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14 Prion Diseases: Meat Safety and Human
Health Implications
N. Hunter*
Neuropathogenesis Unit, Institute for Animal Health, West Mains Road,
Edinburgh EH9 3JF, UK
Introduction
Prion diseases are rare in human beings; however, they are now often the subject of front page newspaper headlines and have had a profound effect on international trade, the food industry, pharmaceuticals and hygiene regulations in hospitals. This group of diseases, also known as the transmissible spongiform encephalopathies (TSEs), includes scrapie in sheep and goats, chronic wasting disease (CWD) of deer and Creutzfeldt–Jakob disease (CJD) and its bovine spongiform encephalopathy (BSE)-related version (variant CJD (vCJD)) in humans (Table 14.1). There is around one new case of CJD per million of the world population per year, but vCJD has been reported in just over 100 cases to date, the vast majority in the UK. TSEs are trans- missible between individuals, either by direct injection – deliberate in the case of laboratory animals or accidental in the case of iatrogenic infection of humans – or by as yet unknown routes in the natural infections in animals. It is thought that the vCJD agent is ingested with food (possibly meat); however, it is not at all clear how the ‘normal’ or sporadic form of CJD is acquired. TSEs have no cure and are characterized by the presence of an abnormal form (PrP SC^ ) of a membrane protein (PrPC) in
infected organs of the body. PrPSC^ is consid- ered by many to be itself the infectious TSE agent and is known as the prion protein. Other researchers remain unconvinced of this hypothesis, or feel it remains unproven; however, at the very least, PrP SC^ is a reliable marker for the presence of TSE infectivity.
PrP Protein and the Nature of
the TSE Agent
The normal form of the PrP protein (PrP C) is attached to neuronal cell surfaces via an anchor made up of sugar molecules. The main differences between the two isoforms of PrP are listed in Table 14.2. The function of PrP C^ is not known; however, there are several intriguing hints that it may have a role in the activation of T cells in the immune system (Mabbott et al ., 1997), in the electrophysiol- ogy of nerve cells (Collinge et al ., 1994; Manson et al ., 1995) and in the maintenance of sleep continuity (Tobler et al ., 1997). The aggregated form of PrP (PrP SC^ ) is partially resistant to proteases and is very closely asso- ciated with infectivity. In one of the major hypotheses on the nature of the TSE agent, PrP SC^ is itself the infecting entity agent or
©CAB International 2003. Food Safety: Contaminants and Toxins (ed. J.P.F. D’Mello) 315
- E-mail: nora.hunter@bbsrc.ac.uk
prion. In this ‘protein only’ theory, PrP SC^ , arising from an infection or from a mutant PrP gene, acts as a catalyst in the conversion of endogenous PrPC^ into yet more PrPSC^ , thus either destroying the normal function of the protein or poisoning the nerve cells and resulting in degenerative disease (Prusiner et al ., 1990). Natural scrapie in sheep tends to be familial in appearance and has been said to result from a recessive gene, the protein product of which causes disease (Parry, 1984); however, as described later, this hypo- thesis has now been discounted. Around 15% of the human TSEs also show a familial pat- tern with a dominant pattern of inheritance (Brown et al ., 1987), and are considered to be genetic diseases (resulting directly from a
mutation). However, genetic disease or not, once a TSE does occur, it is often transmis- sible experimentally to laboratory animals, not the case with simple genetic diseases, for example thalassaemia (Rund et al ., 1991), and so additional explanations for disease spreading mechanisms are required. Because of the heretical nature of the ‘pro- tein only’ hypothesis, in that it invokes an infecting agent carrying genetic information embedded in protein and not in DNA or RNA, the prion theory (Prusiner, 1982) was difficult for many to accept (Chesebro, 1998). TSEs have been shown to exist in many different strains, and so one of the main opposing ideas involves PrP SC^ protein as part of a two- component structure (the virino), including a
