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  • MAD COW DISEAS E The Risks to Humans
  • J . -PHILI . DESLY ` ANDRÉ ICOT MAD COW DISEASE The Risks to Humans 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 85 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 dis- cipline 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 13 The longstanding enigma of prion diseases in humans 13 From cannibalism to kur u 1 7 The classic human forms : Creutzfeldt-Jakob disease 19 Contamination by growth hormone 23 Spongiform encephaiopathies are not new 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 prion 39 Neither virus nor bacterium, a new form of infectious agent 42 The prion protein is present in all mammals 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
  • 5 9 Mad cow disease and the contaminatiohof humans 59 The emergence of mad cow disease 67 A new Creutzfeldt-Jakob disease 7 1 The risks to humans 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 programmes 1 03 Cancluasiori 107 Appendices 109 Notes and references 113 Glossary 121 History of mad cow disease 126 Bibliography 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 çontainina- ted 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 offi- cial embargo was rapidly imposed on all beef by-pro- ducts from the United Mngdom, and this cordon sanitaire, coupled with various protective measures, notably regarding meat-and-bone meal, partially resto- red 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 pro- moted the consumption of meat, whose price dropped, all of which served to boost sales. In late 2000 there was a new development. An increa- sing number of cattle were found to be contaminated more than five years after the ban on the disease-trans- mitting meat-and-bone meal, whereas officially the risk was supposed to have been controlled long before . Investigative reports broadcast on television at peak view- ing times revealed that contrary to what had frequently been asserted, large quantities of contaminated meat- and-bone meal had continued to circulate across Europe with complete impunity. Moreover, screening for B SE had been ineffective, and the protective measures imple- mented 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, fol- lowing 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 precautio- nary 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 fai- led 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 to- gether with the relevant scientific background, so that 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 encephalo- pathies) principally include Creutzfeldt Jakob disease in humans and scrapie* in sheep and goats, and since 1985 bovine spongiform encephalopathy (BSE), 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, consti- tute a biological enigma and the sale example of a trans- missible 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 scra- pie is transmissible to other sheep . D. Carleton Gajdusek (Nobel prize in 1976) later brilliantly demonstrated, nota- bly in studies of kuru*, that human-to-human transmis- sion 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. TSSE 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 deve- lopment 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 . 1 1 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 thou- sands 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 igma of prxÎon 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 paediatri- cian, first described kuru, a strange disease which affec- ted the Fore tribewhich was still living in the Stone Age in the highlands of Papua New Guinea. In the Fore lan- guage, kuru means `shivering or trembling', and was cha- racterized 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 fasci- nated 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 deter- minant factor in the appearance of the disease, even if plants and insects (standard items in ïhe Papuans' diet) see- med not to be implicated. A whole variety of explanations were proposed, including endocrine disorders (hormone- related 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 trans- mitted 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 conta- minating `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 inter- play between the various muscles involved in maintai- ning 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 vic- tim's spirits varied: some laughed constantly, whence kuru's nickname of 'laughing death' ; others were plun- ged 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 prolon- ged 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 simi- larities between kuru and scrapie, and this observation convinced Gajdusek that the experimental inoculations 1 5 should be continued, above all in primates, while exten- ding the period of observation. The first successful trans- mission 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 achie- ved 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 inges- tion 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 conta- inination 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 conta- minated 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 canniba- lism ceased, no child born to, and generally breastfed by, an infected mother (incubating the disease or with full- blown 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 pioneer- ing studies of kurnz,
  • THE CLASSICAL HUMAN FORM S The lassical human for æ Creutzfeldt-Jakob disease 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 iden- tified as one of the TSSEs . The similarity of its histologi- cal 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 iso- lated individuals rather than as an epidemic . Its annual frequency is 1.4 cases per million inhabitants, represen- ting approximately one in 7000 deaths in France, with ï an average of 80 cases a year. CJD usually becomes evi- dent as dementia (defined as a deeply diminished intel- lect; with impaired memory, altered intelligence, temporospatial disorientation and perturbed social beha- viour), which begins progressively, in patients aged 60- 65 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 unre- mitting, 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- 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 indivi- dual 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 sporadic 18 CJD do not seem to be linked to scrapie, since scrapie- free 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 dan- gerous 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 sur- vive). 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 occasio- nally 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 injec- tion 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 clini- cally silent phase or when the first signs are atypical) . Such transmission may be due to the use of contamina- ted surgical instruments, dura mater implantation, cor- neal 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 insom- nia. 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. Host- related 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 hormone 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 for 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#ionalprogrammeswereimplementedtosolvethe 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 1 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.
