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Bovine Spongiform Encephalopathy (BSE)

                      Gordon T. Stewart*.
 

Although this form of spongiform encephalopathy in cattle was not described in professional literature until 1987, it was recognised as an unusual, possibly new disease in the UK in 1985, and might have been recognised elsewhere in Europe by experienced farmers and others before then.  Scrapie, a similar disease causing scratching, incoordination and wasting in certain breeds of sheep, had been well-recognised by shepherds more widely for over two hundred years, and had been intensively studied as a transmissible form of encephalopathy since the 1960' when there was an extensive outbreak in the USA of a transmissible encephalopathy in ranch-reared mink which had been fed with scrapie-contaminated foodstuff.  Similar diseases have been recognised in elk and deer. Less commonly and more recently, other similarly-disabling encephalopathies have been described in various captive and domestic animals including cats though never in free-living animals. Since these forms of encephalopathy can be transmitted experimentally by intra-peritoneal or direct intra-cerebral inoculation and occasionally by feeding to some other animals experimentally, they are referred to collectively as the Transmissible Spongiform Encephalopathies (TSE's). Many species, including some breeds of sheep, are consistently unaffected and appear to be immune. There is to date no explanation of this. 

ANTECEDENTS OF THE EPIDEMIC OF BSE IN CATTLE IN THE UK.

Bovine Spongiform Encephalopathy (BSE) was unknown as such to farmers and veterinarians  and others who worked with cattle until it was observed in a herd of cows in Kent in 1985. This was followed in 1986 by similar observations in the midlands and south-west of England, with a general spread throughout Britain by 1987. Investigations at the Central Veterinary Laboratory at Weybridge confirmed that the new disease was a spongiform encephalopathy, pathologically similar to scrapie, but epidemiologically unconnected to naturally-occurring scrapie. (1) 

The obvious antecedent and likely cause of the sudden occurrence of BSE was a general change in feeding introduced by the Ministry of  Agriculture, Fisheries and Food (MAFF) in the early 1980's. There were two aspects of this change. The first was the scrapping of controls on foodstuffs for farm animals, the second was the introduction of a policy of adding animal offal, which sometimes included meat from sheep with scrapie, to cattle feed to increase milk production. This was known to have occurred beforehand, unofficially, and might have been more widespread than was admitted at the time. But the mandatory change whereby this became a general policy caused a revolution in traditional farming practice in that all dairy herds of herbivore cattle were then artificially and forcibly given carnivore food on a massive scale.  The spread of BSE in dairy herds in the UK was immediate and sensational: from four cases in 1986 to 7137 confirmed cases in 1989 to a peak of 36682 in 1992. With hindsight, this spread is thought to have been aggravated by recycling the causal agent from cattle to cattle in feeds or otherwise. Thereafter, there was a decline at the same rate to 1107 cases in he first half of June, 1998 (2) 

ACTION BY THE GOVERNMENT OF THE UK:

These events gave rise to a hypothesis that an infectious causal agent was being spread from sheep to cattle, from cattle to cattle, from cattle to sheep and to other ruminants. In 1986, it was considered by an advisory committee that the risk of transmission of BSE to humans was remote but a research committee chaired by Dr David Tyrrell, in a preliminary report to the Government, drew attention to the need for further epidemiological and experimental studies. The feeding of offal containing ruminant-derived protein to ruminants was therefore banned in July 1988 (BSE Order No 1039) to arrest this cycle of transmission. The Order also made BSE notifiable and required that cows suspected of having BSE should be isolated when calving.  Amendments to the Order in August 1988 provided for a slaughter policy and compensation to farmers. In November 1988 the feed ban was applied to any use of milk from affected  cattle except for feeding to the cow's own calf. It was also extended to Northern Ireland.  It had by then been shown at Weybridge that BSE could be transmitted to mice by intracerebral inoculation of brain tissue from affected cattle

The European Commission (EC)  imposed a ban on export of cattle born before 18 July 1988 and offspring of affected or suspect cows. In November, Bovine Offal (Prohibition) Regulations became effective throughout the UK banning specified bovine offals for human consumption. Meanwhile it was noted that pedigree beef herds were less affected or unaffected but some beef cattle in mixed herds were affected. As time passed, it appeared that suckler-fed beef herds kept apart from dairy cattle were unaffected. This has now been confirmed. 

Results of further research on BSE became available in 1990. Cases were reported in captive antelopes and cats;  cattle inoculated intracerebrally and intravenously with brain material from cattle with BSE developed the disease and then showed secondary cattle-to-cattle transmission; mice fed orally with similar material  developed the disease, as did a pig inoculated parenterally. The Tyrrell Committee found no evidence of transmission to humans but recommended a ban on specified offals in all forms of animal food, including pet foods. The ban on exported foods from the UK was extended to exclude meat and bone meal from fertilisers.

