmissions in the monkey
were done in the chimpanzee in 196 6 for kuru and i
8 for CJD, with an incubation period ranging fromn196
37
SPONGIFORM ENCEPHALOPATHIES
ARENOT NE W
one to six years . Other primate models, such as the squirrel monkey and macaque, have the advantage that they
reproduce readily and generally develop the diseasés faster . After inoculation by intracerebral route, the macaque
is sensitive to CJD, kuru, scrapie, mink encephalopathy,
and above all to BSE, for which it is the model closest to
hun.ans .
38
An infectious agent of the
third kind : the prion
AN 1NFEC'I"IOUSAGENTOF THE THiRD
KIND : THEPRIO N
(DNA* or RNA*), and so may be a protein. The following year, again in London, a mathematician worthy of
Jules Verne, John S . Griffith, reported that his modelling
suggested that the infectious agent of scrapie is a protein
whose three-dimensional structure is altered andwhich
multiplies by self-association
1970, Raymond Latarget.In of the Institut du Ffadiu m
(now the Institut Curie) in France confirmed by means of
inactivation tests the apparently purely protein nature of
this strange infectious cellular agent.
The Californian neurobiologist Stanley Prusiner reviewed all these data. and demonstrated that these
UTAs, which are insensitive to physical agents (heat9
ionizing radia:tion* . . .) and chemicals (strong mineral
acids, aldehydes* like formol), in practice resist all the
processes that degrade nucleic acids, whereas they ar e
40 sensitive to those which destroy proteins 17 : Since this
fundamental work (19$2), Prusiner has defended the
hypothesis that the infectious agent of TSSEs is an infectious protein, for which he coined the term `prion' from
`I?RC)teinaceous IliTfectious particle' . This hypothesis is
particularly daring, since all known infectious agents
hitherto possess genetic information able to be expressed and to mû .ltiplyin a living organism, like bacteria,
parasites and viruses .
It is commonly admitted that proteins do not carry
genetic infqrmation,btaL rather result from its expression .
Because of this, Prusiner inhisinitial hypothesis proposed that the pathogenic prion is a protein foréigri to the
host which commandeers the host's cellular machinery
in order to multiply?R . As was to be expected, this highly
provocative hypothesis was greeted with great scepticism
NEITHER VIRUS NOR BACTERIU M
by microbiologists, although they were unable to
demonstrate its inexactitude .
In 1984, a protein found in semi-purified brain extracts
from hamsters infected by an experimental strain of scrapie was partially purified and baptised `prion protein',
and then sequenced (i.e . the order, or sequence, of its
amino acids was determined) . This short sequence
enabled the construction of nucleic probes and the
search for the gene responsible for the synthesis of this
prion protein . To everyone's surprise, two teams
(Charles Weismann's in Switzerland and Bruce
Chesebro's in the United States) rapidly identified in
19 85 a génetic sequence corresponding to a cellular protein, the normal prion protein (PrI'c), localized at the surface of healthy nerve cells . In sick animals, this protein
exists in its normal form, l'rE°, which is present in all
healthy animals, and in an abnoran :al, pathological form,
called PrPsc (for scrapie) or F'rPes (for resistant), which is
not degraded by proteases* (enzymes that destroy proteins) and which therefore accumulates in the brain, leading to spongiform encephalopathy. Far from rejecting
the prion hypothesis, this approach redirected it . The
concept of a protein foreign to the host was thus replaced
by a model based on the transformation of the normal
structure of the prion protein, itself naturally synthesized
in the neurones* .
To avoid any semantic confusion, it is wise to distinguish the prion protein, the normal protein present in all
mammals, from the prion itself, the infectious agent responsible for BSE .
41
AN INFECTIOUS AGENT OF THE THIRD KIND : THEPRIO N
The r1 C # i is present
in all mammal s
The hypothesis proposed in 1982 by Stanley Prusiner,
according to which the prion is a protein free of nucleic
acids, is today bacflted by most scientists . Prusiner's group
was the first to show that the normal prion protein (PrP%
omnipresent in the nervous system of healthy animals,
and its abnormal form (I'rPrC5), isolated from animals
with spongiform encephalopathy, were one and the
same protein . The two forms differ just by a change in
conformation*, that is b y a difference in three-dimensional structure . This structural change greatly alters their
respective properties . Thus, I'rlx is soluble in water whereas I'a°1?Te5 is insoluble, even in the presence of detergents
known to solubilize many products .
42 The primary structure* of the protein, that is the order
of the different amino acids 19 which constitute it, has been
known since 19 8 6 . In humans it is a small protein of 253
amino acids . The synthesis of Prll" is programmed in the
D NA by a single gene (called PRNP) localized on chromosome* 20. This protein is located on the outer membrane* of cells, in which its abundance varies depending
on cell type . It is principally present in neurones and, at
much lower concentrations, in glial cells*, which supply
nutrients to the neurones of the central nervous system, as
well as in certain cells of the immune system .
PrPc is synthesized by the cells in a few minutes, but
after three to six hours is degraded by proteases (enzymes
which destroy proteins at the end of their life) . IrPYeg, on
the other hand, remains undegraded for approximately
eighttimes longer (over 24 hours) .
THE PRION PROTEIN IS PRESENT IN ALL MAMMAL S
The cycle of the normal form of the prion protein
seems to be as follows : 1 . synthesis in the protein-producing minifactories calIed ribosomes (the genetic information carried by the DNA of the nucleus* is transferred to
intermediate molecules, the messenger RNAs, which are
translated into proteins in specialized units constituted by
the ribosomes) ; 2 . migration to the cell membrane to
which the protein is fixed by a glycolipid chain (localization on the outer face of the membrane) ; 3 . reintroduction to the interior of the cell by a process called
endocytosis ; 4 . final destruction in the lysosomes* (small
sacs filled with enzymes that degrade proteins) .
The synthesis of normal PrP does not seem to vary
during the course of the disease. In contrast, experimental interruption of the PrP cycle shows that if PrP is not
reintroduced into the cell, the formation of FrFe5 is bloc-
ked . This type of experiment suggests not only that the 43
l'rPLeS derives from the normal PrP but that it also arise s
due to an anomaly in the natural mechanism of destruction of PrP, i.e . in. its catabolism .
The role of the normal PrP is unknown . Because of its
ability to bind copper, it has been thought to play a part
in the biotransformation and storage of this trace element* and an indirect role in protection against the deleterious effects of free radicals20 .
The localization of PrP at the surface of cells suggests it
may act as a receptor. It interacts with a particular target,
the laminin receptor, a key protein of the extracellular
matrix linking the cell membrane to the external structures . This suggests that PrP is involved in the process of
cellular adherence, notably in neurones21 whose outgrowths connect to other cells. A French research teatn22
AN INFECTfCUS AGENT OF THETHfFiL'J KINC :THÉPÉ9IO N
has recently shown that the binding of a molecule to PrP
triggers the sending of a signal to the inside of the cell :
A
. . . in structure alters
i" r rti
The two forms that the prion protein can assume (normal . PrP1 and abnorinal PrPre,, which is considered inféctitrus in the prion hypothesis) have the same amino acid
composition and sequence (order of amino acids, or primary structure) . There are essentially twenty different
amino acids that can be strung together in a chain .
Because of the length of this chain (generally over 100,
and sometimes several thousand amino acids), a protein
has a practically limitless diversity of sequences .
44 Depending on this amino acid sequence, each protein
chain can fold on itself in a well-déterrnined fashion, thereby adopting a secondary structure* (i.e . the conforrnatiçsn, or spatial organization, of the neighbouring amino
acids) . Among thesé secondary structures are alpha
helices* (shaped like a spiral staircase), which explains, for
instance, the stretchable nature of wool (which is made of
proteins), and the beta-pleated sheets*, which have to an
accordion-like conformation, thus explaining, for
example, the great resistance to traction of silk (which
comprises another typ e of protein) . Finally, the proteins
have a tertiary structure*, which is the conformation of the
protein as a whole (for example, a fibrous protein, like collagen or keratin of the skin, or a globular protein, like most
cellular proteins or blood albumin), and a qu.aternazy
structure when they comprise several protein subunits .
A CHANGE IN STRUCTURE ALTERS ITS PROPERTIE S
The two forms of PrP differ in their secondary structure, as revealed by structural analysis techniques .
Among spectral methods, NMR* (nuclear magnetic
resonance) spectroscopy* is as yet the only technique
able to determine the three-dimensional structure of the
prion protein. Kurt Wüdlrich's group 23 in Zurich modelled the three-dimensional structure of the norinal prion
protein (Prpc) in the mouse, and later in humans, the
hamster and cattle . When I'rPc is transformed into prprps
its abnormal form, it acquires a structure of mainly beta
sheets in its globular part (approximately 43°Io of the protein), compared with 3% in the normal protein, whose
structure is mostly alpha helix . It is the acquisition of this
beta sheet structure which confers on prPLeç its properties
of insolubility, aggregability and resistance to degradation . l'rFés can therefore accumulate inside cells, particularly in lysosomes (which normally destroy proteins at 4 5
the end of their lives), and become toxic for the neurones . In the test tube, in the presence of detergents and
proteases, PrPeS condenses in the form of characteristic
fibrils called S.9.F (Scrapie-Associated Fibrils) . In vivo,
deposits are found in the brain, in the form of amyloid
plaques and sometimes fibrils, as classically seen with
other proteins in neurodegcnerative disorders like
Alzheimer's disease .
tr "
rion resist all
?
Unlike conventional proteins (albumin, for example),
the particular structure of I'rpr`'' confers on it exceptional
AFdINFECTIOUS AGENT OF THETHlRD KIAJIJ : THE PRIO N
resistance to thè most of the methods generally used to
destroy these macromolecules* : heat treatments (coagulation), chemical and enzymatic treatments . This allows
prions to be differentiated from conventional infectious
agents, like bacteria and viruses .
The stability of prions when heated (therinostabilit,y) is
quite remarkable : 160°C for 24 hours or even 360T for
an hour in a dry atmosphere is not enough to inactivate
it totally. likewise, prions are partially reesistantunder the
humidconditions usually used, such as boiling or autoclaving at 121°C for one hour (steam treatment under a
pressure of 2 bars) . C3n1y'wet heat at high temperature
inactivates the prion sufficiently to .rneet public health
requirements . The WHO (World Health Or n 'on)
recommends autoclaving at 133T for 18 minutes under
a pressure of 3 bars .
46 However, sensitivity .to autoclaving varies greatly between prion strains, thus complicating the implementation of safety standards . In addition, all prior treatments
that lead to fixation or 'g of proteins (formol, ethanolp even simple drying) protect against the action of
autoclaving . The conditions described above that inæctivate the agent of bovine sporlgiform encephalopathy are
insufficient if the sample has dried on the walls of the
tube and, for the same reason, a temperature of 138° C
seems less effective than 133° C (because the sample is
dried before the steam has time to hydrate it) . In contrast,
a method will be effective if it allows hydration (incorporation of water) or even saponification (transformation
into soap in alkaline medium) of the sample before the
heat acts . Pretreatment with sodium hÿdroxide*, even
diluted to 0 .1 N (one tenth of a normal solution, which
DOES THE PRION
RESIST ALL TREATMENTS ?
contains 40 g of sodium hydroxide per litre), makes autoclaving at 121°C totally effective .
Ionizing radiation is ineffective on prions, and doses
conventionally used in sterilization (25 kGy) have no
effect. Nonionizing radiation*, such as ultraviolet light,
and ultrasound, are also ineffective .
Chemical reagents that generally degrade proteins, are
ineffective, notably the aldehydes, such as formaldehyde
(the formol classically used for decontamination in hospitals) and glutaraldehyde . Likewise, alcohol (ethanol),
ethylene oxide, quaternary ammonium salts, and, in
general, detergents, are wholly inactive on prions - with
the exception of sodium dodecyl sulphate, when heated
and under certain conditions . It is remarkable that several powerful oxidizing agents, such as potassium permanganate in concentrated solution, hydrogen peroxid e
and ozone*, known for their potent ability to destroy 47
living matter, are inactive on prions .
Prions are insensitive to acid conditions, notably in the
stomach (where the pH is 1 .5 to 2), and only very high,
that is alkaline, pHs are effective . Strong mineral bases*,
such as sodium hydroxide at molar concentration (1 N
solution), are considered to destroy prions adequately if
the time of contact is sufficient (at least one hour at room
temperature) . However, resistance has been reported
with certain strains of CJD and of scrapie, and this treatrnent is not effective against other infectious agents, notably spores (microbial) . The most effective chemical
treatment is bleach (sodium hypochlorite*) at a concentration of 20000 pprn (which corresponds to a commercial solution of 6 chlorometric degrees) for one hour .
AN INFECTIOUS AGENT OF THÉTHIRf7 KIND :THE PRIO N
Genetic mut ti in the rÎ
diseases
It is now well established in mammals that the gene
coding for PrP plays an essential role in the prion
diseases . This gene was in fact analyzed in the 1970s by
Alan Dickinson and his team, in Edinburgh. In classical
genetic experiments, they crossed mice and identified a
major gene baptised SINC (for Scrapie INCubation
period) as the principal gene responsible for the susceptibility of the mice to infection . This gene was rediscovered
more precisely by Stanley Prusinorin molecular biology
experiments which confirmed that it was indeed the gene
encoding PrP. Animals that overexpress this gene (transgenic mice) are very sensitive and develop the disease
after a short incubation period. PrP, even when norma l
48 (PrP"),can prove toxic when overexpressed . Certain animals, which synthesize over ten times the normal amount
of PrP, spontaneously develop spon,:iforrn encephalopathy with muscular involvement. There is, however, no
accumulation of abnormal PrP and the transmissibility to
healthy animals remains doubtful . This means that the
same brain disorders can be generated by a very high
level of this protein, due to overproduction of its normal
form (PrPc) or to faulty destruction of its abnormal form
(PrPie5) : In contrast, mice that poorly express this protein
(functional gene on a single chromosome) develop the
disease much later and survive long after the appearance
of the first clinical signs (14 months, compared with one
month for wild-iypernice) . Lastly, mice in which this gene
is not functional, and which therefore do not synthesize
PrI'c, are completely insensitive to prion diseases .
GENETIC MUTATIONS IN THE
PRION
D ISEASE S
The gene that codes for PrP also governs the species
barrier which protects against infection by a UTA of another species, as brilliantly shown in transgenesis experimerats . Genetically modified mice expressing hamster
PrP in addition to mouse PrP become sensitive to hamster UTAs . They then accumulate hamster and not
mouse PrPres As a consequence, their brain becomes
infectious for hamsters and not for normal mice .