  • 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 ~~ ~~T ~R R E77 I I j -~~ 1 Sheep ~~~~ Scrapie around 1730~ Goat .;: Scrapie ?a ^ Kuru around 1900 Man Creutzfeldt-Jakob disease 1920 Gerstmann-Straussler-Scheinker syndrome 1926 Fatal familial insomnia 1992 Mink Transmissible mink encephalopathy 1947 Wapiti and mble deer of the Rocky Chronic wasting disease 1967 Mountain s Cattle Bovine spongiform encephalopathÿ 1985 Nyala 1986 Gemsbok, ~ Spangiform encephalopathy 1987 Cape élk; I~rabian ~~ ~ 1989 oryx, Great kudu Caf Feline spongiform encephâlopathy 1990 Cheetah, puma Spongiform ençephalopathy 1992 Mouflon (wild sheep) Spongiform encephalo pathy 1992 affects other small ruminants, notably goats, and is found in most countries of the world, except for Australia and New Zealand. Principally studied in Suffolk sheep, natu- ral scrapie is an endemic disease and affects animals aged two to five years (older sheep are rarely kept, for com- mercial 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 m- monest,Iéike numerous animal diseases, scrapie posed no particular danger to human health, but was deemed sha- meful 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 hyper- excitability 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 exhaus- tion (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 ani- mals within a flock which could be linked to placento- phagia (when an infected ewe litters, the infectious . placenta is eaten by her or by other ewes) or to contami- nation of pastures by the placenta, or even by faeces. Pastures grazed by infected animals could contaminat enewflocksforovertenyears. It was shown experimen- 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 scra- pie. The sheep model is nonetheless particular, because high levels of infectious agents are found in several peri- pheral organs (tonsils, spleen, lymph nodes, intestine, placenta), unlike what is observed in cattle and in humans, where infectious agents are limited to the cen- tral nervous system (brain, spinal cord) . This particularity could explain the putative contamination of the environ- ment (scrapie-infected pasture) by the placenta or even faeces, and the possibility of horizontal transmission (bet- ween 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 care- fully 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 pas- ture-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 appea- red 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 occur- red 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 hyperexcita- bility and marked aggressiveness, during which the ani- mal 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 monito- ring of food sources . Although the epidemiological data clearly incriminate the animal feed, the actual cause of the disease is still un- known. 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 spon- giform 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 TSSE 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 ruminants 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 wild- life 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 ani- mals, 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 contami- riating the environment and pointless replacement over years of the herd by healthy animals, which may suc- cumb in turn. The systematic search for signs of the disease by micro- scopy 1a has shown that the disease was not clinically diag- nosed 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 fre- quency of CWD in deer rose from 3% to 13% in South- East 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 trans- mission 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 prohibi- ted from donating blood 120 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 repro- ducible 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 fol- lowing 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 disor- ders, 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 peri- pheral nervous systems are completely invaded, regard- less 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 spi- nal 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 32 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 suc- ceeded in detecting any infectious microorganism . A classification of tissues and organs was proposed fol- lowing quantification of the infectious agents in scrapie- affected sheep, and underpins precautionarji measures for the exclusion of high-risk organs . Essentially, the 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, pla- centa) 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 intra- cerebral 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 unde- tectable 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 eli- minated 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 bar- rier. Transmission does not occur through healthy skin, but is relatively effective through damaged skin (scarifi- cation) 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 trans- mission 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 ani- mals), 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 scra- pie, but also of other animal and human TSSEs. It is noteworthy that it has never proved possible to transmit certain scrapie, and above all CJD, 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 domi- 34 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°`Sin cases of transmission. A strain of mouse FITa4 which, is not transmitted to the hamster can be transmitted to a transgenic* mouse which has both hamster and mouse .l'rP, but then willonly be transmissible to the mouse (a-1): In contrast, a hamster strain cannot be transmitted to the mouse, even ajter apassage 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 demons- trated 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 heat treatments. 3G 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 SINC (for Scrapie INCubation period) ,itwassubsequentlyshowntobethegenecodingforthe prion protein (PrP), a normal protein which is neither viral nor bacterial but is a part of the host since it is pre- sent 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 16 which accu- mulates 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 82 in the first purification of the prion protein. The cat The CJD agent istransmissible to the cat by the intra- cerébral 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 ani- mals. 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 n1968 for CJD, with an incubation period ranging from 37