Although the total number of cases notified in cattle in the UK continued to rise through 1992 to a peak of about 37,000 cases and a cumulative total of 172,924 by 1998, the number of confirmed cases in cattle born after July 1988 (the date of imposition of the ban on offal, slaughtering and compensation) fell from 12416 in 1989  to 5279 in 1990, and to three in 1994 (1). For comparison, there were only 262 cases in the Republic of Ireland, 264 in Switzerland and 132 from the rest of the world. Even allowing for under-reporting, BSE was a peculiarly British zoonosis, confined mainly to the UK because of a "Concatenation of risk factors". These were:  1:  a high ratio of sheep to cattle; 2:  a high prevalence of endemic scrapie; 3:  the heavy feeding of MBM to dairy cattle; and 4:  changes in the rendering process whereby meat and bone meal of animal origin were not treated with organic solvents. It seemed likely that a transmissible agent responsible for this outbreak  and resistant to heat and solvents could already  have entered the human food chain (3)

THE NATURE OF BSE:

The neuropathological features of BSE were similar to those of scrapie in sheep and goats, so BSE was at first regarded as a bovine counterpart. But scrapie  was transmissible only within flocks of susceptible sheep and not between sheep and cattle. There was no evidence, in over a hundred years of experience, of any transmission of scrapie to shepherds,  families on sheep farms, butchers or abattoir workers. Nor was there any evidence from 1985 - 87 of cases of unexplained encephalitis in persons exposed occupationally to BSE. On this basis, and having regard also to the history of the  other  spongiform encephalopathies, official Committees and individual experts, with few exceptions, advised the Government that there was no likelihood of transmission of BSE to humans, either through occupational contact or through an infectious agent entering the human food chain. The regulations introduced in 1988 were considered adequate for control of the epidemic in cattle by the ban on feeding of cattle with MBM,  notification, examination and slaughtering of suspect cattle or whole herds, and compensation of farmers. Although the possibility of spread to humans was denied, the need for caution was expressed  by a substantial increase in funding for medical surveillance of all suspect neurological conditions, and for research into the causation and transmission of BSE.  Particular attention was paid to the possibility that, unlike scrapie, BSE might relate to neurological disease, especially Creuzfeldt-Jakob Disease (CJD) in humans, so an intensive national programme of surveillance was organised by the neurological assessment unit at the University of Edinburgh, working cooperatively with the Advisory Committee on Dangerous Pathogens, the Medical Research Council, MAFF, the Department of Health and several Veterinary Schools (1).

SPONGIFORM ENCEPHALOPATHIES IN HUMANS.

TSE's are described (4) in five main forms in man: (i)  Creuzfeldt-Jakob Disease (CJD), a rare syndrome of cerebral disorder with progressive ataxia  and decerebration appearing sporadically in late middle age and causing death within a year or two; (ii)  CJD with a  familial distribution, usually in Askhenazi Jews in the Mediterranean, also rarely; (iii)  Gerstmann-Straussler-Scheinker Disease, occurring sporadically or inherited as an autosomal dominant, fatal within five years; (iv) Iatrogenic, after injections of growth factor from pituitary glands used therapeutically; and (v)  Kuru which was endemic in Melanesian people in the remote eastern highlands of  New Guinea where it was confined to a particular linguistic group (Fore) and to a neighbouring tribe with which this group intermarried. In this limited location,  kuru was a main cause of death in adult women and in children of both  sexes in whom it began insidiously with unsteadiness of stance and gait followed by ataxia, ocular and mental deterioration, leading inevitably to fatality in three months to one year. As in the other TSE's, pathological changes, confined to the brain, were an increase in  astrocytes with degeneration of neurons, leaving spongiform holes more marked in the cerebellum, pons, hypothalamus and basal ganglia. The neurological signs and disability corresponded precisely to the location of lesions in the central nervous system  Although kuru obviously transmissible, there were no signs in brains examined  post mortem of any inflammatory or infectious process.  The pathological changes, like the epidemiological distribution and clinical progression, closely resembled those of scrapie in sheep.  Brain material inoculated into chimpanzees caused a similar disease after 18 months to two years, and was then transmissible spontaneously between chimpanzees. The disease was strongly associated with cannibalism practised apparently as a tribal ritual only by women and children who were encouraged to eat the brains of deceased members of the tribe. Suppression of this practice is said to have led to disappearance of kuru. 

Apart from kuru,  spongiform encephalopathies  in humans are extremely and consistently rare with annual 
incidence that seldom exceeds one case per million in populations with valid data in Europe, Asia and the 
Americas. It is not certain whether they  should include also the various subacute dementias of middle and 
older age and diseases associated with abnormal somnolence such as idiopathic sleeping disease and 
catalepsies.. Symptomatically, there is a considerable overlap with other mental and neurological disorders 
in the premonitory stages but, pathologically, the  spongiform encephalopathies appear to be distinct. 
The best-known is the form of Creutzfeldt-Jakob Disease (CJD).