316 N. Hunter
Human diseases Acronym Types Aetiology
Creutzfeldt–Jakob disease
Gerstmann–Straussler– Scheinker syndrome Fatal familial insomnia Kuru Variant Creutzfeldt–Jakob disease
CJD
GSS
FFI
vCJD
Sporadic Familial Iatrogenic
Familial
Familial Acquired Acquired
Unknown Linked to PrP gene mutations Contamination during surgery or of growth hormone Linked to PrP gene mutation, e.g. codon 102
Linked to PrP gene mutation, e.g. codon 178 Associated with funeral rites ?Diet, related to BSE
Animal diseases Acronym Types Aetiology
Scrapie
Chronic wasting disease
Transmissible mink encephalopathy Bovine spongiform encaphalopathy Feline spongiform encephalopathy Spongiform encephalopathies
Sheep, goats
Deer
Farmed mink Cattle Cats Zoo animals
CWD
TME BSE FSE SE
Natural
Natural
Acquired Acquired Acquired Acquired
Infection, unknown mode of transmission Infection, unknown mode of transmission Contaminated feed Contaminated feedstuff Diet, related to BSE Diet, related to BSE
Table 14.1. Transmissible spongiform encephalopathies (prion diseases).
Characteristic PrP C^ PrP SC
Action of proteinase K enzyme (PK) Molecular weight Molecular weight after PK treatment Detergent Present in normal brain? Present in TSE brain? Infectivity
Degraded 33–35 kDa Degraded Soluble Yes Yes Does not co-purify
Partially resistant 33–35 kDa 27–30 kDa Insoluble No Yes Does co-purify
Table 14.2. Differences between PrP C^ and PrP SC.
PrP gene sequence changes (polymorphisms or mutations) in different affected families (Table 14.3). The sporadic forms of CJD in humans are not linked to any mutations of the PrP gene and, instead, a codon 129 poly- morphism, methionine (M) or valine (V), is associated with differences in susceptibility to disease in that homozygous individuals (either MM or VV) are over-represented in CJD cases and heterozygosity (MV) seems to confer some protection (Palmer et al ., 1991). At the time of writing, all reported vCJD cases are of MM PrP genotype (Collinge et al ., 1996a), which occurs in around 37% of Caucasian populations. Other forms of TSEs in humans appear to be genetic diseases, resulting directly from a mutation in the PrP gene, for example one form of GSS which is linked to a codon 102 proline to leucine mutation (Hsiao et al ., 1989). This mutation, when introduced into the mouse PrP gene in transgenic (Tg) mice and expressed at extremely high levels, resulted in a spontaneous scrapie-like disease which, although no PrP SC^ was detectable by standard methods, transmitted infection to hamsters and other Tg mice and not to normal mice (Hsiao et al ., 1994). This experiment supports the ‘protein only’ hypothesis because appar- ently the only requirement for disease to develop is a single amino acid mutation. However, the interpretation of the results has been disputed on the grounds of the lack of PrP SC^ , the odd transmission characteristics and the high levels of expression needed to see the effect – single-copy transgenes do not make the mice ill (Chesebro, 1998). It is known that high levels of protein produced from normal PrP genes can also result in illness in Tg mice (Westaway et al ., 1994) and so PrP poisoning is a possibility.
There are several other human PrP gene mutations associated with disease, and vari- ous forms of human PrP protein expressed by naturally occurring mutant genes have been studied in cell lines in culture and have been found to be both abnormally processed, for example not appearing on the cell membrane, and to acquire characteristics of the disease-associated PrPSC^ protein isoform (Lehmann and Harris, 1996; Daude et al ., 1997). This suggests that mutations in the PrP gene may cause illness directly through loss of function of the PrP protein by misprocessing or that the mutant protein forms deposits and poisons the surrounding cells. In the familial, or genetic, forms of human TSEs, there is thought therefore to be no need to look for a source of disease other than the aberrant PrP gene itself; however, with sporadic CJD, there is no easy explanation related to genetics, and an environmental source of infection, for example contaminated foodstuffs, may eventually be found to be a risk factor.