  • SPONGIFORM ENCEPHALOPATHIES ARENOT NE W one to six years. Other primate models, such as the squir- rel monkey and macaque, have the advantage that they reproduce readily and generally develop the diseasés fas- ter. 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 follo- wing 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 .In1970, Raymond Latarget 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 re- viewed 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 infec- tious 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 expres- sed 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 propo- sed 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 scra- pie 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 1985 a génetic sequence corresponding to a cellular pro- tein, the normal prion protein (PrI'c), localized at the sur- face 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 pro- teins) and which therefore accumulates in the brain, lea- ding 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 distin- guish the prion protein, the normal protein present in all mammals, from the prion itself, the infectious agent res- ponsible 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-dimensio- nal structure. This structural change greatly alters their respective properties . Thus, I'rlx is soluble in water whe- reas 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 86. In humans it is a small protein of 253 amino acids. The synthesis of Prll" is programmed in the DNA by a single gene (called PRNP) localized on chro- mosome* 20. This protein is located on the outer mem- brane* 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-produ- cing minifactories calIed ribosomes (the genetic informa- tion 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 (localiza- tion on the outer face of the membrane); 3. reintroduc- tion 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, experimen- tal 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 destruc- tion 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 ele- ment* and an indirect role in protection against the dele- terious 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 struc- tures. This suggests that PrP is involved in the process of cellular adherence, notably in neurones21 whose out- growths 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 (nor- mal. PrP1 and abnorinal PrPre,, which is considered inféc- titrus in the prion hypothesis) have the same amino acid composition and sequence (order of amino acids, or pri- mary 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, the- reby adopting a secondary structure* (i.e. the conforrna- tiçsn, 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 type 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 col- lagen 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 struc- ture, 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 model- led 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 pro- tein), 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 degrada- tion. l'rFés can therefore accumulate inside cells, parti- cularly in lysosomes (which normally destroy proteins at 4 5 the end of their lives), and become toxic for the neu- rones. 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 . rion resist all tr " ? 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 (coagu- lation), 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 auto- claving 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 bet- ween prion strains, thus complicating the implementa- tion of safety standards . In addition, all prior treatments that lead to fixation or 'g of proteins (formol, etha- nolp even simple drying) protect against the action of autoclaving. The conditions described above that inæcti- vate 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 (incorpo- ration 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 auto- claving 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 hos- pitals) 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 seve- ral powerful oxidizing agents, such as potassium per- manganate 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 treat- rnent is not effective against other infectious agents, nota- bly spores (microbial) . The most effective chemical treatment is bleach (sodium hypochlorite*) at a concen- tration of 20000 pprn (which corresponds to a commer- cial 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 suscepti- bility 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 (trans- genic mice) are very sensitive and develop the disease after a short incubation period. PrP, even when normal 48 (PrP"),can prove toxic when overexpressed. Certain ani- mals, which synthesize over ten times the normal amount of PrP, spontaneously develop spon,:iforrn encephalopa- thy 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 ano- ther species, as brilliantly shown in transgenesis experi- merats. Genetically modified mice expressing hamster PrP in addition to mouse PrP become sensitive to ham- ster 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 sho- wed 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 cor- respond 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 degra- dation by cellular enzymes. Thus, all the patients who develop a familial form of CJD 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 des- truction 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 neuro- degeneration could prove more complex since at least two studies 25,26 have reported a clear dissociation bét- ween the accumulation of abnormal I'rY, the anatomo- pathological 