CREUZFELDT-JAKOB DISEASE: 

This was described independently by HG Creuzfeldt and A Jakob in the 1920's. It occurs typically as a sporadic occurrence with sudden, worsening muscular twitches followed by then jerks, then ataxia progressing rapidly via loss of memory and somnolence to decerebration, helplessness and death within a year. The number of cases notified in the UK rose from about 30 in the 1980's ( 0.5/million/year) to 54 in 1994 (0.94/million/year) ie during the period when BSE first appeared and spread in cattle.  CJD is a syndrome of dementia, tremors, motor weaknes, cortical blindness and incoordination usually in men aged 40 - 70 occcurring sporadically or in familial form (10%) leading to death within a year. The identifying pathological features are multicentric amyloid plaques and neuronal degeneration with diffuse spongiform changes, similar to those described in kuru. It occurs as a sporadic or familial encephalopathy and has been observed also, with some histological differences, in persons who received injections in childhood of growth hormone prepared from human pituitary glands. Until early in 1994, CJD was regarded as an extremely rare disease with a uniform incidence in developed countries of 0.5 - 1 case annually per million of population. Electro-encephalogram recordings show generalised slowing  of rhythm. It seems clear that, in 1998, there is no increase in notifications of these but the new cases in younger persons reported in March 1994 are different in pathological features which resemble those of BSE. These were identified only after a highly-organised national surveillance in the UK and there is no indication at the time of writing that there are any more. 

Histologically, the brains of patients dying with CJD show diffuse loss of neuronal cells, increase of fibrous astrocytes amid spongiform degeneration in the cortex as in other TSE's. But there is considerable variation in symptomatology according to the distribution of these lesions: blindness if they are in the occipital, deafness in the temporal cortex, ataxia in the cerebellum but always a final decerebration which, occurring as it sometimes does in younger, strikingly healthy persons, attracts horrified attention, the more so because of the unremitting advance to fatality. As with kuru. similar symptoms and pathological changes occurred in chimpanzees about a year after they were inoculated with brain substance from patients with CJD. 

In 1975, Matthews reported the results of a survey of TSE's in man from 1964-73 in the UK. He had observed some geographic clustering and possibilities of contact between cases. In the same year, Slack et al reported that cell-cultures of brains from patients dying with CJD showed sheets and nodules of fibrinous matter and spindle cells after 12 weeks incubation. From 1979 onward Gajdusek and his colleagues in New Guinea and at the Rockefeller Institute in New York City extended their work on Kuru reaching the conclusion - for which Gajdusek was awarded a Nobel Prize - that the transmissible agent responsible for the disease was a "Slow" virus. This view was displaced by the work of Prusiner, showing that the  main  structural abnormality in TSE's is the presence of an abnormal proteinase-resistant Prion protein, PrP, in affected areas of the brain, as described below.

POSSIBILITY OF SPREAD OF BSE TO HUMANS.

The rapid spread of BSE in the UK , the likelihood that a transmissible agent had already entered the human food chain and the increase in notifications of  CJD raised fears that BSE might also be spreading to the human population. This  was discounted oficially until, in 1996, the intensified surveillance organised in Edinburgh revealed eleven cases of spongiform encephalopathy which were atypical of sporadic and familial CJD in that they all occurred in persons below the age of forty and showed post mortem cerebral lesions which resembled those of BSE  rather than classical CJD. These cases were described as a new variant (nvCJD). An unprecedented programme of intensified surveillance world wide since then has produced a total of  27 cases in the UK and one in France. Meanwhile, the Government of the UK accepted advice from medical and veterinary scientists that the most likely explanation of the occurrence of nvCJD was that it came from ingestion of food containing the agent of BSE transmitted from cattle infected before the ban on feeding of scrapie-infected offal. The experts considered that the risk of this happening was small but qualified their caution by refusing to deny the possibility that it might extend eventually to thousands, hundreds of thousands or millions of cases: a record, perhaps, in the annals of hedging bets. 

This advice and decision were made known immediately on all public Media. Details were conveyed to the WHO, European Community and the rest of the world. Further stringent regulations were introduced to extend slaughtering of all suspect cattle and to control procedures in abattoirs, other processing, sale and use of meat and  meat products. The message from the UK was interpreted everywhere as alarming and, together with the restrictive legislation and ban,  halted exports of beef and cattle products from the UK. However, beef with brain, spinal cord and specified offals removed continued to be sold and extensively consumed in homes and restaurants throughout the UK. 

PERCEPTION BY THE PUBLIC AND THE MEDIA.

Confronted suddenly by this unexpected threat from a basic foodstuff which was eaten daily in so many forms and used otherwise in medicines and cosmetics, the general public and newspapers, radio, television and the internet reacted with concern and anxiety. But this was followed within a few days by scepticism when it began to be realised that the immediate risk to human health equated to that of crossing the road.  This led to various shifts in attitudes. On the personal level, most adults accepted that they had already been exposed to the risk but that they should  protect their young children. There was bifurcation between those media which featured the issue as just another item of bad news and those which examined the uncertainties and inconsistencies in the scientific arguments and political response more critically. The bad news subsided when it became obvious to them and to the public that there was no likelihood of an immediate epidemic, or of any epidemic, and that the possibly contaminated roast beef of Old England and everywhere else was being eaten freely by  unalarmed majorities in the land of origin.  The better informed media and sceptic professionals became more critical, calling for more disclosures of scientific evidence and questioning the appropriateness of action to date, especially the damage done to the agroeconomy of the UK which the Economist, voicing widespread criticisms  on 14th March, 1998, considered had been "Bungled".