Genetics of sheep TSEs
The means by which scrapie transmits between sheep is not well understood, although there is good evidence that the most common means of entering the body is by the oral route (Hadlow et al ., 1982; Van Keulen et al ., 1999). Studies of natural scrapie in sheep have confirmed the impor- tance of three amino acid codons in the sheep PrP gene (136, 154 and 171) (Belt et al ., 1995; Clouscard et al ., 1995; Hunter et al ., 1996). (A diagram of the sheep PrP gene structure (similar in all species) is shown in Fig. 14.2 and sheep genotypes are usually
318 N. Hunter
Disease Amino acid number Change Codon 129 a
CJD
FFI GSS
178 200 178 102
Aspartic acid → asparagine Glutamic acid → lysine Aspartic acid → asparagine Proline → leucine
Valine Methionine Methionine Methionine
aCodon 129 polymorphism: the amino acid at this position influences disease type that occurs in
combination with mutation. FFI, fatal familial insomnia.
Table 14.3. Examples of human PrP gene mutations associated with familial TSEs.
represented giving each of the three codons in turn for each allele in turn.) The most resis- tant genotype is ARR/ARR and the most sus- ceptible is VRQ/VRQ, with a range of other genotypes of varying degree of disease risk in between these two extremes (Dawson et al ., 1998). Not all sheep breeds are the same, however, as Suffolk sheep have a very simple linkage with disease: this breed does not have the VRQ allele and scrapie occurs in ARQ/ARQ sheep. Other breeds with the additional VRQ allele, such as Cheviots and Swaledales, are much more complex and, when the highly susceptible VRQ/VRQ genotype occurs, as it is almost always in scrapie-affected sheep, it has been suggested that scrapie may be simply a genetic disease (Ridley and Baker, 1995). However healthy VRQ/VRQ animals can live to more than 8 years of age, well past the usual age-at-death from scrapie (2–4 years) (Foster et al ., 1996a), and susceptible sheep genotypes are easily found in countries that are free of scrapie clinical cases (Hunter et al ., 1997a). The genetic disease hypothesis seems less likely, therefore, than a disease process which involves an infecting agent (prion or virino) causing disease only in susceptible sheep. However, in order to eliminate TSE diseases from sheep in countries where scrapie is endemic, breeding for resistant genotypes
currently is being promoted, rather than elimination of infection – the route used with cattle BSE. A great deal of information on the details of PrP genotype linkage with disease in sheep has come from experimental infections of sheep with scrapie or with BSE under controlled conditions (Goldmann et al ., 1991a, 1994). The genotypes of sheep targeted by BSE are quite distinct, with the shortest incubation periods in ARQ/ARQ sheep and longer incubation periods in ARQ/ARR sheep. Although some sources of experi- mental scrapie (e.g. CH1641) also target these genotypes, SSBP/1 is different and affects Cheviot sheep encoding the VRQ allele (Goldmann et al ., 1994). It is possible, there- fore, that there are also various types or strains of natural scrapie which target either particu- lar sheep breeds and/or different PrP codons. The best way to investigate this at the moment is by passage of natural scrapie into a panel of mouse strains where characteristics of incubation periods and the brain areas which become damaged give distinct profiles or patterns (Bruce et al ., 1994). It is also possible that scrapie strains may produce PrPSC proteins with distinct patterns on Western blots – a method currently under investigation for strain typing in several laboratories throughout the world (e.g. Hope et al ., 1999).
