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ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page i I L S I E u ro p e Report Series T RANSMISSIBLE S PONGIFORM E NCEPHALOPATHY AS A Z OONOTIC D ISEASE R EPORT P REPARED UNDER THE RESPONSIBILITY OF THE ILSI E UROPE E MERGING PATHOGEN TASK F ORCE W ITH THE E NDORSEMENT OF THE I NTERNATIONAL F ORUM FOR TSE AND F OOD S AFETY (TAFS) ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page ii About ILSI / ILSI Europe The International Life Sciences Institute (ILSI) is a nonprofit, worldwide foundation established in 1978 to advance the understanding of scientific issues relating to nutrition, food safety, toxicology, and the environment. By bringing together scientists from academia, government, industry, and the public sector, ILSI seeks a balanced approach to solving problems of common concern for the well-being of the general public. Head-quartered in Washington, DC, USA, ILSI has branches in Argentina, Brazil, Europe, India, Japan, Korea, Mexico, North Africa & Gulf Region, North America, North Andean, South Africa, South Andean, Southeast Asia Region, as well as a Focal Point in China. ILSI’s global branch, the ILSI Health and Environmental Sciences Institute, focuses on global issues of human health, toxicology, risk assessment, and the environment. ILSI is affiliated with the World Health Organization as a non-governmental organization (NGO) and has specialized consultative status with the Food and Agriculture Organization of the United Nations. ILSI Europe was established in 1986 to identify and evaluate scientific issues related to the above topics through symposia, workshops, expert groups, and resulting publications. The aim is to advance the understanding and resolution of scientific issues in these areas. ILSI Europe is funded primarily by its industry members. This publication is made possible by support of the ILSI Europe Emerging Pathogen Task Force, which is under the umbrella of the Board of Directors of ILSI Europe. ILSI policy mandates that the ILSI and ILSI branch Boards of Directors must be composed of at least 50% public sector scientists; the remaining directors represent ILSI’s member companies. Listed hereunder are the ILSI Europe Board of Directors and the ILSI Europe Emerging Pathogen Task Force members. ILSI Europe Board of Directors members Prof. N-G. Asp, SNF – Swedish Nutrition Foundation (S) Prof. P.A. Biacs, Ministry of Agriculture and Regional Development (H) Prof. J.W. Bridges (UK) Prof. G. Eisenbrand, University of Kaiserslautern (D) Prof. M.J. Gibney, Institute of European Food Studies (IRL) Prof. A. Grynberg, INRA (F) Dr. M.E. Knowles, Coca-Cola (B) Dr. I. Knudsen, Danish Veterinary and Food Administration (DK) Prof. R. Kroes (NL) Dr. G. Malgarini, Ferrero Group (B) Mr. J.W. Mason, Frito Lay Europe (UK) Dr. D.J.G. Müller, Procter & Gamble Service GmbH (D) Dr. J. O’Brien, Danone Vitapole (F) Dr. L. Serra Majem, Grup de Recerca en Nutrició Comunitaria (E) Drs. P.J. Sträter, Südzucker AG Mannheim/Ochsenfurt (D) Prof. V. Tutelyan, National Nutrition Institute (RUS) Prof. P. van Bladeren, Nestlé Research Center (CH) Prof. W.M.J. van Gelder, Royal Numico (NL) Drs. P.M. Verschuren, Unilever Health Institute (NL) Dr. J. Wills, Masterfoods (UK) ILSI Europe Emerging Pathogen Task Force member companies Campina Melkunie BV (NL) Danone Vitapole (F) Friesland Coberco Dairy Foods (NL) Kraft Foods Europe R&D (D) Masterfoods (UK) Nestec S.A. (CH) Parmalat (I) Unilever Research and Development (UK) About TAFS The ultimate goal of the International Forum for TSE and Food Safety (TAFS) is to contribute to the safety of food. Its mission is to provide an international and independent platform for different sectors of the food chain including industry, consumers, regulators, and scientists, to openly share, evaluate, and disseminate reliable information concerning the impact of TSEs on the food chain. TAFS member companies Danone, Kraft Foods, Nestlé S.A., McDonald's, Migros, Unilever ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 1 TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE WRITTEN BY PAUL BROWN WITH CONTRIBUTIONS BY: RAYMOND BRADLEY LINDA DETWILER DOMINIQUE DORMONT NORA HUNTER GERALD A.H. WELLS JOHN WILESMITH ROBERT WILL ELIZABETH WILLIAMS PREPARED UNDER THE RESPONSIBILITY OF THE ILSI EUROPE EMERGING PATHOGEN TASK FORCE WITH THE ENDORSEMENT OF THE INTERNATIONAL FORUM FOR TSE AND FOOD SAFETY (TAFS) March 2003 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 2 © 2003 International Life Sciences Institute All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright holder. The International Life Sciences Institute (ILSI) does not claim copyright on U.S. government information. Authorization to photocopy items for internal or personal use is granted by ILSI for libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Services, provided that $0.50 per page per copy is paid directly to CCC, 222 Rosewood Drive, Danvers, MA 01923. Phone: (+1) 978 750 8400, Fax: (+1) 978 750 4470. ILSI®, “A Global Partnership for a Safer, Healthier World.®”, and the International Life Sciences Institute (ILSI) logo image of the microscope over the globe are registered trademarks of the International Life Sciences Institute. The use of trade names and commercial sources in this document is for purposes of identification only and does not imply endorsement by ILSI. In addition, the views expressed herein are those of the individual authors and/or their organizations, and do not necessarily reflect those of ILSI. For more information about ILSI / ILSI Europe, please contact ILSI Press One Thomas Circle, NW Ninth Floor Washington DC 20005-5802 USA Phone: (+1) 202 659 0074 Fax: (+1) 202 659 3859 E-mail: [email protected] Website: http://www.ilsi.org ILSI Europe Avenue E. Mounier 83, Box 6 B-1200 Brussels Belgium Phone: (+32) 2 771 00 14 Fax: (+32) 2 762 00 44 E-mail: [email protected] Website: http://europe.ilsi.org Printed in Belgium ISBN 1-57881-158-9 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 3 4 THE DISEASE FAMILY 5 THE ILLNESSES 7 Scrapie (sheep and goats) 7 Chronic wasting disease (deer and elk) 7 Bovine spongiform encephalopathy (cattle) 8 Variant Creutzfeldt-Jakob disease (humans) 8 ETIOLOGY AND PATHOGENESIS 11 Etiology 11 Pathogenesis 13 GENETICS AND SUSCEPTIBILITY 14 ORIGINS OF BSE: THE ANIMAL FOOD CHAIN 16 ORIGINS OF vCJD: THE HUMAN FOOD CHAIN 20 CHRONIC WASTING DISEASE 24 EVALUATION OF RISK 26 GOVERNMENT RESPONSES AND THE CURRENT SCENE 30 CONCLUSIONS AND RECOMMENDATIONS 33 REFERENCES 35 ANNEX 1: INFECTIVITY CALCULATION FOR A CATTLE FOOD PRODUCT 44 ANNEX 2: INFECTIVITY CALCULATION FOR A HUMAN FOOD PRODUCT 45 ANNEX 3: GEOGRAPHIC RISK ASSESSMENT FACTORS FOR BSE 46 3 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE INTRODUCTION T RANSMISSIBLE CONTENTS 16:56 Page 4 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE 25/04/03 INTRODUCTION T RANSMISSIBLE ILSI Rep BSE/TSE for pdf Although recycling of otherwise wasted carcass tissues makes good sense economically, the transfer of material from one body to another carries a risk of transferring pathogenic passengers. In this case, the passenger was an agent with formidable resistance to ordinary methods of decontamination, including the chemicals and elevated temperatures used in rendering carcasses. Processing changes that were introduced towards the end of the 1970s evidently were sufficient to make the difference between destruction and survival of what must have been an irregular presence of very small concentrations of the infectious agent. T ransmissible spongiform encephalopathy (TSE) is a slowly progressive, uniformly fatal neurodegenerative disease that affects several animal species as well as humans. The prototype TSE is scrapie, a naturally occurring disease of sheep and goats that has been recognized in Europe since at least the middle of the 18th century and was shown to be transmissible in 1936. The most important human TSE is Creutzfeldt-Jakob disease (CJD), which was first described in the 1920s and shown to be transmissible in 1968. No direct causal connection between these two diseases has ever been documented, and the concept of any form of human TSE as a zoonotic disease lay latent in scientific thinking for decades. The landscape changed dramatically in 1996 with the suggestion that several cases of a variant form of CJD (vCJD) in young people in the United Kingdom might have resulted from exposure to bovine spongiform encephalopathy (BSE, or “mad cow disease”), itself probably the result of cattle having been exposed to scrapie-infected sheep carcasses rendered to produce meat and bone meal dietary supplements. Considering the large-scale human exposure to bovine products and the extraordinary commercial and public health consequences of BSE as a danger to humans, the suggested BSEvCJD connection was not proposed without a solid epidemiological basis, and it has since been confirmed by biological and molecular studies. BSE has shifted the engines of TSE research into high gear, and powered an awesome amount of industry and government activity, to say nothing of its interest to the general public. This report places these concerns in the historical context of TSE, and attempts to distill the essence of what has become an intimidating volume of intermingled fact and fiction surrounding the story of “mad cow disease” and its consequences to human and animal well-being, with particular attention to food. 4 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 5 Scrapie was recognized early on as a contagious disease of sheep that was (probably) not infectious for humans, as illustrated in the following passage in a German manual of veterinary medicine published in 1759: “The best solution for the shepherd who notices that one of his animals is suffering from scrapie is to dispose of it quickly, and slaughter it away from the manorial lands for consumption by the servants of the nobleman. A shepherd must isolate such an animal from healthy stock immediately because it is infectious and can cause serious harm to the flock”. Many mammalian species are susceptible to experimental infection by TSE agents, including primates, ruminants, ungulates, felines, and laboratory rodents (Table 1). Natural infections have so far been restricted to sheep and goats (scrapie), and to deer and elk (chronic wasting disease, or CWD). Sheep have transmitted scrapie in “unnatural infections” to other sheep and goats via scrapie-contaminated vaccines, and very probably to mink and cattle as a result of the practice of feeding carcasses to mink, and rendered carcasses to livestock. Recycled BSE-infected carcass tissues have also transmitted disease to a variety of zoo felines and exotic ungulates. It is possible that many other species are susceptible to experimental infection, or that they harbour natural disease, but systematic TSE strain-host species studies are incomplete. If natural disease were to occur at the same one per million incidence as sporadic CJD in humans, histological and immunological examination of brains from several hundred thousand members of each species could be required to determine its presence and such an investigation is unlikely ever to be carried out. Figure 1 Known and suspected inter-relationships between animal and human TSEs. TME = transmissible mink encephalopathy; BSE = bovine spongiform encephalopathy; CWD = chronic wasting disease; CJD = Creutzfeldt-Jakob disease; GSS = Gerstmann-Sträussler-Scheinker disease; FFI = fatal familial insomnia. 5 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE T he family of transmissible spongiform encephalopathies (TSEs) encompasses several entities that have different names as a result of having been first described before their common pathogenesis was recognized. The proven or likely interrelationships among members of the disease family are shown in Figure 1. It is important to understand that all of these diseases are expressions of the same pathological process occurring under different circumstances in different species, and that they share far more biological similarities than their different names would suggest. T RANSMISSIBLE THE DISEASE FAMILY 6 Sheep, goats, cattle, deer, and elk Cattle (bovids) includes domestic cattle and captive nyala, gemsbok, eland, kudu, oryx spp. and bison Pig Domestic cat Other captive felids including lion, tiger, puma, cheetah, ocelot, and Asian golden cat Domestic dog Mongoose Mink, ferret, skunk, martens and raccoon Mink (farmed) Weasels (Mustelids) & Raccoons Hamster (BSE) Mouse, vole hamster, rat, gerbil guinea pig *Resulting from dietary exposure or iatrogenic procedures in humans, or from husbandry of domestic or captive animals. Unsuccessful Successful Apes, old/ new world monkeys & Microcebe spp. Humans ?Lemurs “Unnatural” disease* Sheep and moufflon (ovines) Goats (caprines) Deer and elk (cervids) Dogs Rodents Rabbit Lagomorphs Opossum Marsupials Chicken, duck, goose, turkey, raven Birds Z OONOTIC D ISEASE 16:56 Experimental Transmission Humans Cats Ruminants Non-ruminants Carnivores Ungulates (hooved mammals) AS A 25/04/03 Natural disease Primates TABLE 1 Host susceptibility to TSE infection T RANSMISSIBLE S PONGIFORM E NCEPHALOPATHY ILSI Rep BSE/TSE for pdf Page 6 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 7 T RANSMISSIBLE THE ILLNESSES Scrapie (sheep and goats) Pruritus may be so subtle as to go undetected or so dramatic that an animal will rub off most of its wool, and the areas of wool loss may sometimes be rubbed raw (scrapie acquired its name from the fact that sheep were observed to scrape themselves against fixed objects). Some sheep will pull wool from their sides or bite at their legs or exhibit a “nibble reflex” when rubbing themselves or when scratched by hand over the lumbar area of the back. Affected goats are less likely to rub against fixed objects, instead scratching vigorously with their hind feet and horns. Hypersensitivity is another characteristic of scrapie. An affected animal may appear normal if left undisturbed at rest, but when stimulated by a sudden noise, excessive movement, or the stress of handling, tremors may appear or the animal may even fall down in a convulsive-like state. Scrapie-affected sheep (but not goats) also may lose weight despite retaining a normal appetite. Motor abnormalities often include a high-stepping (trotting) gait of the forelimbs and a “bunny hop” movement of the back legs. This gait is especially exaggerated when the animal is made to run. As the disease progresses there may be severe ataxia of the hind limbs, causing the animal to sway, support its hindquarters against a fence when standing, and have difficulty rising. The duration of illness typically ranges from one to six months. Not all animals exhibit all signs of the disease, and there can be enormous variation in the clinical signs among individual animals. For example, a sheep with severe pruritus may show little if any incoordination and vice versa. There may also be differences between breeds. The clinical signs in most cases of scrapie are quite distinct and can be easily recognized. However, several other conditions should be considered, especially in the early stages of illness, including lice or mite infestation, rabies and pseudorabies, listeriosis, ovine progressive pneumonia (maedi/visna), pregnancy toxemia, and poisoning by various chemical and plant toxins. Chronic wasting disease (deer and elk) Affected cervids are invariably older than 17 months of age, and the majority are three to five years of age. The first signs of disease are usually subtle changes in behaviour that may be recognizable only by caretakers who are familiar with the individual animals, and may involve only changes in interactions with handlers or other herd members. Animals may show repetitive behaviours or periods of somnolence or apparent depression, and carry their heads and ears in a lowered position. Feed consumption is decreased, and there is a gradual loss of weight and general condition. 