In farming and veterinary circles, it was widely known that there was no BSE in herds of  suckler and grass-fed cattle. Although this was not admitted officially, a substantial proportion of the adult population preferred to disbelieve or ignore risk from consumption of beef and beef products as sold in the domestic market in the UK. This means  that  there are several natural though unofficial prospective studies in progress which will, through the passage of time, disclose whether or not there is any real risk of natural transference of BSE from cattle to man or any other species. 

EPIDEMIOLOGY

BSE began as a point-source outbreak in 1985 and rapidly became generalised , reaching a peak of about 37,000 cases in cattle in 1992 (5). Since then, the epidemic has subsided as a result of withdrawal of suspect foodstuffs and slaughtering of animals already at risk from earlier exposure or horizontal transmission of the Prion glycoprotein presumed to be the infectious agent.. The only evidence of possible transmission through the species barrier to man is the occurrence to date of 27 cases similar to classical CJD in clinical features but different in earlier age of onset and in cerebral pathology (nvCJD) which relates in histological and PrP patterns more closely to BSE.

Without exception, these cases have histories which are not suggestive of high or even of any exposure to the agent of BSE, whether infectious or proteinaceous.  If transference across the species barrier is going to occur, it would seem much more likely that the cases now being recognised would be in persons at high, continuous or special risk, namely farm and abattoir workers, veterinarians, butchers and cooks, and that symptoms would be reported or detected in older as well as younger patients. With unprecedented world-wide alarm serving to intensify retrospective and concurrent surveillance, it is most unlikely that cases would occur only in persons at low risk below 40 years of age. 

The population under passive surveillance by competent authorities is enormous: most of northern and western Europe, the USA, Canada, New Zealand, Australia - a denominator of at least 600 million. The intensification of awareness and ascertainment will certainly bring to attention cases of classical CJD which would otherwise have been overlooked. There are many areas, especially in Europe where beef or beef products from Europe have been used freely for many years. In the UK alone, excluding vegetarians, about 40 million people have probably been continuously exposed to the transmissible agent of BSE, if there is one, for 15 years. With only 23 cases to date, this gives a period prevalence of 23/(15x40m)  = 0.038/million person-years (mpy) with zero prevalence to date in about 100,000 persons who are occupationally at much greater risk. Even if nvCJD is only the tip of an iceberg, with only 10% notified, the period prevalence in UK would still be less than 1/mpy, ie lower than the annual incidence of CJD (1/1m) and of deaths from accidents involving motor vehicles (8/1000). When risks are as low as this, operating over a period when people are constantly exposed to greater risks, confidence limits are meaningless.  If TSE's are naturally infectious across species barriers, it is difficult to see how the frequency with low exposure can be higher than that with high  exposure  unless  expression is governed by an idiosyncratic factor or genetic predisposition. This seems to be so in familial CJD and, if a co-factor for the expression of abnormal PrP is required for crossing the species barrier from cattle with BSE to man,  the possibility that this has happened in nv CJD remains. The data are at present too sparse to say more than this.

APPROACHES TO CAUSATION:

Biologically, epidemiologically and clinically, all of these spongiform encephalopathies were occurring in several forms before BSE was recognised in cattle in SE England in 1986.  Neuropathologically, they were all similar in pattern in their original hosts but not necessarily after transmission to experimental animals. The advent of BSE created a new situation in various ways. No other TSE except scrapie spread so extensively and none so rapidly.  But this happened only after herbivore cattle in dairy or mixed herds had been fed, entirely against their natural diet and inclination, with offal-containing  meat which may have included meat from sheep with scrapie. In dairy and mixed herds, BSE spread .like scrapie but in beef herds which had been suckler fed it did not appear let alone spread except when there had been contact via individual animals or herds with cattle already affected by, or incubating BSE. This important demarcation was apparent from the start of the epidemic in the UK in 1986, and is still true. 

Although the neuropathological picture of TSE's was not that of  inflammatory diseases,  the transmissibility within and between species was indicative of an infectious process.  This appeared to be the best explanation of the occurrence of Kuru, and was  supported by earlier work on scrapie in which an agent passing through bacterial filters transmitted disease and could be titred.in experimental infections. Because of the long incubation period, these agents were regarded then as Slow Viruses, but they were unlike "Regular" viruses in several ways. They were partially resistant to inactivation by conventional sterilising processes such as heat, disinfectants and formol fixation. They contained protein but no nucleic acid. They were not self-replicating and could not be grown in cell or tissue culture.  The incubation periods (IP's) of mice inoculated with the agent of scrapie varied, and 20 strains were identified on this basis. The replication of these strains in mice was controlled by a host gene (Sinc). In contrast, the agent of BSE was unique and seemed to be invariable. 