Prion Diseases: Meat Safety and Human Health 319
Fig. 14.2. Diagrammatic representation of the structure of the sheep PrP gene with the positions of the three disease-linked amino acids. Black boxes represent the protein-coding region of the gene.
also during the preclinical phase of the dis- ease during which animals appear perfectly healthy. In one study during development of scrapie, infection was detected first (at 10 months of age) in sheep gut and lymphoid tissue, then much later in the central nervous system (CNS) and brain, the latter of which has greatest amounts of infectivity as the clinical signs develop and the animal becomes ill. In this study, no infection was found in milk, udder or muscle (Hadlow et al ., 1982). However, in a more recent report, peripheral nerves in scrapie sheep muscle were found to be infected (Groschup et al ., 1996). Sheep experimentally infected with BSE have infection present in both brain and spleen (Foster et al ., 1996b), suggesting that, should BSE infect sheep naturally, it would have a similar distribution throughout the body to that found with scrapie. Using blood transfusion between sheep (no species bar- rier), BSE infection has also been detected in blood removed from experimentally infected animals during the preclinical phase of disease (Houston et al ., 2000). In contrast, cat- tle naturally infected with BSE have shown signs of infection only in the brain and spinal cord, and even experimentally infected cattle have only shown additional sites of infection in part of the intestine (distal ileum) (Wells et al ., 1998) and, specifically, milk from BSE cattle was also negative (Taylor et al ., 1995). Using PrPSC^ as a biochemical marker for infectivity, signs of infection can be detected in sheep tonsil as early as 3 months of age in animals destined to develop scrapie at about 2 years of age (Schreuder et al ., 1998). PrP SC^ is found throughout the body of scrapie sheep, with some tissues, such as the liver, being relatively spared and others having higher amounts (spleen, parts of the intestine); how- ever, up to and during the clinical phase, PrP SC^ is found in increasing, and very much higher, amounts in brain and CNS tissues (Van Keulen et al ., 1996). PrPSC^ has also been found in placental tissue, although any involvement of placenta in natural transmis- sion of scrapie from mother to offspring may not be in utero but could result from ingestion of discarded placental tissue by the ewe her- self or by other sheep. In sheep experimentally infected with BSE, lymphoid tissues show
signs of infection early in the incubation period (Jeffrey et al ., 2001) and, by terminal stages of disease, PrPSC^ deposits are found widespread throughout the body (Foster et al ., 2001b). In BSE-affected cattle, it is more diffi- cult to find evidence of PrPSC^ outside the CNS (Wells et al ., 1998). Clearly the pathogenesis of BSE in cattle is different from that in sheep, with more limited involvement of non-CNS tissues. It follows from this, therefore, that in thinking about the risks of infection of humans with BSE, high risk cattle tissues are liable to be fewer in number and easier to elim- inate from the food chain than would be the case if BSE in sheep were to become endemic. Although there is no evidence for the occurrence of BSE in the USA, another related disease does occur: chronic wasting disease (CWD) of mule deer and elk (Williams and Young, 1992; Spraker et al ., 1997). This prion disease occurs in both farmed and wild populations. Although there are precautions in place which test the brain tissue of hunted animals for the presence of PrP SC^ protein (Laplanche et al ., 1999), there are concerns about the occurrence of CJD in a small number of individuals known to have consumed wild venison. Recent epidemiological studies have judged that there was no link between the two diseases but, if more CJD cases occur in hunt- ers, there may be a change of opinion. There is, at the time of writing, no published evidence from outside North America for CWD occurrence on venison farms or in the wild.
Humans
In sporadic CJD cases, signs of infection are not widespread throughout the human body; however, from early studies of vCJD, it was apparent that this new disease was behaving differently. PrP SC^ was detected in tonsils of a number of vCJD victims and in the appendix tissue of another case (Hill et al ., 1997, 1999). Further studies have since been set up to find out which tissues represent a risk of infection for humans and for contamination of surgical instruments. Infectivity was found in mouse bioassays of vCJD brain, as expected, but also in tonsil and spleen at levels between 100
Prion Diseases: Meat Safety and Human Health 321
and 1000 times lower than in brain (Bruce et al ., 2001). Blood fractions (buffy coat and plasma) were negative. In addition, PrPSC^ has also been found in lymph nodes, retina and optic nerve, and at low levels in one vCJD case in the rectum, adrenal gland and thymus
- with obvious implications for contamina- tion of surgical instruments. Other vCJD tis- sues tested for PrP SC^ were negative, including blood buffy coat preparations (Wadsworth et al ., 2001).