7 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Scrapie is a fatal, degenerative disease that affects the central nervous system of sheep and goats. It is thought to spread from ewe to offspring and other lambs through contact with the placenta and placental fluids, although disease symptoms usually do not appear until two to five years after the animal has been infected, and may not appear until much later. The illness characteristically begins with a slight change in behaviour; the animal becomes more nervous or aggressive and may separate itself from the rest of the flock. Some sheep appear to be demented and have been observed pressing their heads against objects, or “star gazing”. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 8 As the illness progresses, weight loss becomes increasingly evident, and many animals display increased drinking and urination, increased salivation with slobbering or drooling, and a progressive incoordination with stumbling, trembling, and a wide-legged stance. Uncontrollable regurgitation, hyperexcitability, and fainting are occasionally observed. The clinical course can vary from a few days to more than a year in unusual cases, with most animals surviving for two to three months. Death may occur suddenly as a result of swallowing difficulty and aspiration, or may occur only after a prolonged period of progressive wasting to a terminal emaciated state. Other conditions that may at times be confused with CWD are internal parasites (especially meningeal worm infections), locoism, elaeophorosis, brain abscess, pneumonia, chronic hemorrhagic disease, trauma, toxicity, dental attrition, arthritis, and starvation. Bovine spongiform encephalopathy (cattle) BSE infections are thought usually to occur during the first year of life, especially from about three weeks of age, when milk is replaced with solid food concentrates; the average age at onset of illness is about five years. The first observable abnormalities may be subtle changes in behaviour or mental status, such as increased nervousness, apprehension, reluctance to enter doorways, and an unusual tendency to kick when being milked. Behavioural signs may also include a change in ear position, teeth grinding and frenzy (the “mad cow”). Initial physical signs include changes in sensation, posture and gait, especially locomotor ataxia. Signs of sensory dysfunction include hyperaesthesia, excessive licking of the muzzle and flank, and head shyness. Ataxia and body tremors are frequent, and changes in posture can be seen in a wide-based stance, low head carriage, arched back and knuckling (falling and recumbency may complicate the differential diagnosis). Pruritus, probably of central origin and a distinctive sign in many scrapie-affected sheep, is not a common feature of BSE. Blindness and circling are unusual. In addition to neurological signs, there is frequently loss of bodily condition, weight loss, and reduced milk yield. Appetite and thirst are normally maintained, but rumination is reduced. No animal shows all of these signs, and none of these signs alone is sufficient to make a diagnosis. There is little clinical variation by breed, sex, geographical residence, or season. The illness progresses over a period of days to weeks to become clearly evident to an experienced clinician, especially if the animal is stressed by unusual restraint, light or sound. Occasionally an animal in apparent good health suddenly exhibits typical signs after transportation or unaccustomed and prolonged stress. Clinical signs last two weeks to more than one year but typically last about two months. Alternative diagnoses can include metabolic disorders, hepatic encephalopathy, cerebral listeriosis, Haemophilus somnus encephalitis, rabies, cerebrocortical necrosis, abscesses, tumours, and a range of toxic conditions. Variant Creutzfeldt-Jakob disease (humans) Variant Creutzfeldt-Jakob disease (vCJD) is a clinically distinctive form of CJD that affects a much younger age group than the sporadic form of the disease, which typically appears in adults between 50 and 70 years of age. The usual onset of vCJD ranges from early adolescence to the mid-30s, with infections having probably occurred 10 to 15 years earlier. The earliest symptoms are also distinct from sporadic disease: instead of the memory loss and incoordination that herald the onset of sporadic CJD, patients with vCJD typically present with psychiatric symptoms such 8 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 9 The diagnosis of vCJD may be impossible to make in the early stages, particularly if the symptoms are purely psychiatric. When neurological deficits develop, the diagnosis is now suspected fairly quickly, and helpful laboratory investigations can be performed, some of which are important because they exclude other potentially treatable disorders. One non-invasive test has a high specificity for vCJD: magnetic resonance imaging (MRI) of the brain shows a characteristically increased signal in the thalamic region in up to 90% of cases. Figure 2 The pathognomonic vCJD “daisy plaque” consisting of a core of amyloid protein surrounded by vacuolar “petals”. This unusual plaque morphology is also seen in CWD-infected mule deer and BSE-infected rhesus monkeys, but not in BSE-infected cattle. Dr. James Ironside, CJD Surveillance Unit, Edinburgh, Scotland. 9 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE After about six months, progressive neurological problems become prominent, usually in the form of increasing clumsiness, slurring of speech, or an unsteady gait. Involuntary movements of the limbs, face and body develop, such as sudden jerking (myoclonus), fidgeting (chorea) or writhing (dystonia) movements. Memory becomes increasingly affected and there is progressive impairment of thinking processes and increasing physical disability. At the end of the illness individuals are usually helpless, unable to get out of bed, incontinent and unable to speak. Death is often due to a pulmonary infection. The average time from the first symptom to death is 14 months, although some individuals survive for nearly three years. T RANSMISSIBLE as depression, anxiety and personality change that often persist for many months before objective signs of brain dysfunction appear. The first clue that the psychiatric symptoms are caused by an underlying brain disease is the development of pain in the limbs, face or body, which may be associated with tingling or numbness. In a minority of individuals these symptoms develop at the same time as psychiatric symptoms or memory loss. 25/04/03 16:56 Page 10 Immunostaining of tonsillar biopsy tissue for the presence of pathognomonic misfolded protein is also a reliable diagnostic method, but the procedure is invasive and entails post-operative pain and potential bleeding complications. Therefore, tonsillar biopsy (like brain biopsy) is not recommended unless persuasive therapeutic or public health considerations require a premortem diagnosis. A definitive diagnosis can only be made by post-mortem examination of brain tissue, which shows the unique presence of many amyloid plaques that stain positive for prion protein and are surrounded by petals of spongiosis (“daisy plaques”) (Figure 2). The question has been raised as to whether the distinctive clinical, neuropathological, and immunohistological features of vCJD identify all patients with BSE infection, and whether atypical cases of apparently sporadic disease might be caused by BSE. Indeed, recent experiments with “humanized” transgenic mice that showed a sporadic CJD phenotype after BSE infection have prompted the question of whether a proportion of human cases with “typical” sporadic CJD might be the result of BSE infection. However, even if this were true, the proportion must be very small, because the number of UK cases diagnosed as sporadic CJD has not increased since the appearance of vCJD in the mid-1990s. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 10 ILSI Rep BSE/TSE for pdf 25/04/03 16:58 Page 11 Etiology However, all known biological viruses contain nucleic acid, and intensive searches during the past half century for a disease-specific nucleic acid associated with TSE have proved fruitless. In the interim, it was discovered that a host-encoded protein (the “prion”) was inseparable from infectivity and that mutations in its encoding gene on chromosome 20 could cause disease. Molecular biological studies showed that in the infected host, the soluble and proteinase-sensitive normal protein was converted into an insoluble and partially proteinase-resistant isoform that had the characteristics of amyloid1. Although we have now begun to think of TSE as a “misfolded protein disease” with similarities to other amyloidoses, including Alzheimer’s disease, the unique and distinguishing feature of TSE is that it alone is transmissible. It is unnecessary here to recapitulate all of the accumulated evidence showing the critical importance of the prion protein to the infectious disease process. The major question that continues to torment scientists is: how is it possible for a protein to encode information that would be needed to account for different strains of the infectious agent? Even allowing for the fact that conformational differences in the protein are alone sufficient to distinguish between different agent strains, how are these differences generated? Theoretically, a misfolded protein could be formed either directly from its unfolded precursor or by transformation of a normally folded mature form. The original “protein only” theory postulated that the misfolding process in which ·-helical domains are converted into a ß-sheet configuration (Figure 3) results from direct interaction and dimerization of the normal and misfolded forms, with or without the help of cellular chaperones. Strain diversity of the misfolded isoform is enciphered within the tertiary structure of the molecule, implying that several stable abnormal conformations of the protein exist in a given genotype and that any one of these multiple isoforms can be transmitted between affected hosts of the same or different species. 1. Over the years, “prion” terminology has accumulated a family of acronyms that can be confusing. The term “PrP” was originally used as an abbreviation for “proteinase-resistant protein”, but later became an acronym for “prion protein”, and it has been used by different authors to indicate all forms of the protein, or just the misfolded form. The normal protein has been abbreviated as PrPc (for cellular) or PrPsen (because it is sensitive to proteinase K). The misfolded protein has been designated as PrPsc (for scrapie), PrPBSE (for BSE), PrPCJD (for CJD), etc., or PrPres (for proteinase resistance). PrPsen/PrPres is the least ambiguous pairing, but even this terminology now requires qualification, as ongoing studies are discovering varying degrees of proteinase resistance of non-infectious intermediate PrP species. For the present, we prefer to use the terms “normal protein” and “misfolded protein” (the fully misfolded amyloid form of PrP that is associated with infectivity). 