In sheep, genetic studies show that susceptibility is also
associated with a gene first called SIP and then found to
be the gene coding for PrP. Genetic selection of resistant
sheep is under way in the United Kingdom. It should be
remembered though that the previous experiments showed that these resistant sheep could be sensitive to an
unusual strain of scrapie 2h, which could presage the
emergence of new strains of scrapie adapted to the sheep
selected intlûs way.
49
In humans, all the familial forms of prion diseases correspond to mutations in this gene, which are reflected in
particular zones of the protein. In vitro experiments
show that these n.iutations favour the aggregation of the
protein into insoluble amyloid fibrils which resist degradation by cellular enzymes . Thus, all the patients who
develop a familial form of CJ D in fact possess a PrP
which has a natural tendency to aggregate more easily
than that in the normal popixlation :
During prion diseases, PrFneS usually accumulates in the
zones of the brain which will later present histological
lesions responsible for the clinical signs observed . Most
of the scientific community considers the accumulation
of the abnormal PrP (PrPres) as responsible for the destruction of t.henervous system during these diseases . This
AN INFECTIOUS AGENT OFTHE THIRD KIND : THE PRIO N
protein, by resisting normal mechanisms of destruction
of proteins (catabolism), accumulates and becomes toxic
for the neurones . Nonetheless, the mechanisms of neurodegeneration could prove more complex since at least
two studies 25,26 have reported a clear dissociation bétween the accumulation of abnormal I'rY, the anatomopathological signs and the appearance of the clinical
signs . The lesions of spongiosis (vacuoles* associated
with the degeneration of certain neurones) and of gliosis
(the reaction of the glial cells of tlle brain in cases of
damage) seem to be linked to pathological PrP, whereas
neuronal death responsible for the clinical signs has another cause, potentially linked to the multiplication of the
causal agents of these diseases .
50 PrIon, virino and. other
f e es
In practice, no one knows the real nature of thése ex traordinary agents . The prian Îa.yp ot.hesis has the advantage of
simplicity . In its abnormal form, the prion protein is considered infectious and alone responsible for the disease.
However, even though this hypothesis finds favour
among most scientists today, and won the 1997 Nobel
Prize for its originator, Stanley Prusiner, it is nonetheless
still hypothetical, and for each argument in its favour a
counter-argument can be developed (see table p. 54) .
The multiplicity of disease forms, all reproducibly
transmissible with specific characteristics, is unquestionably the most troubling feature, and reveals that there is
something lacking in our understan ding of these agents .
Hence, evidence for several strains of TJTfis (unconven-
PRION, VIRINO AND OTHER HYPOTHESE S
tional iransmissible agents) in a given host implies the
existence of strain-specific information which is transmissible independently of the hosi, and therefore of its
prion protein . It should be recalled that on contamination of a mouse with the causal agents of the different
forms of animal or human diseases, the abnormal protein
which accumulates is the host's and not that of the infected animal or human brain tissue used to inject into the
mouse . The idea of a virus was initially the most logical,
especially as the UTA passed through filters that retain
all microorganisms bigger than viruses . However, despite the very large number of infectious agents (in excess
of 10 thousand million units per gram of brain), no structure suggestive of a microorganism, and notably of a
virus, was visible under the most sophisticated microscopes. Furthermore, there was no immune response, a s
if these agents were invisible to the host's defences . 51
The virino hypothesis was developed by Alan Dickinson
and his colleagues in Edinburgh, in the late 1970s . They
envisaged a hybrid structure comprising a very small
infectious naked nucleic acid (like the viroids of plants)
that codes for no viral protein likely to be recognized by
the immune system . This small nucleic acid may, however, be able to bind tightly to host proteins, in this case
PrPc, which it transforms into PrPCeS (resistant to degradation), which would protect it by forming a sort of shell.
In this hypothesis, the UTA would therefore be a nucleic
acid protected by the host's PrPre$, which is particularly
resistant and inaccessible to the molecular biology techniques available today.
Classically, the presence of the infectious agent is
always associated with the detection of normal PrP.
AN INFECTIOUS AGENT OF THE THIRD KIND ; THE PRIO N
Prion Virina Unideniified virus
(amyloidogenic)
PrP, --~ PrPres small exogenous genome ® éxogenous gehom é
+ protein X? coding only for itself coding for its own proteins
+ chaperone protein? protective shell formed
. PrPreS = product
by a protein derived ofviralreplication
s
from the host = PrPre
The principal hypotheses accepted are :
52
1%tepriçrn : the normal protein (PrP`) is transformed into an
abnormal and pathogenicprotein (PrP'BS) in "theprésencé of an
agent whose nature is currently unknown . This then constitutes
a new infeçtious agent able notably to raodify the normalprotéin .
The viriato: the abnormralprotein (PrPYeS) fornzs a protective
shell around a small unidentified nucleic acid which is the actual
infectious agent .
The virus : the accumulation of the abnormalprotein (PrPT05)
in amyloid deposits in the brain is a simfile consequence of the
multiplication of the infectious agent .
However, very curious results were recorded in éxperimental transmission of theBSE agent to themousé26 .
All mice directly inoculated intracerebrally with a hig h
concentration of homogéniaedbraïn from cattle at the
endstage of }3SEdevelopedclinical signs after morethan"
one year of incubation, whereas over half of them did
not have detectable PrpreS . These mice had identical
PRION, VIRINO AND
OTHER HYPOTHESE S
incubation periods and neurological signs, but presented
either undetectable amounts of PrpreS or large quantities
- at least a 10000-fold difference (sensitivity threshold of
the detection method) . They did, however, all develop a
TSSE .
In secondary transmission, from mouse to mouse, starting with mouse brains without FrP`-, the same phenomenon occurred. All mice developed TSSE, but some
had no PrŸ-s (note that transmission was faster from mice
that had accumulated Prhre,) . The histological éxaminaiion provides the explanation : all the mice presented
signs of neuronal death responsible for the disease . But,
on microscopic examination, only the mice possessing
F'rPles showed the signs characteristic of B S E (spongiosis
and glial reaction) . The hypothesis that best explains
these results is that, under our conditions, the B SE agen t
is sufficientlÿ viiulent at the first passage to be able to 53
replicate without being protected by the PrP1eS . More
effective multiplication would then compensate for th e
lack of protection. However, during later passages, there
was selection of variants that had acquired the property of
accumulating Prpre5. This confers a selective advantage
on these variants, because of the resistance of l'rprPS to
degradation. In our analysis, therefore, FBrFCeS seems to
constitute a virulence factor, but not the agent itself, thus
lending weight to the virino hypothesis .
Classical viral hypotheses (animal viroid, conventional
virus, retxovïrus, amyloidogenic virus, i .e . leading to the
accumulation of i'rI'res) suppose that such a virus is not
perceptible by presént-da.y molecular biology techniques .
Certain hypotheses implicate RP"` as a constituent of the infectious agént. The nûcleation* hypothesis, by analogy with
AN
INFECTIOUS AGENT OF THE THIRD KIND : THE PRIO N
Arguments for and against the prion hypothesis
that abnormal PrP is the agent responsible for TSSEs
These are ampioid disease s
Amyloid fibrils are deposited in TSSEs
and may indoçe the polymerization of
precursors .
The other amyioid diseases are not trans mitted by the injection of amyloid fibrils .
Conversion outside the cel l
Abnormal PrP causes the conversion of
normal PrP irrvitro.
The infectivity of this converted PrP ha s
not been demonstrated.
Strains of unconventional transmissible agen t
Abnormal PrP can adopt different structures.
The multiplicity of strains (20) in mice
runs counter to this structural hypothesis.
Mouse without gene encoding PrP (knock-out )
PrP is perhaps a cofactor at the agent or a
receptor and may play a role in the patho physiology .
No disease nor replication of the agent .
Genetics
Prion diseases are both genetic and infec-
tious .
Mutations of PrP alone may cause the dis-
ease .
Retroviruses exhibit both genetic and infec -
tious determinism .
Mutations of PrP may increase susceptibili -
ty to the disease .
Transgenic mice which overezpross Pe P
Overexpression of a mutant PrP causes a
brain disease:
In these mice there is no abnormal PrP ap-
parent in électrophoresis ; no transmissio n
to normal mice, no disease without overex -
pression .
Irradiatio n
Theoretical calculation shows that the target is too small to be a virus .
According to data on inactivation of viru ses of known size, the target is compati-
ble with a small virus :
Composition of the agent
The infectious fractions contain abnormal PrP and perhaps too little DNA for a
virus .
Over 105 molecules of abnormal PrP pe r
infectious unit hâvebeentletected .
Infectious samples contain nucleic acids .
No virus has been found to date .
New viruses are difficult to find (Born a
Saurce : b. caugney ana H. eneseoro.
virus) .
THE SEARCH FOR ANALOGOUS MODELS : YEAST PRION S
crystallizâtion in mineral chemistry, is an interesting alternative to the prion hypothesis, from which it differs in rate
of formation of PrPleS Variants that bring into play chaperone* molecules (proteins involved in correct folding of
other proteins) have also been proposed as models to
account for the rate of conversion of the prion protein .
A unified theory was proposed by Charles Weissmann2i,
with, in addition to the self-replicating protein, a nucleic
acid-containing `co-prion' responsible for the phenomenon of strains . The hypothesis of genetic disease by
somatic mutation of the gene was proposed to explain
the sporadic cases of CJD . A transmissible membrane
anomaly, as in the paramecium (in which the graft of a
foreign membrane leads to the acquisition of new characters transmitted to the offspring), has also come back
into fashion .
The bacterial hypotheses are based on microscope images 55
suggestive of certain bacteria, particularly the spiroplasts ,
or on the possibility of auto-immune disease linked to
other types of bacteria, the acinetobacter, or even to
involvement of mitochondria* . These hypotheses seem
more clearly artefactual, i .e . biased observations unrelated to the phenomenon studied .
Lastly, a new development has emerged with yeast
prions .
ANINFECTIOUS AGENT OF THE THIRD KIND : THE PRIO N
phenomenon related to the mode of multiplication described in the prion hypothésis : Old observations of transmission of information independently of the genetic
material had been put aside since the pre-eminence of
molecular biology and the study of nucleic acidso The
yeast Saccharomyçes cerevisiae (the famous beer yeast) offers
a model of héreditÿwhich doesnot obeythe classical
laws of Mendelian génetics .Iience, under unfavourable
conditions of development, with a lack of nitrogen
intake, the yeast brings adaptation into play. A particular
protein, baptised Ure2p, self-modifies and becomes inactive and releases other genes which will allow the yeast
to grow using poor sources of nitrogen . This seif-modification mechanism, which is transmissible to other Ure2p
proteins without the intervention of the nuclear D NA
genes (which normally are always called into play, bu t
56 involve additional steps), allows a much faster response
to environmental changes . By analogy with the change in
structure of PrP and the hypothesis of a direct transmission of information from protein to protein, this was
renamed the `prion-like phenomenon' . In February
2001, the CNItS 28 at Gif-str,Yvette determined the
structure of Ure2p, and this should enhance understanding of the role of this type of protein .
Lastly, in filamentous fungi, self-transformation of
HET-s protein is also observed and constitutes another
example of heredity independent of the genorne* .
Triggered in cases of viral infection, this self-transformation prevents the development of theinfectiori by inducing cell death which is propagated in a circle and
isolates the contaminated part from the rest of the rnÿcelium of the fungtzs : This circumscribed suicide proves
THE SEARCH FOR ANALOGOUS MODELS : YEAST
PRION S
very effective, as the mechanism of protection is implemented faster than the diffusion of the virus . Given that
filamentous fungi represent the greatest quantity of living
matter per hectare, heredity independent of the genome,
or `prion-like heredity', is among the most widespread
mechanisms on Earth.
Nevertheless, it should not be forgotten that these socalled `analogue' models, however interesting they may
be, correspond to cellular adaptations and are not infectious. In spite of all the possible analogies, it should be
remembered that the causal agents of prion diseases,
whether solely comprising PrP''éS, as in the prion hypothesis, or possessing a small nucleic acid, as in the virino
hypothesis, behave as infectious agents and resist most
methods of decontamination .
b7
Mad cow disease and the
contamination of human s
The emergence
6
of disease
59
MAD COW DISEASE AND THE CONTAMINATION OF HUMAN S
and-bone meal, and the first cases of B SE were diagnosed officially in 19 8 6 . This shows that recycling and
energy saving, which are theoretically excellent from the
ecological and economic points of view, are not necessarily synonymous with protection of health and the environment, nor with profitability.
Soon after the discovery of the first cases, the British
authorities launched a large-scale epidemiological survey
which revealed an astonishing fact : the incidence of BSE
in cattle of the same race vrastotally different injerse
. A single parameter differed : the manu-yandGuers
facture of meat-and-bone meal . This meal could contain
sheep (15%) and cattle (15%) carcasses, and certain
batches seemed to be contanoinatéd b y unconventional
transmissible agents or prions : FroiTw July 19 88 , the
British authorities banned throughout Britain the feedin g
60 of meat-and-bone meal to cattle . This was the first time
that the precautionary principle* was applied on a large
scaTe . In contrast, the export of contaminated meat-andbone meal was not stopped, but continued legally for
almost ayear to the other countries of Europe, principally France, and to the rest of the world .
The detection of the first cases of BSE in 1986 marked
the start of a veritable `epideixa .iç explosion' which peaked in 1992, with over 37000 cases officially recorded,
ie . over 100casesdai[y. Between 1986 and 2001,fôrâ
.5 million, more than 1$ 0 006 bea .catlepouinf1
B S E, over 70°lo of them among dairydhaiefrom
herds .
I.ike sheep and goats, cattle, after a périodof incubation of between two and eight years (on average five),
first exhibit behavioural changes : apprehension, reluc-
THE EMERGENCE OF MAD COW DISEAS E
tance to enter the milking shed, kicking during milking,
and separation from the herd while grazing.
Subsequently, there are neurological disorders of posture; movement and sensation: swaying gait of the hindquarters (reminiscent of the pitching of a boat), high
stepping gait of the hind limbs, erect tail . Another characteristic sign is that the animal overestimates distances
and therefore makes inappropriate movements : it may
jump a simple rivulet as if it were a ditch . This inability
to judge distances explains the animal's anxiety and
several other behavioural traits . The animal also has difficulty getting to its feet from a lying position . It suffers
from hyperaesthesia, an abnormal increase in sensitivity
to noise (such as handclapping), light (for instance, a sudden transition from a dark to light area), and touch, with
excessive licking of the muzzle and flanks and rubbing of
the head.