How are TSEs Contracted?
The route of transmission of natural scrapie in sheep is not known with certainty but, because of the early involvement of the ali- mentary tract (PrP SC^ staining), the oral route is implicated (Van Keulen et al ., 1995, 1996, 1999). Other routes of infection through wounds on the skin or in the mouth are also possible. Infection may be picked up from pasture contaminated with infected placental tissue, or by simple contact with other infected animals. BSE in cattle is most likely to have been spread by oral infection through eating contaminated meat and bone meal (MBM) (Wilesmith et al ., 1991) although in this case the infection seems to by-pass the peripheral tissues and travel straight to the brain. In humans, the route of infection in sporadic CJD is not known. Epidemiological studies sometimes show connections with diet or lifestyle, but these have been thought to be artefactual due to the relatively small numbers of affected individuals (Wientjens et al ., 1996). One study suggested surgery was a risk factor (Kmietowicz, 1999), and there are clear instances of iatrogenic infection follow- ing surgical procedures (Shimizu et al ., 1999) or following injection with contaminated growth hormone used to treat undersized children (d’Aignaux et al ., 1999). There are forms of CJD which appear to be genetic in ori- gin and are linked to the occurrence of specific mutations of the PrP gene (Ghetti et al ., 1995). If this view is correct, no further route need be sought for the familial human TSEs; however, it has been shown beyond any doubt that
the vCJD infectious agent is identical to BSE (Bruce et al ., 1997) and it has been assumed that the disease is picked up via the oral route and eating BSE-infected cattle products. As it is not known how many people are already infected with vCJD and quietly incubating the disease, it is impossible to judge the risk to others from potentially contaminated blood products. Sporadic and genetic forms of CJD are not thought to have infectivity in peripheral tissues, and there is no epidemiological evidence linking blood transfusion with incidence of sporadic CJD. As vCJD involves infection of peripheral tissues and to deal with the potential risk, UK blood supplies are depleted of white blood cells and new US Food and Drug Administra- tion rules will forbid donations from anyone who spent 3 months in Britain from 1980 to 1996 or those who have spent 5 years or more in France since 1980. The rules will also ban donations from anyone who received a blood transfusion in Britain since 1980 and from American military personnel who spent 6 months or more on a European base from 1980 to 1996, when British beef was sold to bases there (Cimon, 2001). Only time will tell if these precautions are justified.
Risk Factors
There have been more than 177,000 cases of BSE confirmed in cattle in Great Britain since the outbreak started in the late 1980s, peaking in 1992 with 36,682 recorded cases. Between 1991 and 1995, there were more than 10, cases per year; however, in 2000, the numbers had dropped to 1270. Other countries are also affected, although at a much lower rate: Northern Ireland (total to December 2000 = 1810, peaking in 1993 with 459); Republic of Ireland (total = 567, peaking in 2000 with 132); France (total = 233, with 153 in 2000); Portugal (total = 489, peaking in 1999 with
- and Switzerland (total = 364, peaking in 1995 with 68) (data from the UKDepartment for the Environment, Food and Rural Affairs (DEFRA)). Many, but by no means all, cases in countries outside the UKare in cattle imported from the UK. Some animals are
322 N. Hunter
features develop in the later stages of the incubation period and it is very difficult to detect (by histopathology) those animals with scrapie which are not yet visibly affected by the disease. In experimental studies of BSE in sheep, the clinical signs are very similar to those of scrapie (Foster et al ., 2001a), and in a field situation it is expected that, should BSE have infected sheep, it would be impossible to use these to differentiate scrapie (presumed to be non-pathogenic to humans) from BSE.