11 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE The disease agents that cause transmissible spongiform encephalopathy were recognized from the start as being rather “special” in that, although they behaved in many ways like a virus, they showed a very long latent period between infection and illness, a correspondingly long duration of illness, an extraordinary resistance to inactivation, and they could not be linked with any identifiable morphological structure. They were for many years therefore known as “slow viruses”, and later as “unconventional viruses”. They can still be considered to be “unconventional viruses” if we think in terms of minimum criteria: a submicroscopic entity with the capacity both to replicate and to transmit information, as in computer “viruses”. T RANSMISSIBLE ETIOLOGY AND PATHOGENESIS T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 12 Figure 3 Schematic structural transformation of the normal (left) to the misfolded (right) prion protein molecule, showing the partial conversion of α-helices (coils) to ß-sheets (arrow ribbons). The structure of the normal protein has been confirmed by NMR analysis; the structure of the misfolded protein is speculative. One modification proposes that the normal isoform is its own chaperone – in other words, that the unfolded protein is either normally folded in the presence of normal protein, or misfolded in the presence of the misfolded isoform, avoiding the need for misfolded protein to act directly on normally folded protein. A further refinement proposes that the protein molecules aggregate by interaction of the globular region of the protein (amino acids 121–231), which has allosteric and regulatory control functions. The increase in ß sheet content is due entirely to misfolding of the flexible tail that starts from the first ß sheet of the normal molecule. This hypothesis also implies that folding is a primary phenomenon that requires no refolding process. Another theory invokes a seeding mechanism (nucleation) similar to what is seen in inorganic crystallizations. In vitro, freshly prepared recombinant protein is monomeric, but over time it transforms itself into dimeric crystals in which helices are “swapped” and which could be the basis for the formation of misfolded crystal polymers. Different protein-mineral-protein interactions within microcellular milieux would account for strain variation. A major limitation of the prion hypothesis and probably of the other “protein only” alternatives is our ignorance of the thermodynamics of the process by which a normal protein molecule is converted to a misfolded isoform. Within the context of protein biochemistry, it is difficult to imagine that the very limited number of stable conformations of a given monomeric protein could be sufficient to account for the observed number of strains of the infectious agent. Much more work on the details of protein misfolding, and especially on the identity of intermediate protein species, will be necessary to decipher this mystery. A growing body of data from both cell culture and in vivo experimental models is gradually advancing our understanding of the intracellular trafficking of the normal protein, and implicating pathways along which the normal protein may undergo misfolding and accumulation in neural cells. Recent observations suggest that unglycosylated protein increases during infection at the expense of glycosylated forms, and that under reducing conditions the unglycosylated protein is vulnerable to misfolding, which triggers a cellular mechanism of retrograde transport to the cell cytosol, where the protein accumulates in an aggregated form that resists catabolic 12 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 13 vagus n. blood IV, IP, IM spleen splanchnic n. blood spinal cord BRAIN intestinal mucosa & LRT blood PNS Neuroinvasive pathways from peripheral sites of TSE infection. The peripheral nervous system (with and without splenic involvement) has been well documented in experimental models. Hematogenous routes have been inferred to occur under some circumstances. IV = intravenous; IP = intraperitoneal; IM = intramuscular; LRT = lymphoreticular tissue; n = nerve; PNS = peripheral nervous system. degradation by cellular proteasomes. The misfolded species has most of the chemical and biological properties of the pathological amyloid protein, is toxic to neural cells, and is even capable of inducing a neurological disease when inoculated into normal mice; however, the disease is not transmissible from these mice to normal mice. Pathogenesis Whatever the final judgment about “prions” as the cause of TSE, there is no argument about the crucial role of the prion protein in the infectious process, or about its value as a marker of infectivity. Thus, the pathogenesis of TSE has been investigated using methods that either infer infectivity from detection of the protein (Western blots or immunohistochemistry) or by direct measurement of infectivity (bioassay by animal inoculation). The itinerary of the infectious agent within the body depends on how infection is initiated (Figure 4). In experimentally induced rodent infections, when the agent is introduced by parenteral inoculation, neuroinvasion is preceded by a phase of replication within the lymphoreticular system followed by transport via the splanchnic nerve to the spinal cord. In the spleen, mature follicular dendritic cells play an important role in replication, and maturation depends on the presence of B cells. When the agent is given by mouth, infection can bypass the spleen and proceed directly from the gut to the brainstem via the vagus nerve. Based on findings of misfolded protein or infectivity in human subjects with CJD, the optic and olfactory tracts have also been identified as potential portals of entry. The role of circulating blood in naturally occurring disease remains uncertain. The few reported disease transmissions from the blood of patients with CJD are far outnumbered by transmission failures, but blood has been repeatedly shown to be infectious in experimental models of TSE, as well as in naturally occurring scrapie infections. Even if neuroinvasion occurs via nerves, blood is the most plausible vehicle by which infectivity in both experimental and natural disease ultimately finds its way to the many peripheral organs in which it is present. It is interesting that the clinical onset of illness after infections introduced directly into the brain (e.g. by contaminated neurosurgical instruments) are indistinguishable from the clinical features of sporadic CJD, but different from the clinical presentations that follow peripheral routes of infection (e.g. from contaminated growth hormone injections). This difference suggests that sporadic CJD may arise within the brain itself, in which case infection would have no neuroinvasive phase. 13 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ORAL blood T RANSMISSIBLE Figure 4 T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 14 GENETICS AND SUSCEPTIBILITY enetic factors play an important role in susceptibility to at least some TSEs. Indeed, during the 1960s a ferocious argument consumed proponents of purely genetic versus contagious etiologies of scrapie. Classical genetic studies defined a hierarchy of susceptibility to scrapie infection according to purebred breeds of sheep, and clearly distinguished between two varieties of the infecting agent, characterized by short and long incubation periods. G In the 1980s, the discovery of the prion-encoding gene made it possible to correlate the results of classical genetics with modern molecular genetics, and the short and long incubation period phenotypes were found to be linked to alternative genotypes of polymorphic codons both in naturally infected sheep and experimentally infected mice. Extensive further studies of breeds of sheep with different susceptibilities to infection have not been rewarded with the same degree of clarity, but it appears that certain allelic combinations of three polymorphic sites within the encoding gene can be broadly correlated with resistance or susceptibility to clinical disease, and perhaps to infection (Table 2). TABLE 2 Molecular genetics of sheep susceptibility or resistance to scrapie Polymorphic coding combinations in sheep “prion” gene Codon 136 Codon 154 Codon 171 A A A A V R H R R R R Q H Q Q A = alanine, H = histidine, Q = glutamine, R = arginine, V = valine Genotypic susceptibility of Suffolk or Cheviot sheep to scrapie Sheep breed Genotype Susceptibility/resistance Suffolk ARQ/ARQ ARQ/ARR ARR/ARR Highly susceptible Occasional occurrence Resistant Cheviot VRQ/VRQ VRQ/ARQ VRQ/ARR ARQ/ARQ ARQ/ARR ARR/ARR Highly susceptible Highly susceptible Occasional occurrence Resistant Resistant Resistant Note: Other breeds of sheep (e.g., Texel) can show still different allelic patterns of susceptibility and resistance. 14 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 15 The human gene contains several polymorphic sites, one of which (at codon 129) has been shown to influence susceptibility to all forms of human TSE. Codon 129 encodes either methionine (M) or valine (V), and in the general Caucasian population the three alternative genotypes are approximately distributed in the proportions of 50% MV (heterozygotes), 40% MM, and 10% VV (homozygotes). The homozygous genotypes are nearly twice as common in human TSE – whatever the cause – as in the general population, and the homozygous MM genotype is the only genotype found in more than 100 tested cases of vCJD. Thus, this genotype appears to be a requirement for susceptibility to infection by BSE, or at least for the expression of disease. There are two caveats to this statement, however. First, it remains possible that codon 129 heterozygotes have a longer incubation period than do homozygotes, and thus would not develop symptoms until after the epidemic was well under way (experience with kuru, also acquired by the oral route of infection, lends support to this idea). Because we do not yet know where we are in the epidemic curve, we cannot know whether heterozygote cases will appear, or whether they will continue to be totally resistant to infection. Second, it is possible that some cases of vCJD might escape detection because instead of having the characteristic vCJD phenotype, they might look like sporadic disease. This possibility has been a kind of shadowy concern from the beginning, and it has recently been given substance by studies of BSE in “humanized” transgenic mice in which two distinct phenotypes were observed: one that was indistinguishable from vCJD transmissions, and another that resembled transmission of the most common form of sporadic CJD. However, if a sporadic-like CJD phenotype from BSE infection occurs in humans, it must be extremely rare, given the fact that the incidence of sporadic disease in the UK has not increased since the first case of vCJD was identified more than eight years ago. A final point concerns mutations. It is curious that whereas in humans more than 30 pathogenic mutations account for some 5–10% of all cases, no pathogenic mutation of any sort has ever been identified in either sheep with scrapie or in cattle with BSE (the same can tentatively be said about deer and elk with CWD and mink with TME, but they are less well studied). This apparent genetic discrepancy between human and animal disease remains entirely unexplained. 15 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Cattle have a different type of genotypic polymorphism, involving the number of octapeptide repeats – either five or six – in the region of codons 50–91 of the prion-encoding gene. The 6-6 homozygous genotype is present in about 90% of cattle, and the 6-5 heterozygous genotype occurs in most of the remaining 10% of cattle (the 5-5 genotype is rare). Several studies have failed to reveal any correlation of genotype with BSE susceptibility. T RANSMISSIBLE These sites are located at codons 136, 154, and 171, and the homozygous allelic combination conferring the greatest resistance encodes the amino acid trio of alanine, arginine, and arginine (ARR). The allelic combination that encodes valine, arginine, and glutamine (VRQ) is associated with susceptibility, both in its homozygous state and in some heterozygous combinations. Some genotypes are susceptible to experimentally induced scrapie but resistant to natural disease, and a further complication arises from the range of genotypic complexity found in different breeds of sheep. However, enough is known to guide breeding programs aimed at producing sheep that are resistant to the development of clinical disease. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 16 ORIGINS OF BSE: THE ANIMAL FOOD CHAIN T he origin of BSE will probably never be known with certainty, but the hypothesis of a species-crossing infection from scrapie-affected sheep is more plausible than the leading alternative hypothesis of a spontaneous case of BSE in cattle. Both hypotheses enlist the mechanism of contaminated carcass recycling as the vehicle by which infection was rapidly amplified to an annual level of over 30,000 new cases within five years of its first appearance in Britain. Any satisfactory explanation of BSE must answer two questions: Why did BSE begin in the mid1980s, and why did it begin in Britain? Both the sheep and cattle origin hypotheses have the same answer to the question of timing, which is that changes in the system of rendering carcasses into meat and bone meal dietary supplements that took place around 1980 permitted infectivity to survive the process and be recycled. At least two such changes could have contributed to the survival of infectivity: increased use of continuous rather than single-batch rendering (which could have created incomplete exposure to heat), and the elimination of a final tallow extraction step consisting of exposure to hydrocarbon solvents under steam. It has since been shown experimentally that neither of these changes had a very great effect, but if the level of infectivity entering the rendering process were near the threshold of transmissibility, small changes could have made the difference between destruction and survival. For example, a simple calculation suggests that a maximum level of input carcass infectivity would have been highly unlikely to exceed 1 log10LD50 (10 mean lethal doses) per gram of tissue, which is the amount that the tallow extraction step was capable of eliminating (Annex 1). More convincing than speculation and arithmetic is the fact that the prohibition of ruminant protein in dietary supplements in 1988 was followed five years later by the start of a downturn in BSE cases, and five years is the average incubation period between BSE infection and manifest illness (Figure 5). It has been objected that if contaminated feed were the only means of spreading disease, no BSE infections should have occurred after the ruminant feed ban was fully in effect by the mid-1990s. How to explain the fact that some cattle born after the ban have come down with the disease? Although maternal transmission has been proposed, the epidemiological evidence favours a widespread but very low-level exposure that in all likelihood represents a kind of “Continental revenge”: the unforeseen importation of contaminated feed from countries to which UK meat and bone meal had earlier been exported, with the result that the carcasses of newly infected cattle began to enter their rendering plants, producing feed for both domestic and international