At the later stages, the behavioural signs are completed
by an abnormal position of the ears and fits . The neurological signs are apparent in trembling, teeth grinding,
lack of coordination (ataxia*) of the hind- and then forelimbs, increasingly frequent falls and prostration (the animal remains lying down), with deterioration of the
general condition (weight loss and reduced milk produ .ction) . The course of the disease generally runs for between one and seven months . The animals were initially
slaughtered because of dangerous behaviour, serious
injury following falls, or deterioration of general condition due to prolonged prostration . ." . =
The cliniçal picture is often incomplete, as only certain
signs are present and bacterial diseases (such as listeriosis) or viral diseases (rabies, Aujesky's disease) can give
61
➢v1ADCOWDISEASE AND THE CONTAMINATION OF HUMAN S
similar symptoms . In practice, the disease may be confused, mainly in the spring, with metabolic* diseases
(hypomagnesaemia, or grass tetany, and a nervous form
of ketosis), andup to 25% of the animals slaughtered in
the United Kingdom at this time of year because of suspected B SE were in fact free of the disease, as shown by
histological examinatïon .
The histological lesions in the brain are principally
found in the sensory and motor nuclei of the vagus nerve
(zone containing all the bodies of the neurones composing this nerve) . These nuclei are situated in the brain
stem, in a structure called the obex29 . The brain stem is
at the base of the brain, beneath the cerebellum, and
links the spinal cord to the evolved brain structures .
62
On microscopic examination, the characteristic
vacuoles, and notably the triad spongiosis-astrocytosisneuronal death, are sometimes difficult to demonstrate ,
and detection of FrF-s by immunohistochemistry* is particularly valuable . Using antibodies, this technique localizes under the microscope the deposits characteristic of
the abnormal form of the prion protein . BSE can now be
diagnosed in a few hours, using rapid tests (see below) to
detect FrPres . Confirmation is possible using another biochemical technique such as the Western blot, with a fresh
or frozen sample, or by histological analysis of a tissue
sample fixed in formol .
It is not known whether the contamination stemmed
from the carcasses of contaminated sheep (1/3 of flocks of
sheep in Britain may be affected by scrapie) or from cattle
with an unknown sporadic form of TSSE. Be that as it
may, the cadavers of animals infected by the BSE agent
were recycled into meat-and-bone meal, thus resulting in
THE EMERGENCE OF MAD COW DISEAS E
SSE distributiun in Europe
Denmark
cases ong 0 .9 million
d Kin
tlom
9
D i .ses/million"
Repuhlic of Ire among 5.3 millio n
1 232 cases amo r st' : cases/millron ~!
i.e. 362 case
Netherlantl ~
~9casé MW
c~~~
.` "
. ~ •~~~;.~x~;~ `
B
eas
6 millio n
milliori
~â ~
~
~~
W~
~
6
r^
.
~
~
~~`~
?~Âp
3 .4 million
P
aa~ on
63
Cattle over two years of age were taken into account .
Data available on June f, 2002
w
~:..
Years when the first case ®i S E
was diagnosed in different European countries
Fnfa~
An~ia
fimubli c
N~iards Qermark Slar~a
y1y~ Be19Nm Spain Slovenia
LaenbairgI ~~Y Græce Polard
UnitetlfGr~dom Irelantl Frar~
Liechltenslein 'aly Israel
1
1986 1 9B7 18$8 1989 1 990 1991 1992 1993 1994 .1995 1996 1997 1990 1 M 2000 200 1 M
the contamination of the whole British beef population .
Only Scotland, which had continued using the old
methods to manufacture meat-and-bone meal, with hightemperature processing, was spared until 1988 .1iie meatand-bone meal was manufactured from unsalable animal
MADCCW DISEASE AND THE CONTAMINATION OF HUMAN S
remains from the slaughterhouse . So, after grinding and
heating, the dried meal was powdered and used as a protein supplement by the manufacturers of livestock feed.
Animal remains were first recommended as a source of
protein for livestock (pigs) in 18 6 5 . In 1908, an industrial
64
process for theinanufacture of carcass meal was described by 0 . Kellner in a book which was translated into
English and published as The Scientific Feeding of
Animals in 1915 .'I'be meat-and-bone meal was deemed
usable in ruminants in 1926 in the United Kingdom and,
from 1930, different reports indicated that it promotes
the assimilation of protein inthé cattle (and even sheep,
in Australia) and so increases the quality of milk production . At the time of the restrictions of the Second World
War, the United Kingdom prescribed the inclusion of at
least 2 . 5% of-meata:nd-bonë meal in feed for ruminants,
and 5% for calves . Use of meat-and-bone meal expanded
to allow different countries to raise their milk production,
notably following the European policy supporting the
The bovine spongiforro erfcépha9opathp (B 5E)epidemic
~ yn ? ~ Sheep
Shéep ? _
Fannetl
Cspec~ liflca6on niminani
sbYre
r~I'ycl i' COw
Gat D Gheetah Puma
(Àotelop)e
Elk Gazelle
Carnivores Wild ruminants
The source of contaanination is unknown (sheep, cow, or other) .
The farst cattle affected were inco ~p orated into meat-and-bone meal
and successive recycling led to an anipli fication of the irfèctious agent,
which in the United Kingdom contaminated over 180000 cattle
and many other sp ecies .
THE EMERGENCE
OF MAD
COW DISEAS E
Transtnissi®n of scrapie
~rM onke,Y
SheE amnivarés
_
_._.. ..
._ Min kJ
~~
Ruminants
Camivores
Rodents
~ Oral transmission
passlble between specie s
Dral transmissio n
Transmission of ffSE
kiuman
betweénspecies
lntracerebral transmissio n
netweenspecles
~~- Monkey
S hoep
~, . pi g
,,,
-
Qmnivore
--"
s6oatcw
Mink
Elk Antelope Cheetah
Ruminants Pum
t aC
MQUSe Carnlvores
Rodents
Unlike scrapie, bovine spongiform encephctlopathy (BS.11`) is transmassible by the oral route to numerous sper.ies of mammals and poses
completely new public health problems .
milk market and limiting the number of animals (with
the aimof maximizing production per animal) .
According to MAFF (the Ministry of Agriculture,
Fisheries and Food, whose functions have since been
65
MA6COWDISEASE AND TNECONTAMINATION OF HUMAN S
taken over by the Department for Environment, Food
and Rural Affairs), the annual production of meat-andbone meal was between 350000 and 400000 metric tons
in -195$, 90% of which was fed to pigs and poultry and
Îa% to ruminants 30 .
To increase profitability, animals found dead (and often
in a state of putrefaction) and even the mud of watertreatment stations were incorporated into meat-and-bone
meal (the dissemination of B S E through meat-and-bone
meal is discussed below. )
Cases of TSSE linked to the BSE agent occurred in
antelopes in zoos and in various carnivores including the
cat (the first cat with spongiform encephalopathy was
diagnosed in May 1990 in England, and beef offal was
banned in pet foodin September 1990) . The bovine origin ofthis new feline disease was demonstrated by cha66 racterization of the strain in the mouse. To date, nearly
90 cats have officially developed sporagifos-rn encephalopathy in the United Kingdom and one in Norway (fed
with various imported foods), only seven of them born
after the official ban on offal . This new strain of UTA
seems more virtalent than the classical strains of scrapie .
The team of the epidemiologist Roy Anderson, in
Oxford, estimate that approximately 900000 British
cattle incubating BSE entered the human food chain
before March 1996, including nearly 400 000 prior to the
ban at the end of 1989 of high-risk offal (specified risk
material), principally the brain and spinal cord . These tissues were not used directly but could be incorporated,
depending on th.e country, into various preparations,
such as meat pies, sauce thickener, sausages (some
contained up to 10%), certain hamburgers, pâtës and
A NEW CREUTZFELDT-JAKOB DISEAS E
more generally all preparations based on mechanically
separated meat (reconstituted meat,raviolis, couscous,
numérous cooked dishes, meat-based sauces, etc) .
re tzte1 t-Jakob diseas e
The first cases of a new form of Creutzfeldt Jakob
disease in humans appeared in Great Britain at the end
of 1994, and 10 cases had been confirmed by March
1996. All these cases were first characterized by an
abnormally young age at onset (29 years on average),
whereas all epidemiological surveys had confirmed that
CJD was extremely rare in people under 40 years of age,
the onset generally occurring around 60 to 65 years of
age. The clinical form was also wholly unusual and
highly stereotypical, with early psychiatric signs and a
prolonged clinical phase (on average sixteen months,
ranging from nine to thirty-eight months, compared with
a mean four months for the classical forms) . In essence,
the disease started with psychiatric signs, followed two
months later by sensory signs, then by lack of coordination from the fifth month, muscle contractions from the
eighth, loss of speech and movements (mutism and akïnesia) from the twelfth, followed by death frequently linked to an episode of bronchial pneumonia .
On examânationof the brain,even more characteristic
signs demonstrated the uniformity of these cases : in addition to the classical signs of CJD (spongiosis, astrocytosis
and loss of neurones), all patients presented numerous
florid plaques consisting of a deposit of Pk-PTeS surrounded
by vacuoles giving them a daisy-like appearance . These
67
MAD CQW DISEASE AND THE CONTAMINATION OF HUMAN S
signs were totally new and re-examination of old sections
of the brains of patients who had died of classical CJ D
confirmed that this appearance had never been observed
before .
It was not possible to demonstrate any particular risk
factor, whether medical (no mutation of the PrI' gene was
detected by molecular biology techniques, nor was there
any history of trcatment with pituitary-derived human
growth hormone), occupational or dietary . The sole risk
factor was living and eating in the country which accounted for 99.7% of all cases of BSE in the world (approximately 165 000 cases of B S E in the United Kingdom in
October 1996, compared with under 500 in the rest of
the world) and in which an estimated 400000 plus contaminated cattle had entered the food chain before brain
and spinal cord were banned .
68 All the patients had a genetic particularity 31 . These
highly compelling arguments pointed to a contamination
of humans by the BSE agent. Two months later, in June
1996 ; the firstexpérimentalproof 32, was furnishedbyth
ediscovryfthamsceislonmaqu
experimentally inoculated with the BSE agent . This was
the first evidence that the B S E agent was the same as that
which is responsible for new cases of CJD in humans
.InOctober196,anewxprimentalsudy3bJohn
Collinge and colleagues in London demonstrated that
the two agents are similar. The biochemical `signature' in
the Western blot of l'rl'rS is characteristic in patients with
so-called new variant Creutzfeldt Jakob disease (nv"CJI))
and similar to that of the BS E agent in other animal species, including macaques . 7hesarne signature was later
found in French patients 34 .
A NEW CREUTZFELDT-JAKOB
DISEAS E
Nuenber of persons affected annuafly
by new variant Grentzfe&dt-Jakob disease
30
25
20
15
10
5
0
1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000
MR, United Kingdo m
" France
Irelan d
One year later, in October 1997, the final demonstra- 69
tion"~' was reported by the team of Moira Bruce in
Edinburgh . The pattern of lesions in the mouse is identical . The agent responsible for nvCJD in humans is the
sarne as that which caused 180000 cases of BS E in cattle
and new cases of encephalopathy in pet cats, and in
various ruminants and carnivores living in captivity.
The only logical route of contamination was dietary,
and the most infectious tissues (brain arid spinal cord)
had been incorporated into a whole range of cheap prepared foods . As an example, the experts of the European
Community have calculated that, depending on the
method of manufacture, with mixtures ranging from kilograms to metric tons, the central nervous system of a
contaminated animal could end up in the plate of fewer
than 50 people in the case of a pkë and over 500 000 fo r
MAD COW DISEASE AND THE CONTAMINATION OF HUMAN S
ravioli. Eating habits, authorization of incorporation of
these highly infectious tissues, and the theoretical and
actual dates they were banned, vary greatly from one
country to another. Brain and spinal cord were banned
for human consumption in November 1989 in the
United Kingdom, in June 1996 in France and in
November 2000 in Germany.
The minimum infectious dose for humans is unknown
and so it is particularly hard to predict the number of
future cases of nvCJD . In 1996, early models indicated a
range of fewer than 80 cases to over 500000 . In 2000
estimations were more optimistic since the range fell to
63 cases to 136000 . Since then, newer modelization studies even came out with an upper limit of 403 cases . This
extraordinary imprecision stems from the fact that we do
not know the average incubation period of nvCJD . If
70 short, the cases observed correspond to most of the
contamination, and the situation will improve . If, however, this period is long (and it is worth recalling that it
can reach forty years for kuru, and that with BSE it may
be extended further, because of the species barrier between cattle and humans), the observed cases would
unfortunately represent just the tip of the iceberg . The
minimum number has already been exceeded since, at
the end of March 2001, 96 cases had been recorded in
the United Kingdom . As the observed increase was 1 .23
a year, the number of cases could be expected to double
every 3 .3 years36 . In such a climate of uncertainty, what
are the risks to humans and what rational solutions ca n
be envisaged?
The risks to humans
71
THE RISKS
TO HUMAN S
since this date3s. Countries like Belgium, th e
Netherlands and also France sharply increased their
imports of British and Irish meat-and-bone meal .
Television reports revealed the numerous possibilities of
illegal sales between Northern Ireland, which was highly
contaminated, like the rest of the United Kingdom of
which it is a part, and 1?'zre ; that is the Republic of
Ireland, officially pronouncedI35E-free by theFuropea
. This authorized the renewe d
nComuity193
export of ineat-and-bone meal, These countries could
legally reprocess the meat-and-bone meal, which thus
automatically received the label of the intermediate
country. A top Belgian dealer sought by Interpol was
even filmed by journalists r in Brussels, but could never
be apprehended. Switzerland, which had virtually never
officially imported British meat-and-bone meal, but had ,
72 so it seems, bought Belgian and French meal, very soon
had the unenviable privilege of counting the largest number of cases of BSE in Continental Europe (118 between
1990and 1994,versus 17 for the same period in the
European Union, barring the United Kingdom, Ireland
and Portugal) .
This well illustrates the complexity ofthe legal and illegal distribution circuits, and the switching of labels that
may have occurred . This information, which was subsequently published in the British press, at the time was
impossible for scientists to oÉatain.