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 ano- ther 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 extra- ordinary agents . The prian Îa.ypot.hesishas the advantage of simplicity. In its abnormal form, the prion protein is consi- dered 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 unquestiona- bly the most troubling feature, and reveals that there is something lacking in our understanding 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 trans- missible independently of the hosi, and therefore of its prion protein . It should be recalled that on contamina- tion 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 infec- ted 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, des- pite the very large number of infectious agents (in excess of 10 thousand million units per gram of brain), no struc- ture suggestive of a microorganism, and notably of a virus, was visible under the most sophisticated micro- scopes. 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, howe- ver, be able to bind tightly to host proteins, in this case PrPc, which it transforms into PrPCeS (resistant to degra- dation), 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 tech- niques 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 from the host = PrPre s The principal hypotheses accepted are : 1%tepriçrn : the normal protein (PrP`) is transformed into an 52 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 éxperi- mental 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, star- ting with mouse brains without FrP`-, the same pheno- menon occurred. All mice developed TSSE, but some had no PrŸ-s (note that transmission was faster from mice that had accumulated Prhre,) . The histological éxamina- iion 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 BSE (spongiosis and glial reaction) . The hypothesis that best explains these results is that, under our conditions, the BSE agent 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 infec- tious 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 The other amyioid diseases are not trans - and may indoçe the polymerization of mitted by the injection of amyloid fibrils. precursors. Conversion outside the cell Abnormal PrP causes the conversion of The infectivity of this converted PrP ha s normal PrP irrvitro. not been demonstrated. Strains of unconventional transmissible agen t Abnormal PrP can adopt different struc- The multiplicity of strains (20) in mice tures. runs counter to this structural hypothesis. Mouse without gene encoding PrP (knock-out ) No disease nor replication of the agent. PrP is perhaps a cofactor at the agent or a receptor and may play a role in the patho - physiology. Genetics Prion diseases are both genetic and infec- Retroviruses exhibit both genetic and infec- tious. tious determinism. Mutations of PrP alone may cause the dis- Mutations of PrP may increase susceptibili - ease. ty to the disease. Transgenic mice which overezpross Pe P Overexpression of a mutant PrP causes a In these mice there is no abnormal PrP ap- brain disease: parent in électrophoresis; no transmissio n to normal mice, no disease without overex- pression. Irradiatio n Theoretical calculation shows that the tar- According to data on inactivation of viru- get is too small to be a virus . ses of known size, the target is compati- ble with a small virus: Composition of the agent The infectious fractions contain abnor- Over 105 molecules of abnormal PrP pe r mal PrP and perhaps too little DNA for a infectious unit hâvebeentletected . virus . Infectious samples contain nucleic acids . No virus has been found to date . New viruses are difficult to find (Born a virus). Saurce : b. caugney ana H.eneseoro.
  • THE SEARCH FOR ANALOGOUS MODELS : YEAST PRION S crystallizâtion in mineral chemistry, is an interesting alter- native to the prion hypothesis, from which it differs in rate of formation of PrPleS Variants that bring into play chape- rone* 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 phenome- non 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 cha- racters 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 unrela- ted 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 des- cribed in the prion hypothésis : Old observations of trans- mission 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 inac- tive and releases other genes which will allow the yeast to grow using poor sources of nitrogen . This seif-modifi- cation mechanism, which is transmissible to other Ure2p proteins without the intervention of the nuclear DNA genes (which normally are always called into play, but 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 transmis- sion 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 understan- ding 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-transforma- tion prevents the development of theinfectiori by indu- cing cell death which is propagated in a circle and isolates the contaminated part from the rest of the rnÿce- lium of the fungtzs: This circumscribed suicide proves
  • THE SEARCH FOR ANALOGOUS MODELS : YEAST PRION S very effective, as the mechanism of protection is imple- mented 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 so- called `analogue' models, however interesting they may be, correspond to cellular adaptations and are not infec- tious. 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 hypo- thesis, 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 humans The 6emergence of disease 59