Treatments
There are as yet no approved treatments which cure TSE diseases. There are possible candidates amongst drugs which prolong the incubation period in animal studies, for example pentosan polysulphate (PS), which if administered 7 h after injection with ME scrapie prolonged the incubation period in mice by up to 66% (Farquhar et al ., 1999). This is not a cure, but is one example of current studies which are aimed at understanding how therapeutics might work. It has also been suggested that anti-PrP antibodies could prevent the conversion of PrPC^ to PrPSC in the body of affected individuals as it seems to do in cell culture (Peretz et al ., 2001). An interesting recent development involves trials in humans clinically affected with TSE disease of the anti-malarial drug quinacrine, not used widely in the UKsince the 1970s, and the related chlorpromazine, which carry additional worries of side effects. At present, the only real defence against these diseases in humans is avoidance of infection, and legislators have been busy trying to ensure that infection is removed from the human food chain.
Legislation and Regulatory Issues
The first laws designed to prevent the spread of BSE in cattle by banning the use of feeding ruminant tissues to ruminants in the UK came into force in 1988, and the rules have continued to be tightened since then. The
European Commission took the first steps in limiting cattle exports from the UKin 1989, which culminated in a complete ban in 1996. In 1989, there was also the banning in human food produced in England and Wales of cattle tissues expected (from previous studies in sheep scrapie) to be infectious. The same legislation was applied in Scotland and Northern Ireland in the following year. For 2 years, from late 1997, meat on the bone was also banned from human consumption. It was clear, however, that the 1988 rumi- nant feed ban had not been completely effec- tive as cattle born after the ban also developed BSE; however, greater control over abattoirs and rendering plants has resulted in a con- tinual drop in numbers of BSE cases in the UK. It is disturbing that the very stringent rules imposed after the announcement of the occurrence of vCJD in 1996 do not seem to be totally effective, as animals born after that date are now showing signs of BSE, albeit in very low numbers. Laws relating to the age of animals allowed into the human food chain (1996) mean that UKcattle over 30 months cannot be used as a source of meat or mech- anically recovered meat (MRM). MBM and ruminant-derived products have been subject to a plethora of regulations in the UKand in Europe about what they can contain and what they can be used for. For example, in 1998, the sale of MBM derived from mammalian tissues was prohibited for use as a fertilizer on agri- cultural land, and the use of certain ruminant tissues in cosmetics was banned in the UKin
- As sheep also became suspect in the BSE audit trail, heads of sheep and goats were pro- hibited for human consumption in the UK in 1996. However, attempts by the European Commission to agree on legislation to control the use of high risk tissues in food and food products have been greatly hampered by the relative difficulty of removal of the spinal cord from sheep and goats compared with the much larger cattle. It also appears that, if BSE does occur in small ruminants, it may be more widespread in body tissues than it is in cattle. Since the introduction of a reliable means of cattle identification by ear tagging and indi- vidual cattle passports with a tracing system, coupled with all of the other control measures in place in the UK, the export market is
324 N. Hunter
gradually opening up again for cattle and meat products (data from DEFRA, UK).
Conclusions
It is too soon to predict accurately the final numbers of people who will become affected by vCJD; however, it is likely that the measures in place to protect humans and animals will mean that, if an epidemic of vCJD does occur, its time span will be limited. Intense efforts are also being made to understand the disease, how it spreads and how it can be treated or prevented in sus- ceptible individuals. It seems, however, that a great deal of the dangerous BSE-infected MBM was exported from the UKto other parts of the world and may have been fed there to indigenous ruminants. Organiza- tions such as the World Health Organization, the European Union, the Office Internation- ale d’Epizootiques and the World Trade Organization have been trying to raise the issue of the dangers of BSE in regions outside Europe and the USA, and it is to be hoped that BSE and vCJD will not become major problems for the rest of the world population.
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