In the absence of suitable protective measures, any
country in the world could have been exposed to BSE,
via British meat-and-bone meal, through products from
countries that recycled British meal and therefore contaminated their own livestock and meal, or via other pro-
MEAT-AND-BONE MEAL FED TO OUR
HERBIVORE S
ducts (bovine and ovine animal fat) imported directly or
indirectly from the United Kingdom or from a country
indirectly contaminated .
Examples of export of British meat-and-bonemeal
in metric tons after the 1 6 July 198 8
bau on their use for câtt9ein the United Kingdom
A
4
~
TMW
~ÇŸfi~kt~7~~
2 555
900
- 65 %
485
7222
15 674
+ 120 %
~ 20
274
1 605
+480 %
740
1
1 826
6 099
+ 234 %
1 689
15 6
2795 ~
559
578
+3%
5
5
92
2 718
+ 2 854 %
9 818
400 8
5
3462
020
20 339
<,
In~ ~~~1 ;
Souwce : lniernet sife o1The Sunday Times .
3
7
The meat-and-bone meal said to be safe as defined in
1996 -- derived solely from animals fit for human
consumption, after elimination of high-risk offal and
treatment at 133° C, under 3 bars, for twenty minutes constituted a feed rich in proteins perfect for pigs, which
are omnivores, and poultry. Its replacement by other
sources of protein, notably transgenic soya, is not necessary if the industry is well regulated . Yet this is precisely
the weak point . Regulation is not assured, as eross-contamination can occur at various levels (manufacturing
plant, transport, storage, : .) and large-scale illegal sales
have not been stopped, and sometimes have even been
sustained or increased . In mid-1996, the United
Kingdom finally banned meat-and-bone meal in all ani-
THE RISKS TO HUMAN S
74
mal industries because it was not possible to stop it entering livestock feed . In France and the rest of the
European Community this meat-and-bone meal was
widely distributed until a ban was imposed on 1 January
2001 . In this way, British meat-and-bone meal, which
allegedly could not be exported far because of the cost of
transport, found its way as far afield as Indonesia . It is
therefore worth distinguishing the health problem from
that of overseeing the application of the recommended
measures and stamping out illegal practices .
That said, once the meat-and-bone meal is banned for
all animals, and therefore no longer of economic value,
there remains the vast problem of waste disposal. France
annually produces 2 . 5 million metric tons of untreated
waste (1/3 ruminants, 1/3 pigs, 1/3 poultry) that before
was usable in animal feed, and 0 .2 inillion metric tons of
specified risk material, notably containing high-risk
waste, such as the brain and spinal cord of ruminants .
Because of the volumes of waste generated, incineration
poses major technical problems, and so waste is stored
under frequently insalubrious conditions and is a cause
for great concern among those living nearby.
In practice, a distinction should bemade between :
® meat-and-bone meal from high-risk tissues (central
nervous system and/or cattle confirmed positive on testing for BSE), which account for a small part of the
volume and shouldbe treated as high-risk waste ;
• meat-and-bone meal from the tissues of animals not
suspected to have BSE but declared unfxt for human
consumption, which can be eliminated as low-risk waste ;
® safe meat-and-bone meal, from tissues declared fit for
human consumption, which can be used in feed for chic-
CAN WE EAT BEEF PRODUCTS ?
kens and pigs (which have always eaten human waste on
farms) . This meat-and-bone meal, which is waste posing
no particular risk, should be treated as such . It accounts
for over 90% of the volume of waste produced . Although
it is a great pity to destroy high-quality products, this
phase is without doubt indispensable as long as we cannot guarantee the amelioration of food production and
distribution practices.
Storage problems can be solved bylrmiting volumes. It
is also more economical and rational to mobilize the
available means to destroy waste that represents a real
danger.
eat beef products ?
It is known that the risks to humans were underestima- 75
ted and without doubt weighed against major economic
interests . Exports to Continental Europe of British liv e
cattle or beef products doubled between 19 88 and 1995
(1.20000 metric tons in 19 88 and 250000 metric tons in
1995) and represented approximately 700 million euros
over the period 1989-19963 9 . The British Prime
Ministers of the time, Margaret Thatcher and then John
Major, openly threatened to block all European
Community business, thereby potentially stalling the setting up of the single market scheduled for 1993 . In
Brussels, misinforming the media was officially sanctioned I so as to hush up the mad cow issue and avoid a spanner being thrown in the works.
In 1989, the Southwood Report, ordered by the British
government, concluded that the transmission of BSE to
THE RISKS TO HUMAN S
Cases of B SE diagnosed in th e United Kingdom
,. .~
ô
40000
35 000 t p Ç
E
30 000
E
° 25 000 E .
20000
E
1500
001
5 000
tvM,
m Q
°
ô
;a
E-Z
1986 1987 1988 1989 1990 1991 1992 1993 1994 1995
Nfe at -and-bone meal exported bytlte United Kingdo m
40 000
~
30 000
ô
200D0 E
10000
1986 1987 1988 1989 190 1991 1992 1993 1994 1995
u~ ExporCedto Continental Europe M Exported outside Europ e
Impact of the ban on nneat -and-bone meal in Continental Europe
(e xa mp9eofSwitz e rland)
~ 100
~
°
9D . v
e
,
ea
ro 80 ~c ° ~
70
K'.É a G m
60
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; .r c C E
50
®
40 m °
30
•
E 20
10
=
I
,,
E
w K
r~'2
1986 1987 1988 1989 1990 1891 1992 1993 1994 1995
Cases of BSE detected In Sw itzerland per year of birth of the cattl e
The animals developed the disease on average fi re years later .
GZ~] Passive diagnosis (clinical diagnosis)
Active screening (tests on at-risk population )
Systematic screening at the slaughterhouse of all cattle aged over 30 months
CAN WE EAT BEEF PRODUCTS ?
Beet products and live cattle exporte d
by the United Kingdom to Continental Europe
300 000
°
250 000
ô 200 000
~
150 000
100 ODD
Correspondence between the appearance of 9SE
in the United Kingdom and contaminations in Franc e
16000- 12 000
1 4 00
0
10000
12
000
~
E
~ 10 000 8000 ~
~
8
6000 ô
00o
® 6000
-
4
000
4
00
200 00°2
É
0
0
1`b 1`JtSr lytfi5 1`~aJ lyyu 1y91 15yL1Jy
É
;S 1994 1995
British oases of BSE in 1988-198 9
British cases of BSE incattle born afterthe 18 July 1988 ban on meat-and-bone rneal
--- British meat-and-bone meal officially exported to Franc e
100
_
90
°
80
E
76
~
-
°
~
v
° a4 a0
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C
~
ro~
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£
c
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° 30 eoEQ mE
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0E
z 10 0
L ..
1986 1987 . 1986' 1989- .1990 1991 1992 1993 1994 1995
Cases of B SE detected in France per year of birth of the cattl
.eThanimlsdvopteanrgfivyesÎat
1: Passivediagnosls (clinical diagnosis )
2 Active screening (tests on at-risk population
) Systematic screening at the sÎaughterhousé of all cattle aged over 30 months
THEFiISKS TO HUMAN S
Change in the method of mahufactura of
So % meat-and-bone meal in the llhlted K6oqdote
7Q
sa -----°-------------50
40
3Q
2Q
iQ
Q
o ~ cu co ~~n w r..
w o~i
le ~2 ?_>
ô o~aô
M
ôo~ ~m m ~ ç Co Co
crs m rn oc w m al o> ?
2
4 t ¢~~~
iR
I Ï'l' I 1"l I I 1Firs
"'
tfirs trecylingsecond etc d
contamination recycling case of BSE
Proportion of rnéat-and-hone meal rnanufaaured --- Proportion of ineat-and-6one meal
using new continuous production methods manufactured using solvent extractio n
humans was highly improbable40, and the powers that
be took the political gamble of assuming that B SE was
not transmissible to htùmaras. The impact was felt at all
levels in the implementation ofprecautionary measures .
What really was the risk? What is the risk now? Can we
still eat meat? What else can We eat ?
What with the mad cow crisis, Belgian chickens and
pigs fed dioxin-tainted animal feed, cheeses and cooked
meats contaminated by Listeràa, and txansgenic plants
(the enignaatic GMOs), it9s a wonder there are any
Europeans left! This situation is all the more paradoxical
as, overall, the life expectancy of the European population continues to rise. Despite the lack of transparency of
the food industry and the worries o€consuahers ; epitomi-
CAN WE EAT BEEF PRODUCTS 7
zed in the notion of junk food, the healthfulness of foodstuffs is constantly improving4l . In France, of 531618
deaths in 1995, only 737 may possibly have been linked
to the consumption of toxic or contaminated food, most
of them (631/o) due to an ill-defined intestinal infection .
It is inadvisable to drown one's sorrows in alcohol,
considered to be responsible for nearly 24000 deaths a
year in France (principally by cirrhosis of the liver and
by cancer of the upper airways and oesophagus) . As for
the pleasures of smoking, the price tag is even higher,
with nearly 60000 deaths every year in France .
In view of these figures, the number of cases of nvCJD
seems derisory, with but three in France since 1996,
compared with 300 deaths over the same period from
the classical form of QJL? (which occurs in all countries
around the world, independently of the presence of BS E
or scrapie) and 120000 deaths linked to immoderate 79
consumption of alcohol .
But is this apparent absence of contamination of the
French population misleading, particularly as the trend
in British cases indicates no improvement with time? As
the length of incubation of nvCJD is inversely proportional to the infectious dose, the cases that we see today
are those in which contamination occurred early and at
the highest doses . Future cases will probably be spread
over several decades, in numbers difficult to predict : tens
to thousands of cases in the United Vingdom, according
to the most recent mode142 ; over 100000 if the mean
incubation period exceeds sixty years, although this
seems exaggerated . In France, forecasting of the number
of future cases is very approximate and is done by dividing the British figures by 10 to 20 to account for the
THE RISKS TO HUMAN S
imports of Briti sh beef products which entered the
French food chain a .p to the embargo of 199 6 . For other
European countries, like Germany, not only should
British imports be taken into account, but also undiagnosed national cases of BSE, bearing in mind that brain
and spinal cord were authorized for human consumption
until the end of 2000 (brief reminder : some 100-gram
sausages contained up to 10 grams of bovine brain) .
Lastly, the circulation of Tritish contaminated .meat-andbone rnealin distant countries may have caused a new
cycle of amplification of the agent and exposure of the
inhabitants of these conntries .
The risks to hm.nans were greatest in the United
Kingdom during the period when tissues known to be
highly infectious entered the food chain . To the end of
1989, brain and spinal cord were used legally, eithe r
80 directly, in numerous prepared dishes, as a sauce thickener, for example, or indirectly, in xneat separated medianically (meat removed from bone, notably the spine, by
mechanical grinding, rubbing ; and high-pressure treatment) . The composition of minced meats and hainburgers, regularly suspected, varied greatly depending on
the country, French regulations being among the strictest . In the United Kingdom, it wasonly frorn April 199 5
onwards that this offal was made unusable by colouring
with an indelible blue stain, and mechanically separated
meat was banned in 1996 .
It is worth remembering that, at the same time, the
British authorities were convinced that BS E presented
no risk to humans . In 1994, John Major, then Prime
Minister, confidently asserted that there was no danger
to humans and, in 1995, the Minister of Agriculture
CAN WE EAT BEEF PRODUCTS ?
made his small daughter eat a hamburger before the television cameras of the entire world .
It is possible to establish a classification depending on
the period of exposure to the contaminated nerve tissue :
® before 1989-1990, brain and spinal cord could legally
enter the human food chain in the United M ngdom (up
to November 19 8 9) and in France (up to February 1990),
whereas the exports of high-risk offal from the United
Mngdom (including therefore brain and spinal cord)
were multiplied 10- to 20-fold from 198713 . The systematic recycling of all usable items raises the question of
how this impacts on the degree of contamination of the
human food chain, in Britain and in Continental Europe .
® between 1990 and 1996, measures excluding nerve
tissue were slowly and variably introduced, while the
number of cases of BS E sky rocketed in the United
Kingdom (from 14407 cases per year in 1990 to 37280 8 1
in 1992 then 24436 in 1994), exports doubled and the
authorities maintained that there was no danger to
humans ;
® from mid-1996, the start of the crisis generated real
awareness, the embargo protected the rest of Europe,
whereas the ban on the consumption of cattle over 30
months of age was costly but effective for the British ;
= since the 1st ofJanuary 2001 in France and Germany,
and from Jtrly 2001 in the other European countries, all
cattle aged over 30 months are tested before they c an
enter the human food chain . The level of guarantee is
very satisfactory for the best cuirent test and will also be
no doubt for future tests . On France's initiative, this test
will soon be applied to all cattle aged over 24 months, as
is the case in Germany. These protective measures have
THE RI9KSTO HUMAN S
been added to the previous ones, and if some prove to be
unnecessary (like the ban on calf's sweetbread), others
must absolutely be maintained (elimination of the brain
and spinal cord) .
In protecting themselves against the BSE agents,
consumers needed to ask which foods could contain
brain and spinal cord . Today, markets no longer offer the
banned nerve tissue (such as amourettes, the gastronomic
name for spinal cord, which were eaten notably in the
Lyon region of France) . Nor can such tissue or mechanically separated meat any longer be incorporated
untreated into prepared dishes. Processed products likely
to contain brain and spinal cord are now strictly controlled, above all those of French origin. There is no risk attached to unprocessed red meat .
It thus becomes possible, in the light of rational exami82 nation, to analyze the outbreak at Queniborough, in
Leicestershire. This small English village was noteworthy
because of the abnormal frequency of cases of nvCJD (5
cases appeared between August 1996 andJanuary 1999) .
The epidemiological survey revealed that all five cases
had a point in conimor►. all the persons contaminated in
the mid-80s patronized the same butcher, just one of
several suppliers of meat . The press announcement of 21
March 2001 pointed to contamination of the knives used
by the butcher, who slaughtered the animals himself .
These knives, which had therefore been in contact with
the spinal cord, would then be used to cut up the meat
served to customers. But this explanation does not bear
scrutiny, because if simple contamination of a knife is
enough to transfer to a steak a dose fatal to humans, what
of quantities 100 000 times greater in the spinal cord and
CAN WE
EAT
BEEFPRCDUCT59
THE RISKS TO HUMAN S
he nonetheless had access to 200 grams of spinal cord
which could be used legally in various culinary preparations . The first cases observed were naturally at the highest
contaminating doses . The first information collected by the
survey commission of the French Senate44 indicated that
this butcher did use the spinal cord in his preparations .