  • MAD COW DISEASE AND THE CONTAMINATION OF HUMAN S and-bone meal, and the first cases of B SE were diagno- sed officially in 19 86. This shows that recycling and energy saving, which are theoretically excellent from the ecological and economic points of view, are not necessa- rily synonymous with protection of health and the envi- ronment, 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 yandGuernsey. A single parameter differed : the manu- facture of meat-and-bone meal . This meal could contain sheep (15%) and cattle (15%) carcasses, and certain batches seemed to be contanoinatéd by 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-and- bone meal was not stopped, but continued legally for almost ayear to the other countries of Europe, princi- pally 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 pea- ked in 1992, with over 37000 cases officially recorded, ie. over 100casesdai[y. Between 1986 and 2001,fôrâ .cattlepopulationof11.5 million, more than 1$ 0 006 bea dhaddiedfromBSE, over 70°lo of them among dairy herds. I.ike sheep and goats, cattle, after a périodof incuba- tion 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 pos- ture; movement and sensation: swaying gait of the hind- quarters (reminiscent of the pitching of a boat), high stepping gait of the hind limbs, erect tail. Another cha- racteristic 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 dif- ficulty 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 sud- den 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 neuro- logical signs are apparent in trembling, teeth grinding, lack of coordination (ataxia*) of the hind- and then fore- limbs, increasingly frequent falls and prostration (the ani- mal remains lying down), with deterioration of the general condition (weight loss and reduced milk produ .c- tion). The course of the disease generally runs for bet- ween one and seven months . The animals were initially slaughtered because of dangerous behaviour, serious injury following falls, or deterioration of general condi- tion due to prolonged prostration . ." . = The cliniçal picture is often incomplete, as only certain signs are present and bacterial diseases (such as listerio- sis) 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 confu- sed, 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 sus- pected BSE 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 compo- sing 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 . On microscopic examination, the characteristic vacuoles, and notably the triad spongiosis-astrocytosis- 62 neuronal death, are sometimes difficult to demonstrate , and detection of FrF-s by immunohistochemistry* is par- ticularly valuable . Using antibodies, this technique loca- lizes 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 bio- chemical 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 Kin tlom 9 cases ong 0.9 million d 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 aa~Pon 63 Cattle over two years of age were taken into account. wData available on June f, 2002 ~:.. Years when the first case ®i SE 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~ 1Liechltenslein 'aly Israel 1986 1 9B7 18$8 1989 1 990 1991 1992 1993 1994 .1995 1996 1997 1990 1 M 2000 2001 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 high- temperature processing, was spared until 1988.1iie meat- and-bone meal was manufactured from unsalable animal
  • MADCCW DISEASE AND THE CONTAMINATION OF HUMAN S 64 remains from the slaughterhouse . So, after grinding and heating, the dried meal was powdered and used as a pro- tein supplement by the manufacturers of livestock feed. Animal remains were first recommended as a source of protein for livestock (pigs) in 1865. In 1908, an industrial process for theinanufacture of carcass meal was descri- bed 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 produc- tion. 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 (B5E)epidemic Shéep ? _ ~ yn ? ~ Sheep Fannetl Cspec~ liflca6on niminani sbYrer~I'ycl i' CO w 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~porated into meat-and-bone meal andsuccessive recycling led to an anipli fication of the irfèctiousagent, which in the United Kingdom contaminated over 180000 cattle and many other species.
  • THE EMERGENCE OF MAD COW DISEAS E Transtnissi®n of scrapie ~rM onke,Y SheE amnivarés __._.. .. ._ Min k~~ J Ruminants Camivores Rodents ~ Oral transmission passlble between species Dral transmissio n Transmission of ffSE betweénspecies lntracerebral transmissio n kiuman netweenspecles ~~- Monkey ~, . pig Shoep ,,, - --" 6 5 Qmnivore s6oatcow Mink Elk Antelope Cheetah Ruminants Pum aCat MQUSe Carnlvores Rodents Unlike scrapie, bovine spongiform encephctlopathy (BS.11`) is transmas- sible 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
  • MA6COWDISEASE AND TNECONTAMINATION OF HUMAN S taken over by the Department for Environment, Food and Rural Affairs), the annual production of meat-and- bone 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 water- treatment stations were incorporated into meat-and-bone meal (the dissemination of BSE 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 ori- gin ofthis new feline disease was demonstrated by cha- 66 racterization of the strain in the mouse. To date, nearly 90 cats have officially developed sporagifos-rn encephalo- pathy 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 tis- sues 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 disease 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 coordina- tion from the fifth month, muscle contractions from the eighth, loss of speech and movements (mutism and akï- nesia) from the twelfth, followed by death frequently lin- ked to an episode of bronchial pneumonia . On examânationof the brain,even more characteristic signs demonstrated the uniformity of these cases : in addi- tion 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 CJD 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 accoun- ted for 99.7% of all cases of BSE in the world (approxi- mately 165 000 cases of BSE in the United Kingdom in October 1996, compared with under 500 in the rest of the world) and in which an estimated 400000 plus conta- minated 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 firstexpérimentalproof32, was furnishedbyth ediscoveryofthesamecharacteristiclesionsinmacaques experimentally inoculated with the BSE agent. This was the first evidence that the BSE agent was the same as that which is responsible for new cases of CJD in humans .InOctober1996,anewexperimentalstudy33byJohn 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 spe- cies, including macaques . 7hesarne signature was later found in French patients 34 .