The work of Prusiner (6) in USA had shown, in 1982, that the agents  of  the  TSE's  were proteinase-resistant, proteinaceous particles with infectious properties (Prion proteins, PrP) but devoid of nucleic acid.  These are present in normal cells as PrP(C) particles but, in sheep with scrapie or in susceptible species injected with scrapie material they are changed into a strain-specific  isoform PrP(Sc) in which the  alpha-helical structure is malfolded into beta-sheets encoded by the sequence of the chromosomal PrP of the mammal species in which it last replicated (7). Experiments in  transgenic mice suggest that the PrP(Sc) acts as a template upon which PrP(C) is refolded into a nascent, abnormal PrP through a process facilitated by another protein.  The change in PrP was confirmed independently in the UK by Collinge and others (8) but they suggested that the self-replicating protein was possibly linked to a nucleotide and could encode the disease phenotype. Neuropathologists in France (9) have suggested that the malfolded version of PrP(C) - which they call PrPres - is involved in the species adaptation and that another, as yet unidentified agent, is responsible for transmission. 

These discoveries, preceding and following the outbreak of BSE, with the alarming possibility that a transmissible heat-resistant agent could already  have entered the human food chain in the UK, as described below, focussed attention on a new  proteinase-resistant PrP as the most likely cause. 

In the search for a causal agent, it is important to recognise differences between encephalopathies occurirng naturally and those induced experimentally. When Pearson et al (10) injected fresh-frozen or formol-fixed brain homogenates from the first cases of spongiform encephalopathy occurring naturally in domestic cats (FSE) into inbred mice, signs of  encephalopathy appeared after an incubation period (IP) of 304 - 573 days with cerebral lesions.similar to BSE but different from scrapie.  The brains of recipient mice showed amyloid plaques which were not present in the donor cats. There were variations in IP's according to which strains of inbred mice were used. The similarities suggested that  BSE and FSE came from a common source but there are differences which await explanation (11)

Inoculation of brain homogenates, variously treated, as the main tools of experimental research for the cause of TSE's , has increased and diversified enormously since the advent of BSE. In some experiments, transmissions from unconnected sources of  cattle with BSE produce remarkably uniform IP's, clinical and neuropathological changes in mice. But this is in contrast to  a  variable pattern observed in many more  transmissions from six other species, including sheep, goats elk and deer. Subsequent experimental studies have shown that the BSE agent has maintained some consistency by keeping its identity through transmissions in six species, and that differences between these species as donors have not changed the pattern of disease in recipient mice appreciably. But this does not mean that a difference in transmission characteristics necessarily means that a source is unrelated to BSE, as the  scrapie agent sometimes changes during passage through different species. It is very difficult to indeed to recognise let alone control all the sources of biological variability and technical error in this experimental field.

THE SPECIES BARRIER.

When they occur naturally, all of the TSE's are sporadic, rare and not readily  cross-infectious in their natural hosts except when they are in captivity or in herds. It has been known since the 1960's that ranch-reared mink became ill when they were fed with meat from sheep with scrapie. Successful transmission to other species usually requires direct intra-cerebral or parenteral inoculation, often with high doses of homogenates of brain material. Transmission by the oral route is unsuccessful unless the parenteral dose is increased by orders of magnitude. It is inconceivable that feedstuffs containing animal material had never been fed to cattle before 1985. The difference then was that potentially scrapie-contaminated MBM was not fed to cattle in concentrated form. In a review of the connection between scrapie and BSE,  Kimberlin (1991) concluded that, even then, exposure.was low, and that the outbreak extended because infected material, recycled in cattle, "passaged" the infection in a manner which bypassed the species barrier.

In a few human diseases (eg measles) and in some zoonoses caused by viruses or virus-like agents, this barrier is remarkable firm though in others, probably the majority, it is in one way or another penetrable. 
Although the TSE's are widely transmissible, there are exceptions and also differences between and within species. Some of these arise from technical factors and others from differences between first and later 
transmissions. Mice lacking an active PrP gene do not develop neurological disease when injected with macerates containing the infectious agents while the main differences in TSE's within species are linked to genetic differences (11). These are revealed only by  cross-breeding, genetic analysis of inbred strains, titrations of inocula, histopathology,  histochemistry and transformation of Prion proteins. All of this, now summated in a considerable data base, has produced many advances in biochemistry and morbid anatomy. 
Despite this (11), there is still uncertainty about the genetic and epidemiological determinants of any of the TSE's,  the immunity of some species, sub-species and herds, the link if any between BSE and CJD or 
nvCJD, or the risks to occupational handlers, consumers of meat and meat products. The over-riding uncertainty is whether or not there is a risk of transmission of BSE to humans but this uncertainty is increasingly muted in official and Media releases of  new information in  which nv CJD (or vCJD, or even CJD) are described as "The human form of BSE". Such releases have served to maintain anxieties which were diminishing with the passage of time, and to raise new  anxieties for instance in recent releases from SEAC about the possibility of widespread transmission of  BSE through transfusions of blood and blood products from British donors.  Scientifically, as described below, the uncertainty remains profound because the nature of the infectious agents, the pathogenesis of the main lesions and the mechanics of transmission await elucidation

EXPERIMENTS ON PENETRATION OF THE SPECIES BARRIER. 