It is important to recall that a simple ban on high-risk
offal in catfood has virtually eradicated the epidemic of
feline spongiform encephalopathy.
All this allows us to stress that the human cases observed currently in the United Kingdom and France are linked to contaminations that occurredbefore the ban on
brain and spinal cord in human food, and so correspond
to patients contaminated by high infectious doses. Since
1996, the United Kingdom ban on human consumption
of cattle aged over 30 months and the embargo on British
84 beef products for the rest of Europe have constituted
additional and theoretically highly effective protective
measures, despite the illegal practicestha,t have certainly
occizr.rred . Henceforth, systematic testing at the slaughterhouse constitutes a safeguard which, if optimally applied
in all countries, will ensure remarkable protection .
In March 2002, Stanley Prusiner's team published
the finding that the abnormal prion protein was present in the muscle of mice contaminatéd . by th.e scrapie
agent, thus almost triggering a new wave of anxiety . In
France this concern was soon put into perspective
when the most sensitive rapid tests showed that the findings in mice changed nothing regarding what we
already knew, that the abnormal protein is not detectable in the muscle, or even in the lymph nodes of cattle
(unlike lymphoid tissues in other animal models) .
FIRST THE MAD COW, NOW THE MAD SHEEP ?
The problems that may arise hereafter will depend on
guaranteed implementation of the most suitable measures and not on any lingering uncertainties .
irst the mad cow5 now the mad sheep ?
British sheep were fed the same contaminated meatand-bone meal as the cattle . As sheep are very sensitive
to oral contamination by the B SE agent, it would be very
surprising if no anim.al developed BSE . But its development in sheep cannot be distinguished from that of scrapie, and so it could easily pass unnoticed . The presence
of the BSE agent in numerous peripheral tissues (lymph
nodes, tonsils, spleen, intestine) raises the fear not only of
a risk of persistence of the disease in an endemic form ,
but also that it exposes humans to a greater risk than with 85
cattle . British sheep continued to be exported around the
world, notably to France, up to the epizooticX of footand-mouth disease, in early 2001 .
On 1 8 October 2001 what had hitherto been just a
theoretical risk suddenly became reality . The media
announced that the BSE strain had been identif"iédby
British scientists in a mixture of brains removed from
scrapie-affected sheep in the early 1990s, and that the
British Government had submitted to the European
Commission a plan for the slaughter of the entire
British sheep population . Models predicting how
many human cases could be expected would now
have to be revised since they did not take into account
the possibility of exposure to BSE in sheep . The
extension of systematic screening to sheep was
THERI3K3 TO HUMAN S
urgently considered in Europe so as to confi .ne to tlhe
slaughterhouse all animals with scrapie and potentially with B SE . Yet on analysis the partial data released proved most surprising, since it would seem
technically impossible to isolate by passage in the
mouse the BSE strain from a mixture of over 2000
sheep brains, unless many of these were contaminate
B SE. And if such contamination were the casedby ,
then previous screening for B SE in British sheep,
albeit very limited, should not have been negative .
The explanation emerged a few days later : it appears
that sornetubes had inadvertently been switched, and
the British researchers had in fact analyzed the brains
of BSE-infected cattle !
However, it would not have been surprising to find
at least one BSE-infeçted sheep brain among the mil86 lions of sheep in the United Kingdom that had bee n
exposed to the same meat-and-bone meal as the cattle
at the end of the 1980s. The number of tests done
today augments the likelihood of finding at least one
positive animal among old samples . Yet the important
point is to ensure that there is no B SE in sheep today
and tlaa.t ad hoc tests are being done .
The t' caut Î ar
sil or umbrella ?
f!
cipl
w
Most of ihe measures taken in France were done so in
the name of the precantionaryprir►çiple : In theory, the
principle is excellent, and allows the required conservative measures to be taken without waiting for irrefutable
THE PRECAUTIONARY PRINCIPLE : SHIELD OR UMBRELLA ?
proof of a danger. It now seems inconceivable that this
principle was not applied in Great Britain by the governments of Mrs. Thatcher and Mr. Major. However, its illjudged application can be especially çounterproductive .
In the beef industry, and shortly the sheep industry, the
application of the precautionary principle leads to regularly announced new measures which fuel a sense of
uncertainty. If new measures are needed, this could be
construed as indicating that the previous measures were
inadequate, despite all the affirmations. Doubt then arises
as to the safety actually provided by the whole system .
Because of this loss of confidence and in spite of the
diagnostic possibilities of the tests, hundreds of thousands of animals over 30 months of age are sacrificedjust
to maintain meat prices in Europe . These spectacular
measures may end by completely ruining the economy
without providing real guarantees for the consumers 8 7
(who may think that the slaughter is proof that they have
been put at risk), and without really protecting the cattle
and sheep inidustries . Certain researchers even predict
that there will be more suicides among farmers who feel
misunderstood and desperate than deaths due to contamination by the BSE agent . Moreover, the money
squandered in this crisis (now hundreds of millions of US
dollars) could have been used much more constructively .
Paradoxically, the great improvement in the control of
the quality of our food and the policy of transparency
regarding anomalies lead to a sense of insecurity, whereas in fact they are the reflection of a marked improvement in food safety . Should we then conclude that it's
best to hide the truth since the consumer is undiscerning?
This would be a gross mistake, compounded by a
THE RISKS TO HUMAN S
somewhat disdainfu.l attitude . Everything always comes
oû.tin the end andisarnplil°red int.he media. Confidence
is precious, often slow and hard to build, and can coanpleteiycollapse overn,ight. Instead, consumers should be
informed and educated to enable them to bring their
own judgment to bear on the risks involved .
Contamination of the environmen t
Apart from the food risks, there is also the problem of
potential contamination of the environment . This has
long been known in the case of sheep,with pastures
contaminated b y Lhe scrapie agent. This natural contamination of the soil can be explained logically in three
ways : b y contacfiwith infected placentas on parturitio n
88 (limited by the fact that the ewe very soon eats the placenta), by an unidentifiéd animal reservoir, such as acarians (hay mites were incriminated, but this experiment
has not yet been reproduced), or by faecal matter.
Experiments have never proved the infectious nature of
sheep and mouse excrement, but were of very limited
sensitivity due, in part, to the large volume of faecal
matter produced daily (whence a great dilütion of the
infectious agent to bé found in the faeces, making detection by our techniques difficult), and in part to the
impossibility of using correctly the methods of detection of prions by inoculation of the mouse (the injection
of concentrated faecal matter into the brain of laboratory mice results in the animal's death within a few
hours) . In contrast, as it is clearly demonstrated that the
scrapie agent is foundthroughout the gastrointestinal
CONTAMINATION OF THE ENVIRONNEMEN T
tract of sheep (it is agreed that PrPLC9 accumulates in the
lymph cells of the digestive tract, that they are dispersed in the walls of the digestive tract, or grouped in
Peyer's patches in the terminal small intestine), it could
theoretically be eliminated in the excrement with the
target cells .
Brown and Gajdusek published an experiment7 in
19 9 1 in which they mixed earth with the brain of a :
hamster infected by a strain of experimental scrapie .
Part of the mixture was stored at -80 °C, and the rest
was placed in a flowerpot, above layers of sand and
earth, and buried in the experimenter's garden at a site
exposed to the weather, notably to rain . After three
years, the flowerpot was dug up, the surface earth recovered, together with the lower layers, and the
samples kept in the freezer were thawed . These three
samples were then diluted and ilajected into hamsters of
different groups . The hamsters developed scrapie when
the samples were infectious. The earth contaminated at
the surface of the flowerpot remained highly contaminated (60-fold decrease in infectiousness for an initial contamination of approximately 100 million of infectious units per gram), whereas nothing was detectable
in the lower layers even though they were in contact
with rainwaier. The prions therefore resist remarkably
well the procésses of natural degradation and can
remain adsorbed (i .e . stuck) to inert matter like clay.
Another underlying problem is the contamination of
water, notably by waste of rendering plants and slaughterhouses processing BSE-affected cattle . Study programmes are under way to assess the risks as a function
of the different types of treatments .
K,
THE RISKS TO HUMANS
Blood #r f
i
Although prions may theoretically be found in blood
and are associated with white blood cells in certain experimental models, epidemiological surveys have never
linked the usual (sporadic) form of CreutzfeldtJakob
disease to a risk due to blood transfusion .
The experimental models of contamination by the oral
route have shown a first phase of replication of these
agents in the Peyer's patches, in the intestine, then the
lymph nodes associated with the digestive tract .
Secondarily, a replication is observed in the distant
lymph vessels, thus implicating lymph and blood recirculation. The phase of neuroinvasion occurs late and is
exponential up to the death of the animal . The levels
observed in the central nervous system are well abov e
90 those seen in the peripheral tissues, in which a plateau is
reached because, it is believed, of a limited number of
replication sites : the cells in question are follicular dendritic cells and certain macrophages .
The transmission of the BSE agent by blood transfusion from a sheep experimentally contaminated by the
oral route served as a reminder of this strain's contaminating potential. In sheep, unlike cattle, the marker of
the infection (prpre; abnormal form of the prion protein)
is found throughout the reticuloendothelial system . This
likely explains why scrapie is endemic in the natural
state (the placenta is infectious) and suggests that it will
be very difficult to eradicate if it turns out that BS E had
also developed in sheep . In addition, the BSE agent is
particularly well transmitted intravenously in primates.
In nvCJD, unlike classical CJD, I'rŸres is found in the
BLOOD TRANSFUSIO N
tonsils, spleen, lymph nodes, appendix, Peyer's patches,
i .e. in all the lymph tissues . The risk of contamination of
the blood, whose cells regularly pass through all the
lymph tissues, is therefore greater than before, but is not
yet quantifiable because of a lack of a diagnostic test .
This risk should be put into perspective in the light of the
number of potentially infected blood donors .
The rapid tests developed to date are sensitive enough
to detect PrPleS in samples of the central nervous system
or lymph tissues, but not in blood . It has been shown
with the murine (mouse) and cricetid (hamster) experimental models that most (approximately 90%) of the
infectiousness is associated with the white blood cells and
can therefore theoretically be eliminated on special filters . In addition, the infectious agents, whose size has
been estimated as 15-40 nm, can in theory be eliminated
by nanofiltration, and validation experiments are under 91
way with plasma derivatives .
Finally, certain countries prefer to screen blood donors
so as to avoid those who have spent a total of over six
months in the United Kingdom between 19 8 0 and 1996 .
As we have emphasized, the nature of the infectious
agent is still not known with precision, even though the
prion hypothesis, which asserts that the agent is constituted solely by FrpCeS, is widely accepted. In practice, the
most sensitive tests to date ensure the safety of the
human food chain (by systematic screening for BSE at
the slaughterhouse and elimination of all animals that
test positive) but not of blood transfusion. Apart from
basic research projects, ongoing work is focusing on the
development of more sensitive tests and exploring new
therapeutic approaches to Creutzfeldt Jakob disease .
THE RISKS TO HUMAN S
Protection of humans . ris
i kmangetr
programmes
The development of rapid screening tests for BSE
constitutes a major advance in the protection of humans .
The new crisis was precipitated because the epidemic
was seemingly uncontrolled, despite the measures
announced, because B SE was being detected in increasing numbers of cattle born over four years after the ban
(1990) on the meat-and-bone meal responsible for the
spread of BSE, and because the scientific uncertainties
concerning the risk to humans are still as great as ever
(the minimum infectious dose by the oral route is nnknown) . The European. Commission took additional precautionary measures by imposing a complete ban o n
92 meat-and-bone meal as well as systematic screening for
BSE in cattle aged over 30 months (i .eo an age when
quantities of infectious agent are detectable and disease
development is possible) before their entry into the food
chain, in order to eliminate all cattle that test positive .
Systematic screening at the slaughterhouse was introduced in France and Germany injanuary 2001 and became
obligatory for the other countries of the European
Community in Julÿ 2001 .
Three tests have been validated to date by the
European Comtnissionh'. All are based on post-mortein
detectionof PrETésin a brain sample . As such, they can
only diagnose the disease oncethé brain is infected, and
not before the end of the incubation period . The Swiss
test (Prionics) is based on the Wéstern blot techrrique
(separation by electtophoresis* followed by irnmuno-
PROTECTION OF HUMAN S
detection) . It was the first used for epidemiological studies
in Switzerland and France and detected previously
undiagnosed animals . The Irish test (Enfer) is based on
the ELISA technique (immunodetection allowing analysis of 96 samples at a time), which is more suited to
largescale screening . The French test (CEA-Biorad) is
based on, purification of the PrPr°5 coupled with ELISA
detection and is the most sensitive .
New tests are being developed and eva.h .tated . The
value of sensitive tests is that they guarantee the elimination of all animals that pose a risk to humans . The efficacy of the most sensitive rapid test has proved
equivalent to that of the reference test, in which potentially infectious material is injected intracerebrally into
mice . Animals that give a negative result with this rapid
test are not dangerous for the mouse (by the intracerebral
route) or therefore for cattle and humans (by the oral 93
route46), because intracerebral contamination of the
mouse has been experimentally shown to be 100 time s
more effective than oral contamination of cattle (so,
100 mg of BSE-infected brain tissue can kill 200 mice by
the intracerebral route but only two cattle by the oral
route), and because the species barrier means that
humans should be more resistant to the BSE agent than
cattle (or at least no more sensitive) .
Systematic screening of all cattle over 30 months of age
involves the testing of approximately 10000 animals
daily throughout France . Tests guaranteeing consumer
safety, if correctly used, should resolve the BS E crisis,
provided that the previous measures are applied, notably
the elimination of high-risk offal (especially brain and
spinal cord) .
THE RISKSTO HUMAN S
Comparison of tests for cattle (European validation)
number of samples that tested positive/total
d6lufio n
of uosiüve control
94
WMINWANNEMW
Test A(i;ritisk, Wallac) is the least sensitive since it only detected
the pure positive sample arad failed to detect anything after the
first dilution of f the positive control. It was not validated, as it
did not detect certain clinically sick cattle .
Test .li (Sw iss, Prionics) is ten times more sensitive.
Test C (Irish, Enfer) is 30 times more sensitive .
7'est ➢ (French, CEA-Biorad) is 300 times more sensitive.