  • A NEW CREUTZFELDT-JAKOB DISEAS E 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 identi- cal. The agent responsible for nvCJD in humans is the sarne as that which caused 180000 cases of BSE 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 pre- pared foods. As an example, the experts of the European Community have calculated that, depending on the method of manufacture, with mixtures ranging from kilo- grams 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 Nuenber of persons affected annuafly by new variant Grentzfe&dt-Jakob disease
  • 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 stu- dies 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, how- ever, 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 bet- ween 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 can 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 nCommunityin1993. This authorized the renewe d 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 num- ber 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 ille- gal distribution circuits, and the switching of labels that may have occurred . This information, which was subse- quently 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 conta- minated 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 1988 bau on their use for câtt9ein the United Kingdom A ~ 4 5 ~2 555 900 - 65 % 485 279 TMW 7222 15 674 + 120 % ~ 20 274 1 605 +480 % 740 1 ~ÇŸfi~kt~7~~ 1 826 6 099 + 234 % 1 689 156 559 578 +3% 5 5 92 2 718 + 2 854 % 9 818 400 8 5 3462 <, ~~~1 ;In~ 020 - 20 339 Souwce : lniernet sife o1The Sunday Times. 7 3 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 neces- sary if the industry is well regulated . Yet this is precisely the weak point . Regulation is not assured, as eross-conta- mination 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 ente- ring 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 tes- ting 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 can- not 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 live cattle or beef products doubled between 19 88 and 1995 (1.20000 metric tons in 1988 and 250000 metric tons in 1995) and represented approximately 700 million euros over the period 1989-199639. 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 set- ting up of the single market scheduled for 1993 . In Brussels, misinforming the media was officially sanction- ed I so as to hush up the mad cow issue and avoid a span- ner 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 BSE diagnosed in th e United Kingdom ,..~ 40000 ô 35 000 t p Ç tvM,E30 000 E m Q ô ;a ° 25 000 E . 20000 ° E-Z E 1500 010000 5 000 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 Nfeat-and-bone meal exported bytlte United Kingdom 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 Europe Impact of the ban on nneat -and-bone meal in Continental Europe (examp9eofSwitzerland) ~ 100 ~ ° 9D .e v , ea ro 80 ~c ° ~ 70 K'.É a G m 60 E 50 ; .r c C E ® 40 m ° = E 30 • I E 20 10 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 cattle 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 ? 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 14 00 0 10000 12 000 ~ E ~~ 10 000 8000 ~ 8 00o 6000 ô ® 6000 - 4 000 4 00 0°20002000 É 0 0 É1`b 1`JtSr lytfi5 1`~aJ lyyu 1y91 15yL1Jy ;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 ° aa40 V C ro~ C~ £ c 1~ P ~ ° 30 eoEQ mE 2 0E 10z 0 L .. 1986 1987 . 1986' 1989- .1990 1991 1992 1993 1994 1995 Cases of BSE detected in France per year of birth of the cattl eTheanimalsdevelopedthediseaseonaveragefiveyearsÎater. 1: Passivediagnosls (clinical diagnosis ) 2 Active screening (tests on at-risk population )SystematicscreeningatthesÎaughterhouséofallcattleagedover30months Beet products and live cattle exporte d by the United Kingdom to Continental Europe
  • 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 ¢~~~i R"'I Ï'l' I 1"l I I 1Firs tfirstfirstrecyclingseconddetected 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 BSE 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 popula- tion 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 food- stuffs 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 propor- tional 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 divi- ding the British figures by 10 to 20 to account for the
  • THE RISKS TO HUMAN S imports of British beef products which entered the French food chain a .p to the embargo of 1996. For other European countries, like Germany, not only should British imports be taken into account, but also undia- gnosed 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-and- bone 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, either 80 directly, in numerous prepared dishes, as a sauce thicke- ner, for example, or indirectly, in xneat separated media- nically (meat removed from bone, notably the spine, by mechanical grinding, rubbing ; and high-pressure treat- ment). The composition of minced meats and hainbur- gers, regularly suspected, varied greatly depending on the country, French regulations being among the stric- test. 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 BSE 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 tele- vision 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 Mngdom (up to November 1989) 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 syste- matic 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 BSE sky rocketed in the United Kingdom (from 14407 cases per year in 1990 to 37280 81 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 fromJtrly 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 mecha- nically separated meat any longer be incorporated untreated into prepared dishes. Processed products likely to contain brain and spinal cord are now strictly control- led, above all those of French origin. There is no risk atta- ched to unprocessed red meat. It thus becomes possible, in the light of rational exami- 82 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
  • THE RISKS TO HUMAN S he nonetheless had access to 200 grams of spinal cord which could be used legally in various culinary prepara- tions. 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 obser- ved currently in the United Kingdom and France are lin- ked 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 slaughte- rhouse 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 pre- sent 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 fin- dings in mice changed nothing regarding what we already knew, that the abnormal protein is not detect- able 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 mea- sures and not on any lingering uncertainties . irst the mad cow5 now the mad sheep? British sheep were fed the same contaminated meat- and-bone meal as the cattle. As sheep are very sensitive to oral contamination by the BSE agent, it would be very surprising if no developed BSE. But its develop- ment in sheep cannot be distinguished from that of scra- pie, 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 foot- and-mouth disease, in early 2001 . On 18 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 to tlhe slaughterhouse all animals with scrapie and poten- tially with BSE. Yet on analysis the partial data relea- sed 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 dbyBSE. And if such contamination were the case , then previous screening for BSE 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 mil- 86 lions of sheep in the United Kingdom that had been 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 BSE in sheep today and tlaa.t ad hoc tests are being done . The t' cautÎ ar f! cipl w sil or umbrella? 