commerce. Whereas the timing of the epidemic is reasonably well explained by changes in the rendering process with consequent survival of infectivity in meat and bone meal dietary supplements, the origin of the contamination is open to discussion: scrapie or the chance occurrence of a de novo case of BSE? Accurate figures for the international prevalence of scrapie are not available, but scrapie is at least as prevalent among sheep in the UK as in most other countries of the world, and the UK has a comparatively large proportion of sheep to cattle. Thus, a widespread potential source of infection was already in existence, whereas BSE was unknown until 1986, when the epidemic began. The argument that unrecognized “spontaneous” cases of BSE could have been occurring in cattle for decades at the same one per million annual frequency as sporadic cases of CJD in humans, and that one such case was a “founder” of the BSE epidemic has a formidable obstacle: sporadic BSE cases cannot have been occurring only in the UK, yet no coincident epidemics occurred in several other countries (for example, the US) in which rendering processes were changed at about the same time 16 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 17 T RANSMISSIBLE Figure 5 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Chronology of the BSE epidemic in the UK. as in the UK. Moreover, the distribution of early BSE cases in the UK (and subsequently in other countries that unwittingly imported BSE from the UK) is more consistent with multiple initiation points than with a single point source of infection. This would favour scrapie as the source of infection, because scrapie was widespread enough to have entered rendering plants throughout the UK, whereas it would have required simultaneous cases of sporadic BSE to achieve the same effect. On balance, the evidence favours scrapie, but it is only fair to point out that the sporadic BSE hypothesis does perhaps more easily explain two curious features of BSE: strain uniformity and human pathogenicity. Like most infectious pathogens, the scrapie agent has many experimentally distinguishable strains. Although rodent models of scrapie provide ample precedent for single strain selection on passage to different rodent species, a bovine origin of BSE eliminates the need for this kind of selection process. As for pathogenicity, all evidence to date indicates that scrapie is not a human pathogen, and if scrapie does not infect humans, why should scrapie passaged through cattle behave any differently? Precedents exist for a strain of TSE in one species that is unable to transmit disease to a different species unless it has passed through an intermediate species; however, because this phenomenon is exceptional, it is not so satisfactory as the explanation implicit in the BSE hypothesis – a new strain of TSE arising in cattle could as easily as not be pathogenic for humans. Cattle were not the only target for dietary supplements: other livestock species, such as sheep, pigs, and chickens, as well as several kinds of zoo animals, laboratory animals, and pets also received feed to which meat and bone meal had been added. Examples of disease transmission to non-livestock species have been reported in ungulates, felines, lemurs in zoos and pet cats. The spectre of BSE-infected sheep has excited a growing concern, because in the process of “backcrossing” to sheep, the agent might carry its newly acquired ability to infect humans, and at the moment there is no practical way to distinguish natural scrapie from BSE in sheep. (The mouse 17 T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 18 Figure 6 Worldwide distribution of UK exports of live cattle during the period 1988–1993. Data obtained from the WHO, courtesy of Dr. Maura Ricketts. Figure 7 Worldwide distribution of UK exports of meat and bone meal (MBM) during the period 1986–1990. Data obtained from the WHO, courtesy of Dr. Maura Ricketts. 18 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 19 TABLE 3 Indigenous cases of BSE arranged by country and date of first occurrence as of 1 January, 2003 Country 1985–89 1990–94 1995–99 United Kingdom Ireland 10,188 10 136,574 78 32,640 342 10 12 118 France Portugal Switzerland Belgium Netherlands Liechtenstein Luxembourg Germany Spain Denmark Italy Slovakia Japan Czech Republic Slovenia Austria Finland Greece Poland Israel 2000 2001 2002 Total 1443 149 1202 246 755 333 182,802 1,158 69 362 215 161 149 33 274 110 42 239 86 24 753 719 432 10 6 2 1 9 2 0 0 46 20 0 0 38 24 0 1 103 52 2 2 7 2 1 125 82 6 106 127 2 238 211 9 48 5 3 2 1 1 1 1 38 6 2 2 1 0 0 0 86 11 5 4 2 1 1 1 4 1 4 1 Note: For some countries, numbers are still incomplete for the year 2002; regular updates of global BSE figures are available on the OIE website: www.oie.int 19 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Bad as it was, the seriousness of the situation was compounded by the continued exportation of live cattle and of meat and bone meal dietary supplements to virtually every country in Europe and a number of non-European countries (Figures 6 and 7). Sometimes, animals sick with BSE were identified before slaughter and did not result in disease outbreaks. However, in countries that imported large numbers of cattle and thousands of tons of meat and bone meal, BSE was able to spread through carcass recycling of both imported and newly infected domestic cattle. By means of systematic epidemiological surveillance and, more recently, immunological testing of the brains of slaughtered animals, most European countries have discovered affected animals within their cattle populations during the past few years, and new countries are added each year. Fortunately, these secondary outbreaks are mere brushfires compared with the original UK conflagration (Table 3). T RANSMISSIBLE panel incubation period and lesion topography “signatures” of BSE require over a year to perform). Pigs are not susceptible to oral BSE infection, and chickens are resistant to infection by any route of administration. Dogs are also apparently resistant to TSE infection, as several animals inoculated intracerebrally with human strains of CJD and kuru did not become ill, and in countries with BSE no dogs have been identified with spongiform encephalopathy, despite the certainty that some dog food, like cat food, would have included bovine tissue. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 20 ORIGINS OF vCJD: THE HUMAN FOOD CHAIN I f ideas about the origin of BSE are less secure than those about the vehicle of infection, vCJD presents the opposite situation of a quite certain origin but a stillspeculative vehicle of infection: dietary exposure to beef products contaminated with central nervous system tissue. The fact that unusual cases of spongiform encephalopathy in young people were appearing in a country still recovering from a vast epidemic of spongiform encephalopathy in cattle was the first clue to the possibility of a connection between the two diseases. Systematic study of the first 10 cases seen within the space of two years (1994–1996) revealed that they did in fact have a distinctive form of illness that had not previously been seen in the UK (and was not occurring anywhere outside the country), and that they had no mutations or recognized environmental causes (such as contaminated growth hormone) to account for the disease. With this evidence, a connection between BSE and the human cases was strongly suggested, and it has since been confirmed by two independent experimental methods. One method used Western blots to show that the misfolded protein extracted from the brains of both BSE-affected cattle and vCJD-affected humans had an identical pattern that was different from the pattern seen in all other TSEs, including sporadic CJD (Figure 8). The other method used inoculations of a panel of inbred mouse strains to show that brain tissue from cases of BSE and vCJD produced the same patterns of latency (incubation period) and brain lesion topography, which also were different from other TSEs, including sporadic CJD and scrapie (Figure 9). These experimental studies, together with the earlier clinical and epidemiological observations, proved the link between BSE and vCJD beyond a shadow of a doubt. Figure 8 Western blots of amyloid protein extracted from the brains of 2 cases of sporadic CJD (type 1 and 2A), and a case of variant CJD (type 2B). Types 1 and 2 differ in their molecular weights; types 2A and 2B differ in the relative amounts of each glycoform. This combination of molecular weight and glycoform pattern distinguishes vCJD from all other TSEs. Courtesy of Dr. Mark Head, CJD Surveillance Unit, Western General Hospital, Edinburgh. 20 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 21 T RANSMISSIBLE Figure 9 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Incubation periods (A) and brain lesion topography (B) in different strains of inbred mice inoculated with scrapie, BSE, FSE (cat infected with BSE), vCJD, and sporadic CJD. Courtesy of Dr. Moira Bruce, Neuropathogenesis Unit, BBSRC, Edinburgh. The source of the human infection was cattle, but by what means was infection acquired? The answer has come chiefly from a process of elimination, and, plausible though it is, it still lacks the kind of laboratory evidence that clinched the identification of BSE-infected cattle as the source of disease. Epidemiological study did not uncover any convincing disease clusters, or point to any regional peculiarities or vectors that might have linked cattle with BSE to human disease, and investigation of “high risk” contact groups such as farmers, slaughterhouse workers, or butchers did not identify any cases of vCJD (although a few cases of sporadic CJD did occur among workers in these professions). 21 T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 22 Figure 10 Age at onset of illness in all cases of vCJD and sporadic CJD seen in the UK during the period 1994 through 2000. If physical contact with infected cattle could not be implicated, the next logical link to explore was exposure to cattle products. This posed an immediate (and continuing) problem because virtually the entire British population was exposed in one way or another to a huge number of bovinederived products, which left little likelihood of implicating any given product as the cause of vCJD. Consumption of meat and dairy products, and exposure to products containing tallow or gelatin (or their derivatives) are universal, and no correlations could be established between vCJD and exposure to any given product. It was particularly important to investigate the consumption of brain by patients with vCJD, because the distribution of infectivity in BSE-infected cows is almost entirely limited to nervous system tissues, but no association was found. The key lay with slaughterhouse practices and meat preparation, about which the scientific community was completely naïve. Before the appearance of BSE, vertebral columns were routinely included in the remains of carcasses from which as much meat as possible had been manually removed. Spinal cords were usually removed, but cord fragments and paraspinal ganglia were certain to remain. The truncated carcasses were then subjected to a process of compression to yield bone fragments (used for gelatin or meat and bone meal) and a paste of “mechanically recovered meat” (MRM) that was permitted to be added to a variety of packaged meat products such as hot dogs, sausages, beef patties, luncheon meats, beef stews, etc., in proportions as high as 30% (but usually in the range of 5–10%). It is now abundantly clear that central nervous system tissues were entering the human food chain through this “unadvertised” vehicle, and that they were the most likely cause of human infection. That said, two very curious questions remain unanswered by any of the hypotheses that have been advanced to explain the phenomenon of vCJD. The most puzzling question concerns the age at which disease appears – there is no satisfactory answer as to why vCJD affects such a young age group (Figure 10). Information about commercial food distribution and consumption, which might provide the best clues, is almost as unreliable as a dietary history obtained from the relative of a patient with vCJD. Thus, although comparatively inexpensive products (including school 22 ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 23 T RANSMISSIBLE Figure 11 cafeteria offerings) that contained MRM have been suspected of being favoured by children and adolescents, hard evidence is lacking. Alternatively, the preference for youth may simply be a trait of the infecting strain of BSE – some other infectious diseases also show preferences for one or another age group – but the word “simply” underscores our ignorance of the cause. The second curiosity involves the relatively small number of cases (currently about 130) in a British population of 60 million that has presumably been ubiquitously exposed to potentially contaminated beef products, and only a handful of cases have appeared in other countries where BSE has been exported (Figure 11). Have only these few people ingested large enough doses of infectivity to overcome the species barrier and an inefficient route of infection? It is hard to imagine that any product would have contained more than a trace of infectivity (Annex 2), and equally hard to imagine that any high-dose product would only infect a random individual rather than produce disease clusters, which have not been seen (in particular, no clusters have occurred in families sharing the same diet). Could it be a question of genetic susceptibility? We know of only one such example – methionine homozygosity at polymorphic codon 129 of the prion gene – and it cannot be the sole answer, because nearly 40% of healthy humans have the same genotype. The most reasonable explanation would seem to be the same phenomenon that operated to cause the limited outbreak of iatrogenic CJD from contaminated human growth hormone – a kind of epidemiological Russian roulette. Threshold doses causing rare and randomly distributed cases were almost surely the explanation for the fact that in the US, for example, only 26 cases of CJD have occurred in a total treated population of over 7000 hormone recipients, which corresponded very nicely with the experimental demonstration that CJD transmitted to only one of two monkeys inoculated with one batch among more than 70 different inoculated batches of growth hormone. If in fact the dose of a TSE agent is usually below but exquisitely close to the threshold of transmissibility, only the rare individual will become infected, and this may be conditioned by numerous factors about which we have little knowledge and less control, as obviously important as an undiscovered “susceptibility” gene, or as apparently trivial as minor inflammations of the oral cavity or gastrointestinal tract. 