The last three tests detected all the clinicallyaffected cattle and
can be used within the regz<latory framemork of each eountry a
The mad cow crisis has brought to the fore a new parameter: the importance of the role of the European
Community, which tends to paralyze all decision-taking
at the national level . There may now be a single market,
but in no way is there a uniform health zone . Laws enacted to promote free exchange of goods run counter to
the confinement measures needed to prevent the spread
of bacterial or viral diseases . This was particularly manifest when the first French attempt to ban British cattle in
1990 had to be abandoned because it flouted the regulations of Brussels . The serious malfunctions revealed by
PROTECTION OF HUMAN S
the commissions of enquiry contributed to the resignaiion of the whole European Commission . It is also significant that the policy of evaluating tests intended to be
used in the slaughterhouse was not drawn up by the
European Agriculture Directorate-General, but rather by
the Health and Consumer Protection DirectorateGeneral, yet the development of a tool guaranteeing
consumer safety constitutes the most logical bulwark
against BS E .
British reluctance to implement measures in a timely
fashion was clearly identified as being largely due to the
government's desire to export its beef products come
what may, and as the consequence of the official blackmail of threatening to block all European Community
business should the embargos be upheld. The highly
reprehensible attitudes of the British political leaders and
senior civil servants were stigmatized in a 16-volume
report of the Commission headed by Lord Phillips (BSE
Inquiry, published in 2000) .
Other blockades were imposed by countries that believed themselves to be unaffected and which above all had
no desire to hear of measures likely to worry their consumers . So, although it was common knowledge that
British meat-and-bone meal had been exported throughout Europe, countries like Denmark or Germany
wished to have no part of measures excluding high-risk
offal and continued to allow the entry of brain and spinal
cord into the human food chain . The discovery of the
first case of mad cow disease in Denmark in 1999 was a
bombshell : a single animal, and overnight all certainties
evaporated. An embargo was declared by neighbouring
countries, while it was predictable that cases would be
95
THE RISKS To HUMAN S
discovered if the means were employed to seek themé In
Germany, two ministers (Agriculture and Health) resigned in late 2000 following the discovery of cases of
BSE, and excessive confidence gave way to irrational
defiance .
The implementation of the systematic slaughterhouse
testing decided by the European Commission should
logically show that B SE is now present throughout
Europe, and probably also in numerous other countries
that imported British meat-and-bone meal and contaminated cattle, or even sheep . Cases of BSE will continue
to be detected for at least another five or ten years,
because of the extensive illicit trading that has certainly
been going on and which may well continue for some
while yet . It could bé,sevcral years yet before promises
to detect the B SE agent in live cattle (blood test) are kept,
96 given the current absence of all concrete scientific data
and the time it takes to develop an industrial test .
Consumer protection and hence restoration of confidence will only be guaranteed by efficient application
of measures throughout Europe, notably full traceability
and elimination of illegal practices . It is pointless to
impose restrictive measures in France if legally imported
products do not offer the same guarantees. Likewise, the
measures proposed must be consistent not only between
the different countries but also all along the food chain,
in terms both of product quality and the protection of
cattle industry employees .
Equally, if consumers are allowed to eat muscle (red
meat) containing nerves and nerve nets that are in theory
contaminated in aBSE-infected animal, what is the point
of banning calf's sweetbread (thymus) which has never
PROTECTION OF HUMAN S
been found to be infectious in BS E? In other words, what
infectious dose is believed to constitute a danger? Neither
muscle nor thymus has ever been found to be infectious,
i .e . no one has yet succeeded in infecting another animal
with these tissues taken from infected cattle, despite
highly effective techniques such as direct inoculation in
the brain . This does not mean that there is no infectious
particle, but rather that any such particle is present at
levels too low to constitute adanger.
This problem of the infectious dose is crucial and must
underpin any rational system of risk assessment . We
must stop being fearful of the slightest infectious particle .
Our environment and our own digestive tract are awash
with thousands of millions of potentially pathogenic
agents, such as bacteria, viruses and other microorganisms, and yet we are in good health because under natural conditions an equilibrium exists with our body's
defences, which protect us . Disease erupts when there is
an imbalance, an abnormal increase in a virulent pathogen and/or a weakening of our defence systems (for
example broken skin, immunodepression after chemotherapy) . Like our parents and grandparents before
them, we have all at one time or another eaten sheep
infected with scrapie without the least harm befalling us.
Yet, if the scrapie agent were injected directly into the
brain of a human, it would probably trigger a disease, as
in the monkey.
As long as it is undetectable by our most sensitive
methods (which does not mean it is not present), the B SE
agent is not dangerous for humans . It is therefore possible to propose a classification based on the detection by
inoculation of the mouse . Whatever is not dangerous for
97
THE RISKS TO HUM .4N S
98
a mouse contaminated directly in the brain is not dangerous for a cow or human contaminated orally. The
amount of infected brain tissue needed to kill one cow by
the oral route can kill 100 mice by the intracerebral
route . We do not know the ininimum dose able to kill a
man by the oral route, but it is evident that we are sufficiently protected by the species barrier not to have to
count the human cases by thousands when we know that
over 400 000 contaminated cattle (at different stages of
the disease) entered the human food chain in the United
Mngdozaa before the ban on the brain and spinal cord .
For the record, it is reckoned than one gram of central
nervous system of a cow with clinical signs of BSE
contains enough infectious agent to kill 1000 to 100000
mice . This means that a single cow, whose brain weighs
around 500 grams and the spinal cord 200 grams, can
theoretically kill 0 .7 to 70 million mice . These figures
explain why it is possible to assert today (just as it was
possible to affirm in 1996 that the BSE agent was transmissible to humans) that the mouse model contaminated
by the intracerebral route is much more sensitive to the
BSE agent than humans contaminated by the oral route .
In consequence, it is possible to analyze the literature
data to determine what is not dangerous for humans,
using the mouse model :
1) The only tissues found to be infectious in the natural
forms of BSE are the brain and spinal cord, and, incidentally, the nerve ganglia along the spine, the trigerninal
ganglion and the retina ;
2) The lymph formations of the small intestine were
only found to be positive in the experimental forms, after
contamination with very high doses of infectious agent
PROTECTION OF HUMAN S
(100 grams of infected brain) . The fact that the infectious
agent was not detected in the natural form of the disease
does not mean that it is not present, but only that it occurs
at levels below the detection threshold and thus under the
theoretical danger limit (this explains why the disease
develops in five years under natural conditions and in
three years following experimental high contamination) ;
3) No other tissue has tested positive . It would be usefiil to measure precisely the quantity of agent found in
the peripheral nerves so as to estimate more accurately
the dilution that exists naturally in the different organs
and in the muscles of contaminated cattle . The fact
remains that this quantity corresponds to the zone defined as not dangerous for the mouse, or therefore for
humans ;
4) After its low-level replication in the intestine, th e
BSE agent travels along the nerves to the spinal cord and 99
brain . It takes a sort of short-cut along the vagus nerve 29 ,
which innervates the whole intestine and ends at the
obex in the brain stem, the part of the brain which is first
affected and is specifically studied in all analyses. In
experimental contaminations, it is this region which tests
positive approximately six months before the appearance of the clinical signs ;
5) The idea that infected cattle cannot be detected
more than six months before the first clinical signs, and
cannot therefore be prevented from entering the food
chain, is a false problem . The important thing is to
remain well below the danger threshold by using a sufficiently sensitive test.
6) The age limit for B SE screening in cattle was lowered from 30 to 24 months in Germany after two cattle
THE RISKS TO HUMATI S
aged 28 months tested positive (at least one animal was
very weakly positive and would not have been detected
without a sensitive test)
. Overall, as there is at least one rapid test as sensitive a s
the mouse test (at present, only the French test is validated, but it is logical to assume that other sensitive tests
will emerge in time), we propose its use to guarantee
consumer protection against BS E, which may be present
in animals entering the food chain, This approach, which
should rationally solve the BSE crisis, has numerous
implications :
1 ) It must first be validated by the scientific authorities
of the European Union ;
2) The age of cattle screened for BSE at the slaughterhouse should be lowered from 30 to 24 months, as originally recommended by the European Commission .
100 Consequently, the British measures, which are applied
from 30 months, should be revised. Nevertheless, the
systematic slaughter of all older British cattle could be
replaced by a simple test ;
3) There would no longer be any logical reason for
maintaining in France the policy of systematic slaughter
of herds, nor that of culling cohorts of animals of the same
age, as practiced in Swi.tzerland IGuarasrteed effective
slaughterhouse testing would avoid the culling of herds in
which 95-991/o of the animals are perfectly healthy
; 4) The problem of meat-and-bone meal could be treated much more sizrAply. The raw materials mostly come
from the remains of cattle that are to be used for human
consumption . Systematic screening at the slaughterhouse, combined with the elimination of high-risk offal
already in force, guarantees the quality of this raw mate-
PROTECTION OF HUMAN S
rial from cattle eaten by consumers . Furthermore, the
additional treatments imposed (133°C, 3 bars) on production guarantee a perfectly safe finished product which
can be distributed to pigs and poultry. Illegal sales and
the impossibility in practice of stamping them out, as
well as cross-contamination, have forced a total ban on
the use of meat-and-bone meal for farmed animals . This
ban should be kept in place as long as we are not absolutely certain that cross-contamination and illicit trading
have ceased . The fact remains that this meat-and-bone
meal, when made safe, is perfectly healthy and does not
need to be destroyed as highly dangerous contaminated
waste . Instead it can be eliminated as normal waste, or
even used to advantage, to limit this loss of perfectly assimilable proteins . On the other hand, every effort should
be concentrated on the cattle that test positive, which
should be destroyed within the beef industry in all countries, and on implementing coherent measures to protect
both people and the environment . Low-risk waste, withdrawn as a precaution, could be subject to intermediate
measures ;
5) The only sheep diagnosed to date have natural scrapie, which has existed for centuries and has never had
any notable impact on humans . The same approach
could be applied to goats, to ensure that no infected animal enters the human food chain . This commonsense
measure may also have the immediate advantage of protecting the consumer against potentially BSE-infected
sheep, and will avoid a major crisis in goat farming when
the first contaminated sheep are diagnosed (it has been
known for years that English sheep have been fed the
same contaminated meat-and-bone meal as cattle and
101
Conclusion
MAD COW
DISEAS E
The tardy decisions to ban meat-and-bone meal totally
and to introduce efficient tests will cost Europe thousand
of millions of euros in an attempt to save the cattle industry and perhaps tomorrow goat farming, without counting the destruction of a vast number of animals, food
that overwhelmingly was perfectly healthy .
The solution to the problem through the development
of tests has not occurred in the framework of official
research programmes in Europe . This reflects on the
inability of traditional systems to react to novel situations
and to provide original answers at short notice .
Applications of scientific research should not be limited
104
to the search for increased productivity or detection of
fraud, as was the case in the past . Instead of the bipartite
relations that bring together scientists and industrialists
or scientists and the authorities, we should strive to set up
tripartite liaising. If scientists manage to work in concert
with the authorities and industrialists, it will become possible to propose solutions reconciling the imperatives of
public health and economic constraints .
The analyses presented in this book are the views of
the authors alone . Based on the experience the authors
have acquired in their respective fields, they reflect their
conception of the public services and of the need to
inform. The mad cow crisis has revealed a whole series
of malfunctions in our society and has wrought changes
in our perception of food as a source of life and health.
We hope that this book will enhance each reader's awareness and will prompt the introduction of systems guaranteeing food safety, thereby contributing to the
pleasure and confidence afforded by quality food .
APPENDICE S
Notes and ~ ~~etences
Glossar y
. ~~~~~~ of mad ~~~~ disease
~~~~~~~~~~hy
tes and reference s
1 . Vache folle : la grande peur, M6, 6 November 2000 .
2 . The es.se historically described by H . Créutzfeidt was in fact of
a 23-year-old woman with a family history of mental disorders .
Given the youth of the patient and the clinical signs, the disease
would doubtless not be considered as CreutzfeldtJakob disease,
which strictly speaking should therefore be called jakob disease .
3 . Clément J.-M., Lalande F., Reyrole L. et al., Rapport sur Phor- 7 67
mone de croissance et la maladie de CreutzfeldtJakob .
Inspection Générale des Affaires Sociales (IGAS), 1992, SA 19 ,
no. 92145 .
4 . The amino acid being a methionine or a valine.
5 . lYlaaâme Schwartz, Comment les vaches sont devenues folles,
Odi%Jacob, 200], p .14
. BSE Inquir}ty, 2000, vol ..6 12, pp . 59-61.
7 Ezxawn P., Gajdusek D .C . "Survival of scrapie virus after 3 years
of interment", Lancet 1991, vol . 337, pp . 269-270.
8. Wisniewski H .-IVI ., Sigurdarson S ., Rubenstein R. et al ., "Mites
as vectors for scrapie", Lancet, 1996, vo1347, p. 1114 .
9. Marsh R. F., Hadlow %V. J., "Transmissible mink encephalopathy', Rev. Sci . Tech . Off. Int. Epiz ., 1992, vol. 11, pp. 539-549.
10 . By detection of FrPre° (abnormal form of the prion protein )
using immunohistochemistry after labelling with antibodies .
11 . Miller M.W, Williams E . S ., McCarty CW, et al.,
"Epizootiology of chronic wasting disease in free-ranging cervids
in Colorado and Wyoming " , J . Wildl. Dis ., 2000, vo136, pp . 67669®.
12 . FDA, the Transmissible ,Spongifoam Encephalopathies
APPENDICE S
Advisory Committee met on 18 artdl9 January 2001 at Bethesda
(Maryland) .
13 . Iasmézas C . L, Deslys J : Ph ., Demaimay R. et al ., "Strain specific and common pathogenic events in murine models of scrapie
and bovine spongiform encéphalopathy", J Gen Virol, 1996, vol .
77, pp. 1601-1609
. Strain 139A in .14 Swiss mice . Kimberlin âZ .H ., Walker C . A .,
"Pathogenesis of mouse scrapie : effect of route of inoculation on
infectivity titres and dose-response curves", Journal of
Comparative Pathology 1978, vol : 88, pp . 39-47.
15 . Mouse strain 87V for Dickinson and hamster strain . 263K for B.
Chesebro and J . Collinge .
16 . Strain 263K
17. There is nonetheless a certain resistance to proteases, which are
enzymes (biological catalysts) specialized in the destruction of
proteins .