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 conserva- tive 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 govern- ments of Mrs. Thatcher and Mr. Major. However, its ill- judged application can be especially çounterproductive. In the beef industry, and shortly the sheep industry, the application of the precautionary principle leads to regu- larly 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 thou- sands 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 conta- mination 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, where- as in fact they are the reflection of a marked improve- ment 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 coan- pleteiycollapse 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 by Lhe scrapie agent. This natural conta- mination of the soil can be explained logically in three ways: by contacfiwith infected placentas on parturitio n 88 (limited by the fact that the ewe very soon eats the pla- centa), by an unidentifiéd animal reservoir, such as aca- rians (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 detec- tion by our techniques difficult), and in part to the impossibility of using correctly the methods of detec- tion of prions by inoculation of the mouse (the injection of concentrated faecal matter into the brain of labora- tory 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 dis- persed 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 1991 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 re- covered, 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 K, different groups. The hamsters developed scrapie when the samples were infectious. The earth contaminated at the surface of the flowerpot remained highly con- taminated (60-fold decrease in infectiousness for an ini- tial contamination of approximately 100 million of in- fectious 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 slaugh- terhouses processing BSE-affected cattle . Study pro- grammes are under way to assess the risks as a function of the different types of treatments.
  • 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 expe- rimental 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 recir- culation. 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 den- dritic cells and certain macrophages . The transmission of the BSE agent by blood transfu- sion from a sheep experimentally contaminated by the oral route served as a reminder of this strain's contami- nating 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 BSE 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) experi- mental models that most (approximately 90%) of the infectiousness is associated with the white blood cells and can therefore theoretically be eliminated on special fil- ters. 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 80 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 constitu- ted 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 kmanagementri 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 BSE was being detected in increa- sing 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 nn- known). The European. Commission took additional pre- cautionary 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 introdu- ced 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 ana- lysis 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 elimina- tion of all animals that pose a risk to humans . The effi- cacy of the most sensitive rapid test has proved equivalent to that of the reference test, in which poten- tially 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 times 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 BSE 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 d6lufion of uosiüve control 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. 94 Test .li (Swiss, 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 para- meter: 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 en- acted 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 mani- fest when the first French attempt to ban British cattle in 1990 had to be abandoned because it flouted the regu- lations of Brussels . The serious malfunctions revealed by
  • PROTECTION OF HUMAN S the commissions of enquiry contributed to the resigna- iion of the whole European Commission . It is also signi- ficant 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 Directorate- General, yet the development of a tool guaranteeing consumer safety constitutes the most logical bulwark against BSE. 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 black- mail 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 belie- ved themselves to be unaffected and which above all had no desire to hear of measures likely to worry their consu- mers. So, although it was common knowledge that British meat-and-bone meal had been exported throu- ghout 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) resi- gned 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 BSE is now present throughout Europe, and probably also in numerous other countries that imported British meat-and-bone meal and contam- inated 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 con- fidence 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 BSE? 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 microorga- nisms, and yet we are in good health because under natu- ral 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 patho- gen and/or a weakening of our defence systems (for example broken skin, immunodepression after chemo- therapy). 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 BSE agent is not dangerous for humans. It is therefore pos- sible 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 danger- ous 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 suffi- ciently 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 trans- missible 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, inciden- tally, 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 use- fiil 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 de- fined as not dangerous for the mouse, or therefore for humans; 4) After its low-level replication in the intestine, the 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 appea- rance 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 suffi- ciently sensitive test. 6) The age limit for BSE screening in cattle was lowe- red 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 as the mouse test (at present, only the French test is vali- dated, but it is logical to assume that other sensitive tests will emerge in time), we propose its use to guarantee consumer protection against BSE, 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 slaughter- house should be lowered from 30 to 24 months, as origi- nally 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)Theproblemofmeat-and-bonemealcouldbetrea- ted 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 slaughter- house, 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 pro- duction 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 abso- lutely 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 assi- milable 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 coun- tries, and on implementing coherent measures to protect both people and the environment. Low-risk waste, with- drawn as a precaution, could be subject to intermediate measures; 5) The only sheep diagnosed to date have natural scra- pie, 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 ani- mal enters the human food chain . This commonsense measure may also have the immediate advantage of pro- tecting 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 104 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 indus- try and perhaps tomorrow goat farming, without coun- ting 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 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 pos- sible 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 awa- reness and will prompt the introduction of systems gua- ranteeing food safety, thereby contributing to the pleasure and confidence afforded by quality food.