23 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Chronology of vCJD occurrence (French and Italian cases had not visited the UK). Data for 2002 are still incomplete. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:56 Page 24 CHRONIC WASTING DISEASE C WD was first identified in the US in the late 1960s in captive mule deer in a Colorado wildlife research facility, but it was not recognized as a spongiform encephalopathy until 1978. Interestingly, its neuropathology included the hallmark abnormality of vCJD (“daisy plaques”) before that disease appeared in the UK. The known occurrence of CWD remained limited to captive mule deer until 1981, but within a decade was discovered in free-ranging mule deer, white tail deer, and elk in Colorado and Wyoming. By 2000, CWD had been identified in both farmed and free-ranging animals in several states near the original endemic area, as well as in contiguous regions of Canada. Intensified surveillance in recent years has identified what appears to be an ever-expanding geographic range, thought to result more from commercial interstate transport of animals than from natural movement: cases have turned up west of the Continental Divide in Colorado, in Wisconsin, in New Mexico, and even in South Korea (from elk exported from Canada that have now caused several endemic cases). The pathogenesis of CWD has not yet received the same attention as scrapie or BSE, but what is known is consistent with the general outlines of TSE: early involvement of the lymphoreticular system, including gut-associated lymphoid tissue, and incubation periods ranging from 15 to 36 months, depending on the species and conditions of infection. Significant differences in the amount and distribution of abnormal protein in different body tissues have been observed in deer and elk, and although the protein has not been detected in either muscle or “antler velvet” – two products consumed by humans – bioassays of these organs have not been undertaken. Comparatively inefficient misfolding of the normal human protein can be initiated by in vitro incubation with a misfolded cervid protein “seed”, and under experimental conditions, CWD can be transmitted to a variety of species, including at least one primate species. Several years ago, in a still unpublished study, two squirrel monkeys were inoculated intracerebrally with brain homogenate prepared from a mule deer dying of CWD, and the disease transmitted to both animals after incubation periods of 31 and 32 months. The clinical features and duration of illness were not recorded, but neuropathological examinations revealed spongiform change without any plaque formation. Immunostaining was not performed. CJD was recently diagnosed in three unusually young patients who had consumed venison, and although epidemiological and laboratory investigation failed to show a convincing link between exposure and disease, the conclusion that these patients most likely had sporadic CJD must be weighed against the fact that we do not know what CWD in humans would look like – it might look like sporadic CJD, or vCJD, or it might have distinguishing characteristics unlike either form of disease. The same may of course be said about human infections with scrapie or mink encephalopathy, but we already know from epidemiological surveillance studies that scrapie does not cause CJD (its incidence is similar in countries with and without scrapie), and mink tissues do not enter the human food chain. 24 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 25 Another potentially dangerous situation would arise if CWD were to find its way into non-cervid animal species. In particular, if CWD were to be introduced and become endemic in livestock species such as sheep and cattle, the animal and human food chains could be put at the same kind of risk as happened with BSE. We know that sheep and cattle can be experimentally infected with CWD by intracerebral inoculation, and tests are under way to determine whether oral dosing with CWD brain tissue, or close contact with CWD-infected deer, can transmit disease to cattle. Although food chain infection would require a series of breakdowns in the system of precautionary measures already taken to prevent a BSE outbreak, including the banning of most mammalian protein for use in ruminant feed, the potential for human error is a real and unpredictable quantity. 25 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Although CWD presents more of problem to individuals (hunters, for example) than to public health, individual infections could have public health consequences similar to those of vCJD: apparently healthy individuals harbouring the infection during its incubation period could possibly transmit disease via cross-contamination of surgical instruments or blood donations, and after death, if disease is unsuspected, their organs could be harvested for donations. Without the ability to establish a diagnosis of human CWD infection, or knowledge of the presence or absence of infectivity in peripheral body tissues and blood, our understanding of the potential for human risk will continue to depend solely on epidemiological inference. T RANSMISSIBLE In brief, CWD is spreading – or is now being recognized in areas distant from its original focus – and may have the potential to infect humans. It is not known whether CWD exists undetected outside North America, but the spectre of a US “mad deer” epidemic has excited much political and media attention, and has brought CWD to the forefront of TSE research. Its unique and troubling feature is that unlike scrapie, TME, and BSE, it occurs in both captive and wild-ranging animals, which poses enigmas both for understanding the means by which it is transmitted from animal to animal, and for devising strategies to prevent its spread. When the disease is diagnosed in captive animals, herds can be culled or entirely destroyed, but this strategy cannot be used for animals in the wild. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 26 EVALUATION OF RISK ssessing the risk of transmitting BSE to animals or humans by products of bovine origin is accomplished by a simple and straightforward set of criteria; the difficulty lies in the fact that data to interpret the criteria are frequently incomplete or non-existent. A The criteria can be summarized as follows: 1. Geographic source of bovines 2. Tissue source of the bovine products 3. Processing effects on infectivity 4. Route of product administration The establishment of a “risk hierarchy” according to the geographic origin of source cattle is by far the most difficult and disputed item in the whole assessment scheme: the criteria used by the European Commission include: adequacy of surveillance to ensure detection of BSE cases; culling schemes; importation of live cattle or meat and bone meal from BSE-affected countries; rendering and other feed preparation practices; and types and implementation of feed bans (Annex 3). Table 4 shows the current placement of all countries evaluated to date; the list continues to be expanded and updated as data become available. For economic reasons, all countries naturally wish to be considered as having as low a risk as possible, and some have disagreed with the category in which they have been placed by the Commission. Moreover, assessment of risk at the national level does not take into account the possibility that regions or even particular herds of cattle in a country that has identified BSE within its borders may be at much lower risk than other regions or herds, or even be risk-free. The reason is not that TABLE 4 Geographical BSE Risk (GBR): categories of evaluated countries as of 1 January, 2003 GBR level Presence of one or more cattle clinically or pre-clinically infected with the BSE agent I Highly unlikely Argentina, Australia, Botswana, Brazil, Chile, Costa Rica, El Salvador, Iceland, Namibia, New Zealand, New Caledonia, Nicaragua, Norway, Panama, Paraguay, Singapore, Swaziland, Uruguay, Vanuatu II Unlikely but not excluded Canada, Columbia, India, Kenya, Mauritius, Nigeria, Pakistan, Sweden, USA III Likely but not confirmed, or confirmed at a lower level Albania, Andorra, Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Israel, Italy, Latvia, Lithuania, Luxemburg, Malta, the Netherlands, Poland, Romania, San Marino, Slovakia, Slovenia, Spain, Switzerland, Turkey IV Confirmed, at a higher level Portugal, UK Notes: The GBR is a qualitative indicator of the likelihood of the presence of one or more cattle being infected with BSE, pre-clinically as well as clinically, at a given point in time, in a country. Assessment of the GBR is based on the assumption that BSE arose in the United Kingdom (UK) and was propagated through the recycling of bovine tissues into animal feed. Later, the export of infected animals and infected feed provided the means for the spread of the BSE-agent to other countries where it was again recycled and propagated via the feed chain. For all countries other than the UK, import of contaminated feed or infected animals is the only possible initial source of BSE that is taken into account. These GBR national categories are current as of December 2002; regular updates are available on the EC Food Safety web site: http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html 26 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 27 It is fortunate that the two most heavily consumed bovine products – meat and milk – do not contain any demonstrable infectivity. Homogenates of bovine muscle have not transmitted disease to intracerebrally inoculated mice and cattle, and milk has not transmitted disease to intracerebrally inoculated mice, or (even more convincingly) to nursing calves of BSE-affected mothers. The absence of detectable infectivity in blood means that organs that are not inherently TABLE 5 The distribution of infectivity in tissues of cattle experimentally infected with BSE Bioassay in mice Months after oral exposure Frontal cortex Caudal medulla Spinal cord Dorsal root ganglia Trigeminal ganglia Distal ileum Bone marrow 2 6 10 14 18 22 26 ≥32 - + - + - + - + - nt - nt - + + + + + (+) nt = not tested. Parentheses indicate a single positive result at 32 months, but negative results at 36 and 40 months after oral exposure. Negative tissues (assayed by intracerebral and intraperitoneal inoculation of mice): pituitary, dura mater, CSF, peripheral nerves, skeletal muscles and tendon; non-ileum GI tract, liver, pancreas, salivary glands; spleen, tonsil, lymph nodes, blood clot, buffy coat, serum; kidney, lung, trachea, heart muscle/valve/ pericardium; testis, prostate, ovary, placenta; skin, fat, bone; milk, urine, feces. Bioassay in cattle Months after oral exposure Caudal medulla/spinal cord Caudal medulla Spinal cord Distal ileum Tonsil 6 10 18 22 26 32 nt ot ot + ot nt ot ot + + nt ot ot + ot ot nt nt nt nt ot ot ot nt ot + nt nt ot nt nt = not tested; ot = on test (assay in progress and negative to date: survival range as of 1 January, 2003 is 46-59 months after intracerebral inoculation). Tonsil has transmitted to only 1 of 5 assay animals. Other tissues for which assays are in progress and negative to date (7-33 months after oral exposure): skin, skeletal muscle, and peripheral nerve; mesenteric, pre-scapular, and popliteal lymph nodes; liver, kidney, and spleen; tonsil, thymus, bone marrow, and buffy coat. 27 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Evaluation of risk according to the bovine tissue source of the product is on more solid ground, because experiments have been conducted that define which tissues are infectious and which are not. Early infectivity bioassays employed strains of mice for tissue inoculations, but the partial species barrier effect was understood to reduce the assay sensitivity. Shortly thereafter, parallel bioassays were initiated in cattle, and still preliminary results confirm the earlier results in mice: as shown in Table 5, they indicate a distribution of infectivity largely limited to nervous system tissues – and much less widespread than occurs in other TSEs, such as scrapie in sheep and CJD in humans. T RANSMISSIBLE such variability is unappreciated, but rather that it is a practical impossibility to collect reliable data from which to assess risk in smaller geographic units than entire countries. In special cases, commercial firms have requested individual risk assessments in order to be permitted to import and sell bovine products from isolated source herds in countries that would not qualify under the nation-by-nation risk assessment scheme. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 28 TABLE 6 Bovine materials used by humans (not exhaustive) CONSUMABLES1 NON-CONSUMABLES1 Used directly (or after minimal processing) Meat on the bone2 (T-bone, ox tail) Deboned meat Offals3 (e.g., liver, heart, kidney, thymus, and brain) Fat4 (suet) Bone (soup and broth) Brain and endocrine powders in some unregulated “health food” supplements Bone, heart valves, pericardium, and trachea used in biologicals and medical devices Used after processing Milk and milk products (e.g., butter, lactose and casein) Rennet (chymosin and pepsin derived from abomasum) used in the production of cheese, whey and whey products such as lactose Bone2 and skin to make gelatin, gelatin derivatives, and collagen Meat, including tongue, meat extracts used as flavouring, and “mechanically recovered meat”5 Tripe (fore-stomachs) Tallow6 (used for frying and in food as shortening) and to make tallow derivatives7 Fat4 (suet), beef drippings Blood and blood products (e.g., hemoglobin to clarify wine) Intestines (duodenum to rectum) for natural sausage casings Milk and milk products (e.g., skim milk, lactose from whey – used as excipient and stabilizer) Bone2 and skin to make gelatin (injectables) and gelatin derivatives (used for amino acids and blood expanders) Meat and meat products (used in media to grow bacteria for vaccines) Blood products: (e.g., fetal calf serum used in cell culture growth media for viral vaccines, stem cells, and recombinant products; thrombin used as a hemostatic agent) Tallow6 (e.g., soaps) Tallow derivatives7 (e.g., buffers, cosmetics, and suppositories) Other tissues Lungs (e.g., surfactant injections) Intestines (e.g., catgut) Endocrines (e.g., pancreatic insulin) Heart, arteries, trachea, tendon, joints used to prepare collagen, elastin, and various chemicals8 Brain, marrow, and viscera9 used in cosmetics 1 With the advent of BSE, safe sourcing has been practiced by avoiding tissues that are known to harbour or replicate TSE agents, eliminating ruminant materials, or where there is no satisfactory alternative, sourcing them from countries, herds and animals with an acceptably low geographical BSE risk assessment. This is therefore not a table of risk, but rather a reminder of the extent to which humans are exposed to products of bovine origin. 2 Some bone material, such as skull and vertebral column (excluding the tail) from cattle over certain ages are classified as specified risk materials (SRM) in the EU. 3 SRM include skull, brain, ganglia, eyes, tonsils, intestines and spinal cord, and in the EU and some other countries these are compulsorily removed for destruction. 4 Some fat (e.g. mesenteric fat) is classified as SRM in the EU. 5 Production of mechanically recovered meat from ruminant animals in the EU is banned. Elsewhere, its inclusion in meat products usually does not exceed 5–10% by weight. 6 Tallow (bovine rendered fat) is one of the two major end products from rendering bovine carcass waste. Tallow is not used in regulated medicines, but can be used in soaps, topical creams, cosmetics, and suppositories. Tallow from SRM is not permitted for use except under license as a fuel. 7 Tallow derivatives include glycerol, fatty acids and their esters, stearates, polysorbates, and sorbitan esters. Tallow derivatives are produced from tallow by hydrolysis at temperatures and pressures that have been shown to sterilize TSE infectivity. They are used in the manufacture of some medicines and cosmetics and may also be used in plastics (food wrappers). 8 Glucagons (replaced by recombinant technology in some countries), corticosterol,chondroitin sulfate, vitamin D3, cholesterol, and cholic acid. 9 Liver, thymus, heart, mammary glands, placenta, ovary, and spleen. 28 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 29 Although the minimum infective dose for zoonotic forms of TSE has not been established, the route of administration of products for human use certainly plays a role in the determination of risk. TSE is not easily transmitted; it usually requires some form of tissue penetrating event. Direct inoculation into the brain is by far the most efficient method of transmitting disease, but apart from therapy for certain types of neurological disorders (for example, CNS mycoses), this route is irrelevant to human exposure. Peripheral routes of inoculation are much less efficient: five to ten times more infectious material is needed to transmit disease by the intravenous than by the intracerebral route, and other peripheral routes (intramuscular, intraperitoneal, and subcutaneous) are even less efficient. Experimental transmission has also been accomplished by contaminating dermal, ocular, and gingival abrasions, although application of infectious material to unbroken skin does not transmit disease. Although the oral route of infection is at least 100 to 1000 times less efficient than intracerebral inoculation, ingestion is certainly the means by which non-bovine species became infected with BSE, and it may well be the natural route of contagion for scrapie and CWD. How does all of this information bear on the question of risk of individual products? For example, would ingestion of a frankfurter made with MRM from cattle in Japan be more or less risky than the application of an anti-aging skin cream made with tallow from cattle in Belgium? How to compare the risk from a vaccine containing fetal calf serum from Italy with that of a capsule made from gelatin obtained from cattle in the Ukraine? Has BSE silently crossed back into sheep, carrying its newly acquired pathogenicity for humans, and should we be concerned about ovinederived consumables? (In contrast to BSE-infected cattle, experimentally infected sheep show the pathological form of prion protein in lymph nodes and in both the large and small intestines, and blood transfusions can transmit disease to other sheep). The answers to these teasing kinds of questions are of course unknown, and merely reinforce the common sense conclusion that the risk inherent in any foods or other products should be minimized as much as possible by appropriate selection of animals, avoidance of tissues known to be infectious, and use of production steps known to eliminate (or at least reduce to an untransmissible level) any infectivity that might be present in the unprocessed tissue. 29 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE The third issue in assessing risk – the degree to which processing may reduce any infectivity that might be present in the tissue – requires separate data for each product in question (i.e. validation testing of infectivity clearance), which are available for only a fraction of the hundreds if not thousands of products containing, or exposed to, bovine tissues (Table 6). As it happens, the processing of gelatin (from hides and/or bones) and tallow (from fat), which are the starting materials for a multitude of products, ranging from pharmaceuticals to foodstuffs to cosmetics, have been found to reduce infectivity by a much greater level than could ever possibly be present in source material. The processes by which other tissues, such as pancreas (for insulin), lung (for surfactant) and stomach (for rennet), are prepared have not as yet been individually validated for infectivity reduction, but none of these source tissues appears to be infectious. Excellent up-todate risk assessments of various products of bovine origin are available from the Scientific Steering Committee of the European Commission, an independent body that advises the Commission on matters related to consumer health and protection (http://europa.eu. int/comm/food/fs/sc/ssc/outcome_en.html). T RANSMISSIBLE infectious are unlikely to be contaminated by “passenger infectivity” in circulating blood. Thus, the only tissues outside the central nervous system (brain and spinal cord) that pose a risk of infectivity are the trigeminal and paraspinal ganglia, the distal ileum, and possibly bone marrow and tonsil. The anatomic isolation of ganglia and of lymphatic elements within the distal ileum minimizes the risk of cross-contamination of neighbouring tissues, and careful removal of the tongue similarly minimizes its cross-contamination by tonsillar tissue. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 30 GOVERNMENT RESPONSES AND THE CURRENT SCENE G overnments worry when the power of public concern is brought to bear on issues with which they may be involved in a regulatory capacity, and many governments have acted to minimize risk from BSE both to animals and to humans, and to defuse what has at times been a media-induced panic. Government responses have varied from urgent to desultory, as outlined in Table 7. The single most important measure taken by the UK was decreed as soon as contaminated meat and bone meal were proposed as the most likely vehicle of infection: their use in ruminant feed was prohibited in July 1988. The same prohibition was not instituted by the European Union until six years later, when it had became painfully obvious that exported BSE was causing indigenous outbreaks via the same carcass recycling mechanism that had spread the original epidemic in the UK. During this same period, numerous import-export regulations were also initiated that had the effect of preventing further international transfer of infection, and the prohibition of high-risk tissues and tissue products from entering either the animal or human food chain essentially “finetuned” the earlier, more limited restrictions. Some of these have since been deemed unnecessary (for example, the short-lived ban on the exportation of bone-in meat) as experimental data have accumulated to indemnify many products as having a negligible potential for transmitting disease. Responses varied from country to country: the US, which had the good fortune to be nearly selfsufficient with respect to breeding animals and producing dietary supplements, imported very few animals and only one small shipment of feed during the 1980s; it prohibited importation of bovines and bovine products in 1989. Japan, which imported hundreds of tons of MBM from the UK and continental Europe during the same period, did not institute any bans or surveillance programs and learned to its chagrin in 2001 that it was host to some BSE-infected cattle. It seems self-evident today that any country that imported significant quantities of cattle or MBM from the UK during the period from 1980 through 1991, when all exports were banned, or from countries of continental Europe through the end of the century, must consider itself at some risk of having cattle with BSE. Despite the issuance by many governments of a package of prohibitions that could reasonably be expected to eliminate the veterinary and public health risks of BSE, loopholes often exist that could allow the spread of disease. The situation in the US is illustrative: • No rules exist that require the rendering process to be capable of sterilizing BSE infectivity. Most feed mills render carcasses under conditions that cannot be guaranteed to sterilize. • Feed for ruminants and non-ruminants can be processed in the same feed mills, creating the potential for cross-contamination. Thus, rendered carcasses of deer and elk with chronic wasting disease could conceivably be fed to cattle. • If farmers or ranchers mistakenly or deliberately use non-ruminant feed for ruminants, the mammalian to ruminant feed ban would be bypassed. Spontaneous TSE occurring in nonmammalian species, if it occurs, would also escape the mammalian to ruminant feed ban. 30 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 31 Great Britain2 Ban on ruminant protein in ruminant feed Ban on export of UK cattle born before July 1988 feed ban Ban on export of UK cattle >6 months of age Ban on SBO3 in human (1989) and animal (1990) nutrition July 1988 Ban on export of SBO and feed containing SBO to EU countries High-risk waste to be rendered at 133°C/3 bar/20 min Ban on export from UK of SBO and feed containing SBO to non-EU countries Ban on mammalian MBM4 in ruminant feed Ban on mammalian protein in ruminant feed5 Rendering methods must sterilize BSE SBO ban broadened to include the bony skull MRM6 from bovine vertebral column banned and export prohibited Removal of lymph nodes and visible nervous tissue from UK bovine meat >30 months exported to EU Ban on export of all UK cattle and cattle products except milk Slaughtered/dead cattle >30 months (except certain beef cattle >42 months) ruled unfit for any use (hides for leather excluded) Mammalian MBM prohibited from all animal feed/fertilizer Mammalian MBM and MBM-containing feed recalled Mammalian waste (except fat) to be rendered at 133°C/3 bar/20 min BSE cohort cattle in UK ordered slaughtered and destroyed Replace human plasma and plasma products for use in the UK with imported sources Slaughter and destruction of offspring born to BSE-affected UK cattle after July 1996 Leukodepletion of whole blood donations from UK residents Ban on cattle and sheep SRM7 throughout the EU Ban on MRM production from any part of cattle, sheep and goats Ban on mammalian protein in all livestock feed Ban on slaughter techniques that could contaminate cattle carcasses with brain emboli (e.g., pithing or pneumatic stun guns) Immunologic brain examination on all slaughtered cattle >30 months of age 1 2 3 4 5 6 7 European Union2 July 1989 Mar 1990 Nov 1989/ Sept 1990 Sept 1990 Nov 1990 July 1991 June 1994 Nov 1994 Jan 1995 Aug 1995 Dec 1995 Jan 1996 Mar 1996 Mar 1996 Mar/Apr 1996 June 1996 July 1996 Jan 1997 Aug 1998 Jan 1999 July/Nov 1999 Jul 2000 Jan 2001 Jan 2001 Jan 2001 Jan 2001 The most important measures are shown in boldface type. In Northern Ireland and Scotland, dates of implementation were sometimes different than those shown for England and Wales; also, individual EC countries often adopted different measures on different dates. SBO = Specified bovine offals (brain, spinal cord, thymus, tonsil, spleen, and intestines from cattle >6 months). MBM = meat-and-bone meal (high-protein residue produced by rendering). Some exemptions, e.g., milk, blood, and gelatin. MRM = mechanically recovered meat (residual meat derived from bones, including vertebral column with dorsal root ganglia and possibly spinal cord in situ). SRM = specified risk materials (all tissues shown to be infectious in cattle, sheep, or goats; where infectivity is limited to animals over a certain age, the ban applies to animals over that age. The definition of SRM changes as new information is acquired). 