Inoculation of homogenates from scrapie- as well as BSE-affected brains to mice have shown differences according to donor, recipient, route of inoculation, dose and previous or subsequent passage. These 
differences may be absolute or graded.  They express themselves in IP's, symptomatology, histopathology and varying susceptibilities of inbred strains of mice. As would be expected, IP's are longer with 
intraperitoneal, intravenous and subcutaneous than with intracerebral inoculation, and very long indeed in the surprisingly few feeding experiments which are on record.  The pathogenesis is also different in that with the oral and peritonaeal routes replication follows the pattern of natural infection, beginning  in the lymphoreticular tissues of the spleen, lymph glands and thymus, and spreading from there along non-medullated nerves to the spinal cord and brain. The salivary glands are also affected. According to Kimberlin and Walker, the dose required for intragastric transmission is about 40,000 times that required in the intracerebral route but, by the same token, the oral route, round the clock in natural feeding must convey enormous inocula to visceral tissues and intestinal contents which must in turn become infectious. These findings are respected in recent regulations which attempt to make meat safe for consumption.

Mice lacking genes for PrP are resistant to scrapie. With both scrapie and BSE, the PrP of the host influences the IP, pathogenesis and outcome, but so does the PrP of the inoculated material which, by itself or in close association with an unidentified agent-specific molecule, appears to be an essential component of the "Infectious" agent. This in turn may depend upon compatibility between the molecular configuration of the agent and the PrP genotype of the host. To obtain consistent experimental results, it is advantageous to use specially-bred transgenic "Null" mice into which genes for compatible PrP have been injected. With repeated passage under these ideal experimental conditions, IP's shorten and the transmitted disease assumes a consistent, reproducible neuropathological profile. In this way, many different phenotypic strains of BSE have been identified. In a recent review, Haywood (11) postulates that such variety in infectious 
strains is difficult to explain unless the agent contains nucleic acid., as suggested also by Collinge et al (8).. 

According to Bruce et al (12), BSE material produces a characteristic pattern of disease (a BSE signature) in inbred mice which is retained after experimental passage through intermediate species. In further experiments, mice were inoculated intracerebrally with material from six typical cases of sporadic (sp ie classical) CJD aged 55 - 82 and three  nvCJD aged 29, 30 and 31 years. The brains of the cases of spCJD had widespread spongiform vacuolation with few or no amyloid plaques whereas those of the nvCJD had extensive  deposits of PrP amyloid and prominent cerebellar involvement. But clinically-affected mice injected with nv CJD showed vacuolation without amyloid plaques though similar otherwise to mice injected with material from BSE and other TSE's, excluding scrapie. Mice given material from spCJD did not become ill in 600-800 days. On these grounds, and in combination with surveillance data (not quoted), Bruce et al concluded that nvCJD was a new condition occurring exclusively in the UK with a compelling link with BSE. Independently, Hill et al.(13), working with transgenic mice, found that the attack rate of nvCJD was lower than that of "Typical" ie spCJD but the clinical course longer as in (younger) patients. They reported also striking similarities in PrPdeposits between BSE- and nvCJD- inoculated mice, leading them independently to the conclusion that BSE and nvCJD are caused by the same Prion strain, and that nvCJD is caused by exposure to BSE. This explains the exclusion of scrapie and other animal prion diseases in which fibrils containing protease-resistant, pathological forms of membrane-

associated, host-encoded.proteins serve as unique molecular markers, and deposition of gross amyloid as a common pathological feature.  If this hypothesis is correct, it could mean that nvCJD is more likely to be a 
new form of encephalopathy  with extensive amyloid change in the cerebellum, and not a variant of classical CJD. 

Fibrils similar to those of scrapie are present in Alzheimer's disease and other degenerative cerebral dementias but the fibrils in scrapie contain a protease-resistant 27-30kd protein (PrP) with a unique N-terminal amino acid sequence which enables its gene to be identified. This gene produces a highly specific PrP which, on biochemical and genetic grounds, is now  regarded as the infectious agent in scrapie. 
Repetition and further development of this work with material from animals with BSE since 1986 has shown that a PrP with a different structure and genotype is present in the cerebral lesions (8,11). 

Experimental evidence from transmissions and expressions of PrP's from TSE's therefore support the possibility of a causal connection between BSE in cattle and nvCJD in humans, perhaps mediated by interactions with genetic factors in the host. Biochemical studies indicate that the role of  prion proteins variously modified in the pathogenesis of all these conditions is not in doubt but the epidemiological evidence that this is an infectious process remains unconvincing. Of 839 referrals to the surveillance unit in Edinburgh of brains taken post-mortem from cases diagnosed as CJD in the UK from 1990 to 15th June 1998, only 26 were listed by MAFF in July 1998 as being definite or probable  nvCJD. All of these  were referred in the years 1995-98 but there was a numerically-
greater increase in referrals of all other forms of CJD from 1990-1998 resulting presumably from heightened interest and ascertainment (2). 