18 . Prusiner S. B ., "Novel proteinaceous infectious particles cause
scrapie",Science,1982, vol . 216, pp .136-144
. The small building blocks of proteins all of.19 which have on th e
same carbon atom a carboxylic acid function, - COOH, and an
amine* function, - NH2 1 whence their name of amino acids .
108 20 . By regulation of a copper-dependent superoxide dismutase, an
enzyme whose role is to destroy an active form of dioxygen at the
interface between neurones .
21 . Neurite outgrowth is the prolongation of neurones which
contact other neuronal cells.
22 . Mouillet-Richard S ., Ermonval M ., Chebassier C. et al ., "Signal
transduction through prion protein", Science 2000, vol . 289,
pp .1925-1928.
23 . Billeter M., Wnthi-ich K., "The prion protein globular domain
and disease-related mutants studied by molecular dynamics
simulations", Arch. Virol ., 2000, vol .16, pp . 251-263.
24 . Strain CH1641. Depending on the animal, there are variations
in three amino acids (at positions 136, 154 and 171) that are associated with variable sensitivity to different prion strains.
25 . Büeler H., RaéberA ., Sailer A. et al . "High prion and PrPsc
levels but delayed onset of disease in sërapie-inoculated mice
heterozÿgous for a disrupted PrP gene", Molecular Medicine,
1994, vol. 1, pp . 19-30.
26 . Lasmézas C. I ., Deslys J.-Ph, Robain O :, et at, "Transmission
of the BSE agent to mice in the absence of detectable abnormal
prion protein", Science, 1997, vol . 275 , pp . 402-405 .
NOTE S
27 Weissmann C . A., "Unified theory of prion propagation",
Nature, 1991, vol. 352, pp . 679-683
. Bousset L ., Belrhali Ii . ; Jaiiin .28 J ., et al ., "Structure of the
Globular Region of the Prion Protein Ure2 from the Yeast
Saccharomyces cerevisiae ", Structure, 2001, vol . 9, pp . 39-46
.29
. The vagus nerve, or pneumogastric nerve, which corresponds
to the tenth cranial pair, is a complex nerve which has vital autonomic functions. In particular, it innervates Meissner's plexus
(the submucosal plexus) comprising fibres and ganglion cells of
the autonomic nervous system, which regulates the movement of
the intestinal villi (the folds of the intestinal mucosa which ensure
a large surface area in contact with foodstuffs) and Auerbach's
plexus (myenteiic plexus), which is a ganglion cell group between
the two smooth muscle layers which control intestinal peristalsis
the contractions of the intestine that propel the food bolus
through the gut) . This anatomical feature explains why this particular zone of the obex is the first to become positive after oral
contamination. The vagus nerve can be contaminated by any of
the sensory branches along the intestine, and the infectious agent
then reaches the neuronal bodies in the nuclei of the obex .
30 . BSE Inquiry, 2000, vo13, pp 101-102 .
31 . Homozygotes* for the methioninc at codon 129 of the gene 109
encoding PrP, like 401/6 of the normal population, so, as for
growth hormone, heterozygous patients seem resistant to disease
development
. Lasmêzas C.32 . I ., Deslys J.-Ph ., Robain 0 ., et al . "BSE transmission to macaques", Nature, 1996, vol . 381, pp . 743-744.
33 . Collinge J, Sidle KCL, Mea .dsJ, et al. "Molecular analysis of
prion strain variation and the aetiology of `new variant CJD' ",
Nature, 1996, vol . 383, pp . 685-690.
34 . Deslys J .-Ph:, Lasmézas C . I., Streichenberger N., et al., "New
variant CreutzfeldtJakob disease in France", Lancet, 1997, vol .
349, pp . 30-31 .
35. Bruce M . E ., Will R . G., Ironside J. W, et al., "Transmissions to
mice indicate that `new variant CJD' is caused by the BSE agent",
Nature, 1997, vol . 389, pp . 498-501 .
36 . Talk by Robert Will given at a meeting on "Transmissible spongiform encephalopa .thies" (Ctu-rent understanding), Institut de
P'rance, Académie des Sciences, 14 to 16 March 2001 .
37. Report no. 3291 of 15 January 1997 by the Mission d'information commune sur l'ensemble des problèmes posës par le développement de I'ESB .
APPENDICE S
3 8. Because of illegal trading and the absence of traceability,
ongoing surveys are hampered by the difficulty of reconstituting
the events and will no doubt only reveal part of the trarth.
39. BSE Inquiry, 2000, vol 10 pp 51 and 60 .
40,1lTotably because of experience with scrapie .
41 . Risques et Peurs alimentaires, Edited by Marian Apfelbaum,
(3dile Jacob, 1998 .
42. Ghani A . C ., Ferguson N. Ivi .,l3onnéliy C . A ., Anderson R. M.,
"Predicted vCJD mortality in Great Britain", Nature, 2000, vol :
406, pp. 583-584.
43 . Data kindly provided by Professor Jeanne Brugèré-Picoux of
the Ecole Vétérinaire de Maisons-Alfort.
44 . Senate Report of 11 May 2001 . The Assembly released a report
on 13 June 2001 .
45 . Moynagh J ., Schimmel H., "Tests for BSE evaluated . Bovine
spongiform encephalopathy", Nature, 1999, vol. 400, pp . 105 .
46 . Iieslys J Ph., Comoy E ., Hawkins S ., et al ., "Screening slaughtered cattle for BSE", Nature, 2001, vol . 409, pp . 476-478.
110
Glossar y
Aldehyde : organic compound possessing an aldehyde group
whose structure includes a carbon atom, bound to a hydrogen
atom and attached by a double bond to an atom of oxygen . Several
aldehydes are disinfecting agents, such as formaldehyde (formol) .
.9lpha helix : polypeptide chain which assumes a çlockwise (righthanded) helical confôrmation and is stabilized by low-energy
intrachain chain bonds called hydrogen bonds .
11 1
An-tine : nitrogen-containing organic compound derived
from ammonia (NH3) by replacement of its hydrogen atoms by
one or more organic groups . Certain neurotransmitters have an
amine fignciion (dopamine, adrenaline) .
Antyloid : a polymerized glycoprotein which resembles starch and
which in certain diseases is deposited in the form of plaquebuilding fibrils (amyloid plaques) in various organs (spleen),
including the central nervous system .
Antibody: defence protein produced by the cells (lymphocytes B)
of the immune system of an animal, following interaction with a
bacterium, virus or xenobiotic substance .
Apoptosis : genetically programmed cell death (suicide) .
Anomalies in apoptosis play a role ixi certain diseases, including
degenerative brain diseases . ;
Ataxia: loss of control of the limbs due to damage to the nervous
system .
Axon : long extension of a nerve cell which ensures the
transmission of the nerve signal along the neurone.
APPEN D ICE S
Beta-pleated sheet : chain of polypeptides arranged in parallel
and forming a folded, accordion-like, highly stretched
conformation.
Central nervous system : the principal system that processes
nervous information in vertebrates . It includes the brain,
cerebellum and spinal cord.
Chaperone (chaperone protein) : a protein which ensures the
correct folding of another protein thus enabling it to fulfil its
function, as a catalyst, forinstance .
Chromosome : structure formed by a DNA molecule and
associated proteins (histones . . :) which carries the hereditary
information of an organism possessing a nucleus . The number of
chromosomes is characteristic of a given species .
Codon : sequence of adjacent nucleotides in a nucleic acid (DNA
or messenger RNA) which constitntes the code that instructs the
incorporation of a specific amino acid in a protein chain being
formed .
Conformation : three-dimensional shape of a macromolecule .
1
12
Cytoplasm : contents of a cell delimited by a plasma membran e
DNA (deoxyribonucleic acid) : high molecular weight nucleic
acid containing deoxyribose as sugar, which serves as the genetic
information carrier (genome) of living organisms . In eukaryotic
cells, DNA is found in the nucleus.
Electrophoresis : technique of fractionating proteins based on the
ability of electrically charged molecules to migrate in an electric
field
, Enoephal.opa:t4ay: a disease affecting the encephalon, part of the
central nervous system comprising the brain, tlie cerebellum and
the medulla oblongata.
Epidemiological : pertaining to epidemiology, the field of
medicine concerned with the development of a disease in a large
number of indivïduals in a given region .
Epizootic : an outbreak of disease affecting an unusually large
number of animals throughout a given region .
Eukaryote : living organism comprising one or more cells whose
GLOSSAR Y
genetic material (DNA) isconcentrated in the nucleus . Animals,
plants,fungi and yeasts are eukaryotes.
Ea.a%a.x•gotic cells : organisms comprising one cell (yeasts) or several
cells (animals, plants, fungi) whose genetic material (DNA) is stored
in the nucleus .
Gene: unit of heredity corresponding to a region of DNA which
controls a particular character. A gene governs the synthesis of a
single protein or of a single RNA and thus conditions the
transtnission or manifestation of a given hereditary character.
Genetic : inherited . Genetics is the science of the heredity of living
organisms .
Geraetic engineering : the set of techniques used to alter the
genetic material of a cell or living organism.
Genome : the sum total of the genetic material specific to each
species of organism .
Genotype : genetic constitution of a cell or individual organism.
Glial cells : cells that supply nutrients to the neurones and which
in the central nervous system include astrocytes and
oligodendrocytes .
irieteroz ygote : an individual possessing different alleles of a gene
for a given character.
Heterozygosity: the state of possessing different alleles of a gene
at a given locus .
Histologi.cal : pertaining to histology, the science of the tissues of
living organisms
: tissue
.Homgenat
that has been homogenized .
Homozygote : diploid cell (containing two copies of each gene) or
an organism with two identical alleles (alternative forms of the
same gene) of a given gene . This contrasts with a heterozygote, in
which the two alleles are not identical
Horizontal transmission : transmission between generally adult
individuals (cf vertical transmission : from mother to child) .
Ia.tr ogenic : resulting from a medical act or medication .
Imrrtagnohistoehernistry : technique of using labelled antibodies to
11 3
APPENDIGE S
label a specific protein in a tissue section (examined under the
microscope) .
Io '° g radiation: radiation (X-rays, gamma rays, etc .) whose
energy is enough to ionize, that is to strip electrons from the outer
layers of atoms or molecules .
Kuru: neurodegenerative human disease of the spongiform
encephalopathy type, which for over a century affected solely the
Fore tribe in Papua New Guinea (Oceania) .
Lysosomes: small organelles in eukaryotic cells that are rich in
hydrolytic enzymes (peptidases, amidases . . .) .
Macromolecule : molecule of high molecular weight (above
several thousand Daltoris) . Many macromolecules are organic in
nature and are essential to life . Among biological macromolecules
are proteins (structural proteins, transport proteins, enzymes . . .),
polysaccharides (complex sugars), nucleic acids (DNA and RNA) .
Macrophages : white cells thatdô not circulate in the blood o r
114 lymphatic system and play a vital role in our defence systems
against airborne chemical (dust) or biological (bacteria, viruses° . .)
agents.
Membrane : double layer of lipoprotein macromolecules
(phospholipids containing unsaturated fatty acids associated with
proteins) which envelop prokaryotic cells (bacteria, algae . : .) . In
eukaryotic cells, biological membranes separate the ce ll from the
outside world (plasma membrane) and also delineate the different
organelles (nucleus, mitochondria) within the cell's cytoplasm .
Metabolic: pertaining to metabolism, which in living organisms is
the sum of all chemical transformations catalyzed by enzymes and
allowing the transformation of biological moleculés .
Mineral base : inorganic molecule able to fix a proton (>Ei+) .
Sodium hydroxide (NaOH) and potassium hydroxide (KOH) are
strong, highly conosive bases which attack many materials and
destroy living matter,
Pliftochoradria: small organelles in the cytoplasm which are the
cell's power sources .
Mutation: change in a gene that can be transmitted to other
generations, resulting in a hereditary change.
GLOSSAR Y
Myoclonia: involuntary contractions of the muscles .
Neurone : nerve cell comprising a cell body containing the
nucleus, an axon (long membrane outgrowth) and nerve endings :
the dendrites, which at the synapse ensure axonal communication,
i.e . transmission of the nerve signal.
Neurotransmitters: chemical messengers released by the nerve
cells subject to an electrical influx (action potential) which is
created by the movement of ions across the plasma membrane of
the nerve cell . These chemical molecules act as messengers to
transmit the messages produced by the nerve cells .
NMR (nuclear magnetic resonance) : spectral technique based
on the capacity of atomic nuclei for resonant absorption of
electromagnetic radiation . NMR can be used to determine the
three-dimensional structure of small water-soluble proteins .
10Tonioiti .2iig radiation: radiation (ultraviolet, infrared, visible
light) whose energy is insufficient to ionize atoms or molecules .
Nucleation : acting as a nucleus for, in a process of formation (of
crystals, for example) .
Nuclaic, acids : biological macromolecules formed by the 115
combination of simpler nitrogen-containing molecules nucleotides, elementary units containing pyrimidine and purin e
bases, a sugar of five carbon atoms (pentose) and a phosphate .
Nucleus : in eukaryotic cells (animals, plarits yeasts . . .), large
organelle containing the chromatin formed from DNA and nuclear
proteins .
E9 olle : well-characterized element isolated by a membrane
within the cytoplasm of a eukaryotic cell.
Oz one : trioxygen (O3) gas which is extremely oxidizing and toxic
and is used as a bactericide (disinfection of water . . .) .
Peptide : molecule of moderate size formed by the association of
amino acids linked together by peptide bonds . Peptides form
proteins.
Polypeptide : small macromolecule formed by the chains of
several peptides themselves co mprising amino acids linked
together by peptide bonds.
APPENDICE S
Precautionary prinoiple : the absence of certainty, = in view of
current scientific and technical knowledge, should not delay the
adoption of preventive measures .
Prirraa.ry structure :sequence of simple units in a polymer,fo r
. ,examplthofincdsapethinor
Prion : protein found in animals, but also yeasts, which can exist in
two forms : one normal, the other altered.
Prokaryotic cells : living organisms of relatively simple structure
characterized by a cytoplasm containing dispersed D NA not
delimited in a nucleus . Bacteria and blue algae are prokaayotes .
Protease : proteolytic enzyme which degrades proteins by
hydrolyzing, sometimes very selectively, some of their peptide
bonds.
Protein : macromolecule generally of high molecular wéightwhich
comprises several polypeptide chains, each of which has a
characteristic sequence of amino acids . Certain proteins have a
structural role incells (biomembrane, cytoskeleton . . .), others play
a part in transport (haemoglobin . . :), catalysis (enzymes . . .) or
communication (hormones, cytokines . . .) .
l~ 6 l~riateolysis :degradation of a protein, generally by hydrolysis of at
least one of its peptide bonds .