31 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Precautions1 T RANSMISSIBLE TABLE 7 Principal governmental measures taken to protect human and animal health T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 32 • Organs known to be infectious in cattle with BSE (including brain) are not prohibited from human consumption. • Plate waste from restaurants, which could contain bovine brain or paraspinal ganglia in the uneaten remains of some cuts of meat, could be recycled to cattle in feed produced by rendering plants. • Mechanically separated meat expressed from crushed carcasses can be added to cooked and uncooked meat products, up to a concentration of 30% by weight. Since 1997, spinal cord has been removed, but the vertebral column, including the paraspinal ganglia, can still be processed and used in many products – hot dogs, sausages, canned beef, luncheon meats, and soups and stews. A recently introduced and much safer process, “advanced meat recovery”, has not yet completely replaced the crushing method. • Glandular dietary supplements containing various animal organ powders, including cattle brain, were often imported from the UK or countries in continental Europe until the US Department of Agriculture import ban in 1989. The ban relies on proper labelling of the shipment and can be abused. Government regulatory agencies have also addressed the problem of possible “secondary” disease transmission, that is, the transmission of vCJD from an individual during the unsuspected pre-clinical phase of disease to other humans via blood and organ donations, or from crosscontamination of instruments used in surgical or other invasive procedures. In particular, blood and blood products have received ongoing attention in many countries, and donor deferral policies have been implemented that have become almost arcanely complicated, as more and more countries report BSE (or vCJD). In the US, for example, deferrals differ for prospective donors with different length of residence times in the UK and continental Europe, and for military personnel stationed either north or south of the Alps (“Hannibal’s line”). Consider the plight of a soldier stationed in Turkey who en route had stayed three weeks at a base in the UK, and also had vacationed in Switzerland. The situation with respect to deferral of organ donors is less well defined, depending as it does on rules adopted by individual organ bank associations. In general, it can be said that these associations are aware of the potential risk posed by patients with vCJD, and of policies applied to blood donors, and that they tend to issue rules that are in line with these policies. The situation with respect to instrument cross-contamination is entirely unregulated, and it is likely that many if not most hospitals continue to employ standard decontamination procedures, which are insufficient to guarantee the sterility of instruments used on patients with vCJD (or even sporadic CJD). 32 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 33 S It stands to reason, therefore, that the elimination of scrapie could be the single most effective means by which further outbreaks of TSE could be avoided. As used by epidemiologists, the word “elimination” implies repeated successes in reducing infection to undetectable levels in various regions of the world – like putting out brush fires – and is sharply distinguished from the term “eradication”, which is defined as the total global disappearance of a disease. Examples of elimination are measles and polio; the only example of eradication is smallpox. While it may never be possible to eradicate scrapie, its elimination is now a practical goal. This idea is neither new nor particularly original, but it is sometimes lost from view in the imbroglio of dealing with the secondary diseases. Nor has it escaped the attention of governmental agencies in some countries, which have made sporadic efforts to eliminate or at least reduce the burden of disease among their sheep populations. Alas, the disease has proved more durable than the will of its antagonists, and all past efforts have eventually languished and disappeared. Today, however, we have tools that did not exist even 15 years ago, and it would seem more than timely, in view of recent events, once again to take up the campaign. Immuno-detection of pathological protein in the third eyelid of sheep has proved to be a very useful method for the random screening of flocks for the presence of scrapie, and biopsy of the olfactory epithetium may be an even better index of infection. Equally important is the association of specific prion genotype with post-exposure risk of developing clinical scrapie, which makes possible the selective breeding of sheep, so that flocks are eventually composed of the most scrapie-resistant genotypes. National control and eradication schemes have been developed or are under development in several countries (the Netherlands, Great Britain, France and the US). In Great Britain a national voluntary scheme has started with pedigree and pure-bred flocks: breeding eligibility is limited to rams with the more resistant genotypes, and rams with the most susceptible genotypes are slaughtered or castrated. With respect to BSE and vCJD, it is evident that strict compliance with the following three principles should eventually make these diseases a matter of history: 1. Identification and destruction of all clinically affected cattle 2. Prohibition of all mammalian protein for use in ruminant livestock 3. Prohibition of all high-risk tissues for use by humans Plugging loopholes to these rules (when they exist or are identified) will accomplish the task more quickly, but compliance with the basic rules is the more important issue, and experience shows that compliance is never perfect. Also, a continued awareness of the unlikely but still possible vertical or horizontal BSE transmission within herds, and of the backcrossing of BSE to sheep (or its spread to other species) is needed, but it is made difficult by the absence of any practical test to distinguish BSE from scrapie. Efforts to develop such a test should receive high priority. 33 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE crapie, recognized since at least the 18th century to be a contagious disease of sheep, may lie at the root of all other forms of animal TSEs. It has certainly been the cause of at least some outbreaks of TME in mink, it was probably the cause of BSE in cattle (in turn spreading TSE to other species by exposure to contaminated recycled livestock feed), and it may be the cause of CWD in deer and elk. T RANSMISSIBLE CONCLUSIONS AND RECOMMENDATIONS T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 34 Continuing attention also needs to be given to the possibility of secondary vCJD infections from blood or tissue donations and surgical procedures. Donor deferral policies should be tailored to estimated national risks of vCJD infection or, where vCJD has not occurred, to estimated national risks of BSE infection, and should be regularly reviewed and revised as new data accumulate. In particular, as risk diminishes, deferral criteria should be relaxed in a timely manner. Prevention of secondary cases from surgical cross-contamination is easier in principle and more difficult in practice. Effective sterilization procedures are well defined (exposure to NaOH and autoclaving at 134°C) but need to be advertised and possibly mandated by national infection control organizations. Research priorities should include efforts to develop a rapid and reliable laboratory method to distinguish different strains of TSE irrespective of the affected species, to develop an immunological screening test to detect preclinical TSE infection, and to explore new technologies for reducing infectivity in processed meat products (for example, heating under high pressure has recently been shown to conserve the quality of processed meat while substantially reducing TSE infectivity). The whole question of a spreading danger from CWD needs the same kind of urgent attention that was given to BSE when it first appeared, including knowledge of its host range, its possible occurrence outside North America, and its characteristics in experimentally infected primates (assuming human infection remains unidentified). It could be argued that unrecognized TSE in other animal species should also be systematically investigated, although the huge investment of resources needed for such studies might be better spent on more immediate concerns. On a more fundamental note, molecular genetic studies might be profitably directed at demystifying the unique association of human susceptibility to BSE with the homozygous methionine codon 129 genotype, and renewed attention paid to the carbohydrate moieties of the prion protein, which appear to constitute the principle biochemical distinction between the BSE/vCJD and other strains of TSE. An important practical concern is the potential risk to travellers and temporary residents of countries in which BSE has already occurred or is thought likely to occur but be unrecognized. In view of the delays of many nations to implement precautionary measures and surveillance programs to detect disease, it would probably be prudent for travellers to countries that imported significant quantities of cattle or feed from Europe to avoid products that contain processed meat for at least the next few years. Finally, a difficult issue must be addressed. Does the risk of BSE (or other as yet undetected types of animal TSE) warrant the permanent elimination of animal protein from livestock feed? The rendering industry as presently constituted would disappear, and the incineration and landfill industries would expand in conjunction with the production of nutrition crops such as soybeans. Would the increased incineration and landfill disposal have adverse environmental effects? Would plant protein be as effective as animal protein, and would its increased production result in a shortage of arable land for other purposes? Is the public prepared to pay more for meat or eat only as much as can be produced from range-fed animals? And would such a policy compromise the already marginal status of animal and human nutrition in developing countries? These are questions that must be decided, not by scientists and government regulators, but in national debates by society as a whole. 34 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 35 The Disease Family Chesebro, B. (1999). Prion protein and the transmissible spongiform encephalopathies. Neuron, 24:503-506. Gajdusek, D.C. (1990). Subacute spongiform encephalopathies: transmissible cerebral amyloidoses caused by unconventional viruses. In: Fields, B.N., Knipe, D.M., and Howley, P.M., eds. Virology, 2nd Edition, Raven Press, New York, 2289-2324. Gajdusek, D.C., Gibbs, C.J.Jr., and Alpers, M. (1966). 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Nature, 383:685-690. Lasmézas, C.I., Deslys, J.-P., Demalmay, R., et al. (1996). BSE transmission to macaques. Nature, 381:743-744. Scott, M.R., Will, R., Ironside, J., et al. (1999). Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proceedings of the National Academy of Science (USA), 96:15137-15142. Will, R.G., Ironside, J.W., Zeidler, M., et al. (1996). A new variant of Creutzfeldt-Jakob disease in the UK. Lancet, 347:921-925. Risk Evaluation Bradley, R. (1999). BSE transmission studies with particular reference to blood. Developments in Biological Standardization, 99:35-40. Brown, P. (2001). Afterthoughts about bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. Emerging Infectious Diseases, 7(June Supplement):598-600. Brown, P. (2003). Variant CJD transmission through blood: risks to predictors and predictees. Transfusion, 43:425-427. Donnelly, C.A., Ferguson, N.M., Ghani, A.C., and Anderson, R.M. (2002). Implications of BSE infection screening data for the scale of the British BSE epidemic and current European infection levels. Proceedings of the Royal Society of London, Series B, Biological Sciences. 269:2179-2190. 41 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Taylor, D.M., Fernie, K., McConnell, I., Ferguson, C.E., and Steele, P.I. (1998). Solvent extraction as an adjunct to rendering: the effect on BSE and scrapie agents of hot solvents followed by dry heat and steam. Veterinary Record, 143:6-9. T RANSMISSIBLE Stevenson, M.A., Wilesmith, J.W., Ryan, J.B.M., et al. (2000). Temporal aspects of the epidemic of bovine spongiform encephalopathy in Great Britain: individual animal-associated risk factors for the disease. Veterinary Record, 147:349-354. T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 42 Ferguson, N.M., Ghani, A.C., Donnelly, C.A., Hagenaars, T.J., and Anderson, R.M. (2002). Estimating the human health risk from possible BSE infection of the British sheep flock. Nature, 415:420-424. Foster, J.D., Hope, J., McConnell, I., Bruce, M., and Fraser, H. (1994). Transmission of bovine spongiform encephalopathy to sheep, goats, and mice. Annals of the New York Academy of Science, 724:300-303. Foster, J.D., Parnham, D.W., Hunter, N., and Bruce, M. (2001). Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission. Journal of General Virology, 82:2319-2326. Ghani, A.C., Ferguson, N.M., Donnelly, C.A., and Ferguson, R.M. (2000). Predicted vCJD mortality in Great Britain. Nature, 406:583-584. Hilton, D.A., Fathers, E., Edwards, P., Ironside, J.W., and Zajicek, J. (1998). Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease. Lancet, 352:703-704. Hunter, N., Foster, J., Chong, A., et al. (2002). Transmission of prion diseases by blood transfusion. Journal of General Virology, 83:2897-2905. Hunter, N., Foster, J., Chong, A., et al. (2002). Transmission of prion diseases by blood transfusion. Journal of General Virology, 83:2897-2905. Kao, R.R., Gravenor, M.B., Baylis, M., et al. (2002). The potential size and duration of an epidemic of bovine spongiform encephalopathy in British sheep. Science, 295:332-335. Kimberlin, R.H. (1996). Bovine spongiform encephalopathy (BSE) and public health: some problems and solutions in assessing the risks. In: Transmissible Subacute Spongiform Encephalopathies: Prion diseases, Court, L. and Dodet, B., editors. Elsevier, Amsterdam, 487-502. Lasmézas, C.I., Fournier, J.-G., Nouvel, V., et al. (2001). Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt-Jakob disease: implications for human health. Proceedings of the National Academy of Science (USA), 98:4142-4147. Race, R., Raines, A., Raymond, G.J., Caughey, B., and Chesebro, B. (2001). Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans. Journal of Virology, 75:10106-10112. Scientific Steering Committee of the European Commission for Food Safety. http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html Sources of BSE infectivity. Project MO3108 for the Food Standards Agency, London, October 2002. www.foodstandards.gov.uk Valleron, A.