If nvCJD is indeed the  human form of BSE, it is strange that  it should express itself only in the younger population with shorter  exposure. This would suggest either that the older population was immune from long 
 exposure to a related but less pathogenic agent or that the new variant was selecting a minority of susceptible  individuals from an immense popoulation at equal or greater risk but so far unaffected.On the other hand, if sporadic CJD was an unrecognised, pre-epidemic form of BSE, the increase in spontaneous referrals might correspond to the spread and IP of epidemic BSE. But the experimental and biochemical evidence does not support this possibility. A large case-control study of risk factors of CJD in Europe concluded (14) that there was little evidence for an association between the risk of CJD and either animal exposure, or consumption of processed bovine meat or milk products in the period 1993-95. The findings suggested that genetic factors other than the 
known CJD mutations might play "An important part" in pathogenesis. It was noted that the first cases of nvCJD were homozygous for methionine at codon 129 of the gene for PRNP and that no cases had been identified in  valine homozygotes who are more common among cases of sporadic CJD and possibly in those considered to be at risk of iatrogenic CJD than in the unaffected population..

ALTERNATIVE HYPOTHESES.

Alternative sources of infection:  Hoinville et al (17), looking for evidence of direct transmission of BSE to cattle born after imposition of the feed ban, found that there was a significant increase in animals born up to three days after a subsequently affected cow calved but no other difference between the offspring of cases and cows that were subsequently affected by BSE. This held even when adjustment was made for exposed animals that would have been culled before enough time elapsed for development of clinical signs. Comparisons between herds and even within herds show differences in the proportion of exposed animals which develop BSE. Although the pathology and dynamics generally of BSE point to a single infectious disease with a common cause, the possibility of alternative routes and mechanisms cannot be excluded.

Pesticide-initiated modification of Prion protein:  At about the same time as the introduction of protein-containing feeds, lipophilic formulations of organophosphates were used compulsorily for pest control in sheep and cattle throughout the UK. No other European or American country had any such regulation. It is known that these compounds traverse the placental barrier and form covalent bonds with foetal prion protein. Some of the compounds contain phthalidimide, which could be neurotoxic and teratogenic. 

The onset, distribution and epidemic of BSE coincides with the use of these substances.   Mr M Purdey, a farmer in Somerset, England (where BSE is prevalent), has advanced a hypothesis that widespread use of these compounds in the UK may explain this, and also account for the 23,000 cattle born after the 1988 feed ban that developed spongiform encephalopathy (18). The reasoning is that phosphorylation of the Prion protein blocks the access of protease and creates an undegradeable, malfolded Prion protein (PrPsc) which is deposited in the central nervous system, causing the clinical, neuropathological and biochemical changes which are thought to be specific for BSE. Others besides Mr Purdey have drawn attention to this possibility. The biochemical reasoning in not implausible but pathologically the lesions associated with organophosphate poisoning are oedema affecting white matter, synapses and nerves, different from those of BSE. The suggestion may therefore relate only to a temporal symptomatic coincidence, and can be tested experimentally. If correct, it might explain why cattle treated with organophosphates developed neurological illness but it would not explain why, in a procedure which was nation-wide and compulsory, so many others escaped. 

Genetic susceptibility:  Although homogenates of brains from animals with bovine and other spongiform encephalopathies have transmitted identical or similar disease within species and through species barriers, this usually requires massive, artificial inocula and identification of a susceptible host. To obtain predictable results with BSE inocula in mice, closely inbred or transgenic recipients are required. This, together with the many other genetic variables which have emerged from intensive experimental research, leaves no doubt that genetic factors are  involved as determinants of the extent and pattern of susceptibility. Research to date identifies genetically-mediated changes in Prion proteins as the main mechanism, and the similarity or identity of the  PrP in BSE to that found in nvCJD leaves no doubt that this transformation is an essential correlate of the specific neuropathological changes and might be the direct cause. It is difficult to see how this transformation is encoded in the absence of nucleic acid in the transmissible agent, and also how activity withstands  formalin, heat and proteolytic enzymes. With plenty of specific PrP now available, it should be possible (a) to test its transmissive and other properties directly with purified formulations and attachments;  (b) to look for evidence of modification of this prion in patients with nvCJD; and (c) to compare TSE-specific amyloid chemically and biologically with other forms of amyloid. With more cases of TSE's now being notified, it should be possible compare the distribution of polymorphism at codon 129 of genes  coding for TSE-specific amyloid in families of patients with any form of CJD. 

In a cohort study of offspring from known dams in cattle with BSE, Ferguson et al (17) found that onset of disease could be explained  better or entirely by genetic predisposition as opposed to vertical (maternal) transmission., given some differentials in the exposure of diseased and unaffected dams to contaminated cattle feed. This could explain differences in susceptibility of host species and herds.

Co-factors:   Working in the neurological service of the French Commissariat on Atomic Energy, Lasmezas et al (1997) found that all of the mice injected intracerebrally with BSE brain-homogenates showed  neurological  abnormalities and neuronal death, more than 55% had no detectable protease-resistant protein (PrPres). But this appeared during subsequent serial passage. Their conclusion was that PrPres might be involved in species adaptation while another, unidentified agent was responsible for transmission of BSE. Diringer ( ) has suggested that this might be an animal virus interacting with aggregates of misfolded prion protein on the surface of nerve cells.