Pruritus : itching of the skin due to cutaneous or systemic disease .
A (ribotxucleic acid) : nucleic acid, generally of inediurn
molecular weight, based on ribonucleotides whose sugar is ribose .
Ribonucleic acids are mainly,found in the cytoplasm of eukaryotic
cells, in the ribosomes, which synthesize proteins . RNAs decode
the genetic information carried by the DNA for subsequent
synthesis of proteins .
Scarification: superficial incision in the skin.
Scra.pfe : a. transmissible nervous disease in sheep and goats with
an incubation period of between two and four years . I{nown in
Europe since the 17t1i century.
Secondary structure : motif of regular local folding of a polymeric
molecule . In proteins, there are two types of secondary structure :
alpha helices and beta-pleated sheets .
Soclltitrn hydroxide : strong, extremely corrosive mineral base .
GLOSSAR Y
Sodium lrypochlorite (NaOCl) : sodium salt of hypochlorous
acid (HOCI) . Bleach contains mostly sodium hypochlorite whose
oxidizing properties make it a powerful disinfectant .
Spectroscopy : technique for measuring the absorption of
radiation of various wavelengths (IR, visible, UV) .
Spongiosis : spongelike appearance of nerve tissue due to the
presence of vacuoles in the neurones .
Sporadic ( disease) : diseas e that affects a limited number of
unrelated subjects.
Strain : all individuals (bacteria, etc .) from the same colony of
microorganisms . A strain of prions results from the transmission of
a prion disease to several successive hosts .
Syngeneic : individuals or tissues that have an identical genetic
makeup, following successive controlled crosses .
Tertiary structure : three-dimensional shape of a macromolecule .
In proteins, their tertiary structure corresponds to the conformation
of the polypeptide chains.
Trace element: mineral element essential to living organisms i n
very small amounts and whose lack results in severe nutritional 117
deficiencies .
Transgenic : plants or animals in which one or more genes o f
another cell or organism have been stably incorporated
experimentally and which can be transmitted to subsequent
generations .
Transmissible agent: element which is responsible for a disease .
TSSEs (transmissible subacute spongiform encephalopathies) :
group of diseases affecting the central nervous system . They are
characterized by the progressive destruction of the neurones of
certain zones of the encephalon, following the accumulation of a
pathological protein, PrPreS, thus giving the nervous tissue a
spongelike appearance .
Unconventional transmissible agent (LTTA) : name of the
biological agent responsible for transmissible spongiform
encephalopathies .
Vacuole : cellular storage organelle .
AFPEPBDICE S
1/° ° o : very small infectious particle composed of genetic material
(L9iVA,1dNP,) surrounded by a lipoprotein envelope belonging to
the host.
t% : particle consisting of a nucleic acid (based o n DNA or
RNA, as in retroviruses) surrounded by a protein envelope and
which can reproduce inside a host cell .
118
History of mad cow diseas e
Around 1732 : first cases of scrapie in England, according to a 1772
text (T. Comber) which dates the appearance of the disease forty
years before.
1883 : description in France of a case of scrapie in a bullock, which
after veterinary diagnosis was sold off as cheap cuts (ref . M.
Sarradet, Revue mëdicale v4ft6rinaire, vol . 7 : pp . 310-312) .
1920-1921 : Hans Creutzfeldt then Alfons Jakob, two German doc- 119
tors, described the first cases of human spongiform encephalopathy, which was later named after them .
1936: two veterinarians in Toulouse, Jean Cuillé and Paul-Louis
Chelle, demonstrated that scrapie is transmissible from one sheep
to another, but also to goats, and that it is caused by an agent smal1er than a bacterium .
1957 : Vincent Zigas and D . Carleton Gajdusek described kuru, a
neurodegenerative disease affecting the Fore tribe in the centre of
Papua New Guinea. This was a new type of human spongiform
encephalopathy.
1967 : the mathematician John Griffith hypothesized that an infectious protein may be responsible for scrapie .
1966-1968 : kuru and then Creutzfeldt Jakob disease were transmitted to chimpanzees by Gajdusek .
1973 : first oil crisis and bankruptcy of the less efficient industries .
Acceleration of changes in the processing of meat-and-bone meal
in England, which are heated less . Production of meat-and-bone
tneal rises .
APPENDICE S
1976 : D . Carleton Gajdusek received the Nobel Prize for Medicine
for twenty years of work on degenerative brain diseases, which at
the time he attributed to slow viruses .
1982 : on the basis of inactivation experiments, the neurophysiologist Stanley Prusiner developed the hypothesis that the agent responsible for transmissible subacute spongiform encephalopathies
(TSSEs) is not a virus, but an infectious protein, which he named
`prion'.
1985 : description in Great Britain of the first cases of bovine spongiform encephalopathy (BSE ) , which were only diagnosed as such
from November 1986 onwards . It is probable that the first cases of
this new `mad cow' disease date from the 1970s .
1987 : steep rise in cases of mad cow disease in Great Britain : 442
cases identified by 31 December . Meanwhile, the principal
concern of the British officials in charge at the time was to avoid
adverse publicity that could harm British exports (BSE Inquiry,
vol . 3, notably pp . 33-36)
1988: in April 1988, the English epidemiologistJohn Wilesmith, of
the State Veterinary Service (SVS), showed that meal from sheep
and cattle was the source of the epidemic of bovine spongifor m
120 encephalopathy (BSE) . While 421 cases had been diagnosed in
352 herds across the country, a front-page article in Farming News
accused MAFF (Ministry for Agriculture, Fisheries and Food) of
seriously underestimating the extent of the BSE epidemic and voiced concern for human health . The Minister of Agriculture in Mrs
Thatcher's cabinet continued to refuse to consider any idea of mandatory slaughter and of financial compensation for farmers .
20 June : first meeting of the working party headed by Sir Richard
Southwood (which included no expert on prions diseases) charged
by the British Government with assessing all the implications of
BSE. The members learned that animals with BSE had been slaughtered and fed into the human food chain (BSE Inquiry, vol : 1, p. 47) .
18 July : MAFF at last bans the distribution of meat-and-bone meal
to cattle in the United Kingdom : This meal was then exported, Mrs
Thatcher's government considering that it was the responsibility of
the importing countries to ban it once they learned that it was prohibited in the United Kingdom .
1989 - 27 February : publication in England of the Southwood
Report, which concluded that there is no proof of a danger to
humans posed by offal from BSE-contaminated cattle . The report.
HISTORY OF MAD COW DISEAS E
asserted that the estimated risk does not justify consumer-specific
information on products containing brain . In its conclusion, the
report's statement that `if our assessment of these likelihoods are
(sic) incorrect, the implications would be extremely serious', was
soon forgotten and this report was considered as the scientific
demonstration that BSE presents no danger to humans (BSE
Inquiry, vol. 1, pp . 48-55)
: France decided
.13Augst to ban the import of British meat-andbone meal, except in cases where the company undertook not to
use it for ruminants! Description of the first French cases of
Creutzfeldt Jakob disease in children treated with pituitary-derived
human growth bormone from contaminated batches .
13 1Vovernbex° : the British Government banned in England and
Wales the human consumption of certain beef offal : brain, spinal
cord, but also spleen, thymus, tonsils and intestine .
1990 : the ban on offal was extended to Scotland and Northern
Ireland in January 1990 . The export of cattle offal to other countries of the European Union was banned in March 1990, and to
developing countries in July 1991.
10 May : announcement of the first spongiform encephalopathy,
apparently linked to BSE, in a pet cat . Strangely, on the 7th of 121
June, the European Veterinary Committee considered that animal s
with BSE pose no danger to human health !
24 July: France banned the use of meat-and-bone meal in cattle
feed. Its use in feed for pigs, poultry and fish is still authorized .
24 September : announcement in the United Kingdom of the first
transmission of BSE to laboratory pigs . The next day, the ban on
high-risk offal was extended to all animal feed .
12 October: the Spaniard Fernando Mansito, the representative of
the Agriculture Directorate-General in Brussels, advised European
experts to play down the mad cow disease affair through a policy
of misinforming the press (ref. report on television M6 in
November 2000, Vache folle : la grande peur) .
1991 : the first case of mad cow disease was discovered in France on
2 March 1991 in Côtes-d'Armor (Brittany) . The French
Government decided that all herds should be slaughtered if they
included one BSE-affected animal, a very wise measure in the
absence of targeted means of protection . In the United Kingdom,
description of the first `naive' case of BSE, that is in an animal born
after the ban on meat-and-bone meal .
APPERFDICE S
1992 : in Great Britain, mad co- disease peaked at over 100 cases
a day, which constituted a genuine epizootic . In France, INSERM
set up a department to monitor Creutzfeldt Jakob disease .
1994 - 27 June : six years after Britain, the European Union banned in all member states the use in cattle feed of protein from the
tissues of ruminants or the meat of unidentified mammals . The
use of this meal was still authorized for other ruminants, poultry,
rabbits and pigs. It was as if the European Veterinary Committee
was unaware that the agent of mad cow disease crosses the species barrier.
1995 : death from a new variant of CreutzfeldtJacob disease of a
young woman of 18 (R. Vicky) . The average age of people affected
by the classical disease is 65 .
1996 - 20 March : Stephen Dorrell, the British Secretary of State
for Health, revealed that ten young Britons had Creutzfeldt-jakob
disease (CJD) . These adolescents seem to have been contaminated
by eating beef from BSE-affected cattle .
21 March: France, then the European Union on 27 March, ordered an embargo on the import of British beef products and cattle .
The admission for the first time that inad cow disease can be tr•ans122 mitted to humans immediately caused a wave of panic in Europe,
which has still not subsided .
April : creation in France of an Interdepartmental Committee on
TSSEs (the Dormont Committee), chaired by Dr Dominique
Dormont, Head of the Neurovirology Department at the CEA.
June : through experiments with maca .ques,the CEA funii.shed the
first experimental proof that BSE can be transmitted to humans .
July : the European Union imposed new technological conditions
for the manufacture of meat-and-bone meal which ensure that the
pathogenic prions are inactivated (133C, 3 bars, twenty minutes) .
In October 1996, the first case in France of new variant CreutzfeldtJakob disease was confirmed .
1997: the Nobel Prize for Physiology and Medicine was awarded in
October 1 997 to Stanley Prusiner for his work on prions. The use
of meat-and-bone meal in cattle feed was banned in the United
States, where officially the situation was as follows : there were no
éases of BSE, but meal may have been imported, scrapie was endemic, Chronic Wasting Disease was continuing to spread and affected five States, the processes used to manufacture meat-and-bone
meal were similar to those in the United Kingdom.
HISiORY OF MAD
GOVN DISEAS E
2000 - 26 October : publication in the United Kingdom of the
report of the official inquiry into bovine spongiform encephalopathy and new variant CreutzfeldtJakob disease (BSE I:nquiry, 4000
pages in 16 volumes) . This inquiry, diligently carried out by Lord
Phillips, lasted three years and covered the period before 201VIarch
1996, the date when the Government of John Major announced
that the BSE agent is transmissible to humans .
The mad cow disease crisis has resulted in the contamination of
over 180000 head of cattle, the slaughter of about 4.5 million cattle,
and close to seven thousand million euros have been spent on
compensation and the destruction of waste . Certain observers
consider that this report is too lenient towards the officials concerned and does not answer the real questions on the origin of BSE .
1[qoveanber. discovery of the first cases of BSE in Spain and
Germany. From 1 January 2001 in France, a ban on meat-and-bone
meal for all species and systematic screening in the slaughterhouse
of cattle aged over 30 months . Announcement in France that fimding of prion research is to be tripled (32 million euros) .
2001 : discovery of the first cases of BSE in Italy . The European
Union imposed a moratorium on meat-and-bone meal from the 1s t
of January, and from July introduced the systematic screening in 123
the slaughterhouse of cattle over 30 months of age . The first results
indicated similar low levels of contamination in the countries that
performed the first large-scale testing (17 positive tests out of
500000 tests in France), but implementation was slow in some
countries and the notion of guarantee provided by the doubl e
safety standard of sensitive testing + elimination of high-risk offal
was not yet accepted.
24 April : death in France of Arnaud Eboli, aged 19, the third `offacial' victim of new variant Creutzfeldt,Jakob disease (nvCJD) . Thus
far, there have been just over one hundred cases of nvCJD, 97 of
them in Great Britain .
APPENDICES
Bibliograph y
APFI,r.anuM M . (director.), Risques et Peurs alimentaires, Odi3e Jacob,
1998 .
KoviuLsKy Ph ., Viney G ., Le Principe de précaution ; rapport au
Premier ministre, Odile Jaeob, 2000 .
Lord PxrLrrs BSE Inquiry (in 16 volumes), 2000.
Sefnt,ARra M ., Çomment les vaches sont devenues folles, Odile Jacob,
124 2001 .
Selection of Internet sites providing
information on prion disease s
,SUes in French
AFSSA : www.afssa .fr/dossiers
Bureau vétérinaire suisse : www.bvet .admin.ch
CEA: www.cea .fr
Centre de ressources Infobiogen (Génopole d'Évry) :
www.infobiogen .fr/people/dessen/prp
CNRS : ivww.cnrs .fr
Commission Européenne :
http://wwweuropa.eu
eu.int/cornm/food/index_fr.htm]
.int/comrn/food/index-fr.htTn]
INRA : www.inra .fr
INSERM : www.inserm.fr/serveur/prions .nsf
Ministère de l'Agriculture: www.agriculture .gouv.fr/esbinfo .htm
Ministère de la Santé :
BIBLIOGRAPH Y
www.sante .gouv.fr/htm/pointsur/vache/info .htm
Statistiques ESB : perso.infonie.fr/vetolavie/bse .hthn
Vache folle en ligne (INRA) :
www.inra.fr/InternetJProduits/dpenv/vchfol00 .htm
Sites in English (United Kingdom)
BSE Inquiry : w,Aw.bseinquiry.gov.u k
Department for Environment, Food and Rural Affairs :
http ://-,vwnv.defra.gov.uk/
Department of Health : www.doh.gov.uk/cjd
European Commission :
www.eirropa.eu.int/comm/food/fs/bse/index_en .htm l
Meat and Livestock Commission: http ://wuryv.mlc .org.uk/
Mad cow disease: www.Mad-cow .org
125