-J., Boelle, P.-Y., Will, R., and Cesbron, J.-Y. (2001). Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science, 294:17261728. 42 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 43 Brown, P., Will, R.G., Bradley, R., Asher, D.L., and Detwiler, L. (2001). Bovine spongiform encephalopathy and variant Creutzfeldt-Jakob Disease: background, evolution, and current concerns. Emerging Infectious Diseases, 7:6-16. WHO Health Topics. http://www.who.int/health_topics/en WHO Regional Office for Europe. Information on Transmissible Spongiform Encephalopathy (TSE). http://www.euro.who.int/eprise/main/WHO/Progs/FOS/MainActs/20010913_1 The European and Allied Countries Collaborative Study Group of CJD (Euro CJD) plus the Extended Collaborative Study Group of CJD (NEUROCJD). http://www.eurocjd.ed.ac.uk The UK Creutzfeldt-Jakob disease surveillance unit. http://www.cjd.ed.ac.uk OIE (World Organization for Animal Health). http://www.oie.int Department for Environment, Food, and Rural Affairs (DEFRA). BSE. http://www.defra.gov.uk/animalh/bse/index.html The European Commission (EC): Food safety: Bovine Spongiform Encephalopathy (BSE). http://europa.eu.int/comm/dgs/health_consumer/index_en.htm 43 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Scientific Steering Committee of the European Commission for Food Safety. http://europa.eu.int/comm/food/fs/sc/ssc/outcome_en.html T RANSMISSIBLE Government responses/current scene T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 44 Conclusions and Recommendations Brown, P., Cervenáková, L., and Diringer, H. (2001). Blood infectivity and the prospects for a diagnostic screening test in Creutzfeldt-Jakob disease. Journal of Laboratory and Clinical Medicine, 137:5-13. Brown, P., Meyer, R., Cardone, F., and Pocchiari, M. (2003). Ultra-high pressure inactivation of TSE (prion) infectivity in processed meat: a practical method to prevent human infection. Proc. Natl. Acad. Sci (USA), in press. Detwiler, L., and Baylis, M. (2003). The epidemiology of scrapie. In: Risk Analysis of BSE and TSEs: Update on BSE and Use of Alternatives to MBM as Protein Supplements. Revue Scientifique et Technique. Office International des Epizooties (OIE), Paris, 2003, in press. O'Rourke, K.I. (2001). Ovine scrapie. New tools for control of an old disease. Vet Clin North Am Food Anim Pract, 17:283-300. O'Rourke, K.I., Duncan, J.V., Logan, J.R., et al. (2002). Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming. Clinical and Diagnostic Laboratory Immunology, 9:966-971. WHO (1999) Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva, Switzerland, 23-26 March 1999. Department of Communicable Disease Surveillance and Response, Document WHO/CDS/CSR/APH/2000.3 Zanusso, G., Ferrari, S., Cardone, F., et al. (2003) Detection of pathologic prion protein in the olfactory epithelium in sporadic Creutzfeldt-Jakob disease. New England Journal of Medicine, 348: 711-719. 44 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 45 The titer of infectivity in sheep brain/spinal cord (CNS) is ~ 5 log10LD50/g, as bioassayed by intracerebral inoculation of mice (mouse intracerebral LD50). The weight ratio of CNS to carcass ranges from 1:100 to 1:400; if we use the lower ratio (10-2) in order to approximate the additional small infectivity contribution from peripheral tissues, the total carcass infectivity is ~ 3 log10 LD50/g. A typical proportion of ovine to total tissue from all animal species in a batch of carcasses (in the UK) is ~ 10-1, reducing the input infectivity to ~ 2 log10 LD50/g. Exposure to steam heat during processing would reduce infectivity by at least another 1 log10LD50/g, such that any remaining infectivity in the heated carcass mass would not have exceeded 1 log10LD50/g. The weight ratio of carcass to meat and bone meal (MBM) is ~ 5:1, and if all infectivity segregates with MBM, the amount of infectivity in 5 g of heated carcass tissue (≤1 log10LD50) would be present in 1 g of MBM. A final tallow extraction step would further reduce infectivity by about 1 log10LD50/g, yielding an MBM infectivity level hovering close to zero, which because of the comparative inefficiency of oral infection would have very little chance of transmitting disease. Elimination of the tallow extraction step would have preserved the ≤1 log10LD5/g level of infectivity in MBM. A calf consumes ~ 2 kg of feed per day, of which ~ 4.5% (90g) consists of MBM. It follows that 90g x ≤1 log10LD50 ≤ 900 (mouse intracerebral) LD50, = infective dose that might have been consumed by a calf on any given day, assuming the MBM had come from a batch of carcasses containing an infected sheep (the estimated overall incidence of scrapieinfected sheep in the UK is about 1%). The point of this arithmetical exercise is not so much to establish exact limits of infectivity before and after rendering as to illustrate the fact that at infectivity levels near the threshold of transmissibility, small reduction effects – usually dismissed as insignificant – may have large consequences. 45 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Calculation of the amount of TSE infectivity that might have been present in meat and bone meal before and after elimination of a final tallow extraction step during the rendering process (ca. 1980) T RANSMISSIBLE ANNEX 1 T RANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 46 ANNEX 2 Calculation of the possible amount of BSE infectivity in a product containing mechanically recovered meat (MRM) MRM is produced from carcasses from which heads, limbs, and offals have been removed, consisting of shoulder girdle/ribs/vertebral column/pelvic girdle skeletons with associated untrimmed muscle, fat, and connective tissue. The only infectious tissue that would invariably have been present was paraspinal ganglia embedded within the vertebral column. The titer of infectivity in BSE tissues has been measured only in brain, which has a titer of 6 log10LD50/g, as assayed by intracerebral inoculation of cattle. Thus, ganglia infectivity must be estimated by analogy with scrapie. In scrapie-infected sheep, spinal cord infectivity is ~ 1 log lower, and sciatic nerve ~ 3 logs lower, than brain. Assume that the titer of ganglia is intermediate between these two neural tissues, or ~ 2 logs lower than brain, giving a BSE ganglia titer of ~ 4 log10LD50/g. The total weight of paraspinal ganglia in adult cattle is ~ 30 g, and thus the total amount of infectivity in the ganglia is 30 x 4 log10LD50 = 3 x 105 LD50. MRM is produced in batches of ~ 5 tons (= 45 x 105 g). Assuming one BSE carcass is included in the carcasses used to produce a batch of MRM, the concentration of infectivity in the batch would be 3 x 105 LD50/45 x 105 = 3/45 = 0.07 LD50/g. An average hot dog weighs 2 oz (60 g) and may contain up to 10% MRM. The amount of infectivity in such a hot dog would be 0.07 LD50/g x 60 g x 10% = 0.42 (intracerebral) LD50, an amount unlikely to transmit disease by the oral route, but consumption of several hot dogs over a day or two could increase the chances of transmission. However entertaining it may be to play with numbers in this way, the fact is that the conclusions to be drawn from this exercise are even more fragile than those from estimating infectivity in meat and bone meal, because: 1. The estimate of infectivity in paraspinal ganglia from BSE cattle is a hybrid of incomplete data from BSE–infected cattle and scrapie-infected sheep and, although reasonable, could be wrong by at least a factor of 10 in either direction. 2. If spinal cord had not been removed from the vertebral column, the amount of infectivity in the resulting batch of MRM would increase at least 10-fold (spinal cord weight being about 300 g, and possibly having a higher titer than ganglia). 3. The magnitude of a bovine-human species barrier (if any) is entirely unknown. 4. The efficiencies of oral versus intracerebral infection in humans is also unknown, but by analogy with scrapie would probably be at least 1:100, and still incomplete oral dosing studies in cattle suggest a ratio as high as 1:100,000. 46 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 47 Factors considered in assessing the geographic BSE risk (GBR) Results of BSE surveillance: - Number of cattle, by origin (domestic/imported), type (beef/dairy), age, method used to confirm the diagnosis, and reason the animal was examined (central nervous system symptoms, BSE suspect, BSE-related culling, other) - Incidence of reported BSE cases by year and birth cohort BSE-related culling - Culling schemes, date of introduction, and criteria used to cull - Information on animals already culled in the context of BSE Import of Cattle and Meat and Bone Meal (MBM) - Imports of live cattle and/or MBM from UK and other BSE-affected countries - Information that could influence the risk of imports to carry the BSE agent (BSE status of the herds of origin of imported cattle, precise definition of the imported animal protein, etc.) - Main imports of live cattle and/or MBM from other countries - Use made of the imported cattle or MBM Feeding - Domestic production of MBM and use of MBM (domestic and imported) - Domestic production of composite animal feed and its use - Potential for cross-contamination of feed; measures to reduce and control it, results of the controls MBM bans - Dates of introduction and scope (type of animal protein banned for use in feed in different species; exceptions, etc.) - Measures taken to ensure compliance - Methods and results of compliance control Specified Risk Material (SRM) bans - Dates of introduction and scope - Measures taken to ensure compliance - Methods and results of compliance control Rendering - Raw material used (type; annual amounts by type) - Process conditions and their share of the annual total domestic production 47 SPONGIFORM ENCEPHALOPATHY AS A ZOONOTIC DISEASE Surveillance of BSE Measures in place to ensure detection of BSE cases: - Identification system and tracing capacity - Date when BSE became notifiable and criteria for a BSE suspect - Awareness training (when, how, who was trained) - Compensation (since when, and how much in relation to market value) - Other measures taken to ensure notification of BSE suspects - Specific BSE-surveillance programs and actions - Methods and procedures (sampling and laboratory procedures) used for the confirmation of BSE cases T RANSMISSIBLE ANNEX 3 ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 48 Acknowledgments ILSI Europe and the Emerging Pathogen Task Force would like to thank the main author of this report, Dr. Paul Brown, National Institutes of Health, Bethesda (USA) as well as the other authors who helped the members of the Emerging Pathogen Expert Group on TSE: Dr. Ray Bradley (UK), Dr. Linda Detwiler, USDA/APHIS Veterinary Service (USA), Dr. Dominique Dormont, Commissariat à l'Energie Atomique (F) and Prof. Robert Will, National Creutzfeldt-Jakob Disease Surveillance Unit (UK). ILSI Europe and the Emerging Pathogen Task Force would also like to thank the International Forum for TSE and Food Safety (TAFS) for their scientific review and endorsement of the report. ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 49 Other ILSI Europe Reports • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Addition of Nutrients to Food: Nutritional and Safety Considerations An Evaluation of the Budget Method for Screening Food Additive Intake Antioxidants: Scientific Basis, Regulatory Aspects and Industry Perspectives Applicability of the ADI to Infants and Children Approach to the Control of Entero-haemorrhagic Escherichia coli (EHEC) Assessing and Controlling Industrial Impacts on the Aquatic Environment with Reference to Food Processing Assessing Health Risks from Environmental Exposure to Chemicals: the Example of Drinking Water ß-Carotene, Vitamin E, Vitamin C and Quercetin in the Prevention of Generative Diseases – The role of foods Detection Methods for Novel Foods Derived from Genetically Modified Organisms Exposure from Food Contact Materials Food Additive Intake – Scientific Assessment of the Regulatory Requirements in Europe Foodborne Viruses: An Emerging Problem Food Consumption and Packaging Usage Factors Food Safety Management Tools Functional Foods – Scientific and Global Perspectives Markers of Oxidative Damage and Antioxidant Protection: Current status and relevance to disease Method Development in Relation to Regulatory Requirements for the Dectection of GMOs in the Food Chain Overview of Health Issues Related to Alcohol Consumption Overweight and Obesity in European Children and Adolescents: Causes and consequences – prevention and treatment Packaging Materials: 1. Polyethylene Terephthalate (PET) for Food Packaging Applications Packaging Materials: 2. Polystyrene for Food Packaging Applications Packaging Materials: 3. Polypropylene as a Packaging Material for Foods and Beverages Recycling of Plastics for Food Contact Use Safety Assessment of Viable Genetically Modified Micro-organisms Used in Food Safety Considerations of DNA in Foods Salmonella Typhimurium definitive type (DT) 104: A multi-resistant Salmonella Significance of Excursions of Intake above the Acceptable Daily Intake (ADI) The Safety Assessment of Novel Foods Threshold of Toxicological Concern for Chemical Substances Present in the Diet Validation and Verification of HACCP To order ILSI Europe Avenue E. Mounier, 83, Box 6 B-1200 Brussels Belgium Phone: (+32) 2 771 00 14 Fax: (+32) 2 762 00 44 E-mail: [email protected] ILSI Rep BSE/TSE for pdf 25/04/03 16:57 Page 50 The International Life Sciences Institute (ILSI) is a nonprofit, worldwide foundation established in 1978 to advance the understanding of scientific issues relating to nutrition, food safety, toxicology, and the environment. By bringing together scientists from academia, government, industry, and the public sector, ILSI seeks a balanced approach to solving problems of common concern for the well-being of the general public. Head-quartered in Washington, DC, USA, ILSI has branches in Argentina, Brazil, Europe, India, Japan, Korea, Mexico, North Africa & Gulf Region, North America, North Andean, South Africa, South Andean, Southeast Asia Region, as well as a Focal Point in China. 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