  • APPENDICE S Notes and ~ ~~etences Glossary . ~~~~~~ of mad ~~~~ disease ~~~~~~~~~~hy
  • tes and references 1. Vache folle: la grande peur, M6, 6 November 2000. 2. The 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 .6. BSE Inquir}ty, 2000, vol. 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 encephalopa- thy', 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 . 676- 69®. 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 spe- cific and common pathogenic events in murine models of scrapie and bovine spongiform encéphalopathy", J Gen Virol, 1996, vol. 77, pp. 1601-1609 .14. Strain 139A in 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 .19. The small building blocks of proteins all of 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 asso- ciated 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 .28. Bousset L ., Belrhali Ii .; Jaiiin 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 auto- nomic 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 parti- cular 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 .32. Lasmêzas C. I., Deslys J.-Ph ., Robain 0., et al. "BSE transmis- sion 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 spon- giform encephalopa.thies" (Ctu-rent understanding), Institut de P'rance, Académie des Sciences, 14 to 16 March 2001 . 37. Report no. 3291 of 15January 1997 by the Mission d'informa- tion commune sur l'ensemble des problèmes posës par le déve- loppement de I'ESB .
  • APPENDICE S 38. 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. GhaniA. 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 slaugh- tered 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 (right- handed) 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 plaque- building 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. irieterozygote: 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 . pertaining to histology, the science of the tissues of living organisms .Homogenate: tissue 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) . 11 3 Ia.trogenic: resulting from a medical act or medication. Imrrtagnohistoehernistry: technique of using labelled antibodies to
  • 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 or 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 cell 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. Ozone: 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 comprising 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,for ,example,thatofaminoacidsinapeptidechainoraprotein. 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 DNA 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): disease 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 on 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 encephalopa- thy, 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 smal- 1er 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 infec- tious protein may be responsible for scrapie . 1966-1968: kuru and then Creutzfeldt Jakob disease were transmit- ted 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 neurophysiolo- gist Stanley Prusiner developed the hypothesis that the agent res- ponsible 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 spon- giform 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 voi- ced concern for human health . The Minister of Agriculture in Mrs Thatcher's cabinet continued to refuse to consider any idea of man- datory 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 slaugh- tered 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 pro- hibited 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) .13August: France decided to ban the import of British meat-and- bone 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 coun- tries 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 ofBSE, 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 ban- ned 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 spe- cies 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, orde- red an embargo on the import of British beef products and cattle. The admission for the first time that inad cow disease can be tr•ans- 122 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 Creutzfeldt- Jakob disease was confirmed . 1997: the Nobel Prize for Physiology and Medicine was awarded in October 1997 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 ende- mic, Chronic Wasting Disease was continuing to spread and affec- ted 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 encephalopa- thy 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 concer- ned 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 fim- ding 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 `offa- cial' 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 Bibliography APFI,r.anuM M. (director.), Risques et Peurs alimentaires, Odi3e Jacob, 1998. KoviuLsKy Ph., Viney G., Le Principe de précaution ; rapport au Premier ministre, OdileJaeob, 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 diseases ,SUes in French AFSSA : www.afssa .fr/dossiers Bureau vétérinaire suisse : CEA: Centre de ressources Infobiogen (Génopole d'Évry): www.infobiogen .fr/people/dessen/prp CNRS : Commission Européenne:]] INRA: INSERM: .nsf Ministère de l'Agriculture: .htm Ministère de la Santé:
  • BIBLIOGRAPH Y .htm Statistiques ESB: Vache folle en ligne (INRA) : Sites in English (United Kingdom) BSE Inquiry: w, Department for Environment, Food and Rural Affairs : http://-, Department of Health : European Commission : .html Meat and Livestock Commission: Mad cow disease: 125