 Disordered nutrition.  Cattle are obligate herbivores. They do not like, need or want preformed protein from other animals or any other source because their anabolism, eating and survival are entirely preoccupied during daylight hours at least with the formidable metabolic task of converting grass and other plants into unique bovine proteins which include their own, species-specific cerebral glyco- and lipo-proteins. When forcibly fed as they were with an artificial overload of a proteinaceous miscellany, or mess, of substance which they could not metabolise, they tend to become sick as any animal does when given food which is blatantly inappropriate. They obviously experienced particular difficulty in ridding themselves of a structurally-altered, malfolded protein which was protease-resistent. Some of this protein was excreted but the remainder was absorbed and moved onward from intestinal lymphatics to other viscera, including the brain where localised deposits led to spongy degeneration and neurological malfunction, analagous to that seen in other forms of spongiform encephalopathies. When this unwanted protein, now PrPSc or whatever is inoculated in brain homogenates into other species intracerebrally, the brains of recipients experience similar problems and extracts show similar, malfolded Prions, with or without alteration by passage and perhaps with accession of other potentially-harmful passngers such as latent murine viruses. If cattle receive neurotoxic chemicals in the form of lipophilic organophosphate and other pesticides at the same time, a brainstorm resulting in a mad cow would seem to be a very likely outcome.

Bureaucracy and Politics.    Whatever the biological explanation, the fact remains that BSE occurred as a consequence of flawed advice given to bureaucratic mediators, and then to decision-making politicians by scientists working either as specialists in laboratories advising quasi-autonomous non-governmental organisations (QUANGOS), or as members of Quangos debating only with each other, and then advising a Government. This is a commonplace and perhaps a necessary scenario in public affairs but it is not one in which prior experience, mature judgement, immunity to political pressures and intellectual independence are necessarily operational.  There was, in the case of BSE, considerable debate, administrative and political action, scientific research and exchange of information all targetted hopefully on correction or damage-limitation. Instead, the outcome was a panic which all but destroyed the agricultural economy and related livelihoods of the UK, created wave upon wave of anxiety in  the domestic market and food habits, monstrous losses and expenditure, immense international repercussions and the slaughter of two million well-bred, outstandingly healthy cattle. The only members of British society who seemed undisturbed by this unprecedented mass slaughter were the Animal Rights Activists.

When the outbreak of BSE was confirmed, it was obvious that the transmissible agent or process had already entered the food chain but the possibility of transmission to humans was at first discounted, possibly because of the weight of negative evidence from scrapie. Then a few cases of  CJD were identified in brains referred post-mortem to the surveillance centre in Edinburgh where they were confirmed in 1996 as having  neuropathological lesions resembling those of  BSE rather than of classical CJD. For reasons discussed above, the expert advisers decided that the most likely explanation of these cases, occurring atypically in persons below the age of 40, was ingestion of the transmissible agent in food. This advice was accepted immediately, without wider consultation, and the outcome -also immediate - was the world-wide ban on British beef and beef products, with knock-on effects as listed above. Official caveats that the risk, if any, was minimal were ignored internationally though unofficial doubts in the UK allowed the domestic market and eating habits to survive. Thus milk from low-risk cattle is deemed to be safe though blood from near-zero risk healthy humans for transfusion is not. To date, there is no evidence of any appreciable increase in any form of TSE, even in those at highest risk of exposure to a transmissible pathogenic agent or process.

Limitations of scientific advice.  It is important to recognise that neuropathology is a descriptive discipline providing plenty of examples, especially in encephalitis and encephalopathies, where similar changes arise from dis-similar causes. A disease such as CJD which is excessively rare and therefore unfamiliar, overlapping with age-related dementias, incurable and diagnosable only post-mortem is more likely than not to be overlooked in routine clinical work.  The neuropathologists  realised that the referral of cases regarded as atypical could be a consequence of intensified ascertainment but they rejected the possibility that  nv CJD  occuring  in younger persons, might be more likely to resemble that in young cattle rather than that in old men.  Meanwhile, the statisticians, ignoring this in their calculations, considered that the occurrence of nvCJD raised the general risk from one in a million to a higher frequency with confidence limits of zero to perhaps 1:100,000, meaning that the risk of  acquiring  nvCJD from the agent of BSE in food could not be excluded.  In practical terms, risks with these limits of confidence are excluded constantly in daily life, otherwise people would never ride bicycles, cross roads or drink water, but this logic does not apply when there is political pressure for damage limitation, avoidance of blame, and therefore for zero risk. With few exceptions (See the TIMES, 25th March 1996), the Media in the UK continually refer to CJD as the human or British form of BSE while in France, where all forms of meat except British are freely consumed, Figaro noted (14 June, 1998) with ironical interest that experimental research had shown that sheep (in Britain only) were apparently contaminated with the BSE agent during a period when sheep were exported and lamb and mutton were on sale. 

CONCLUSION.

Through 1997, the possibility of risk to humans was supported by research which showed that inocula of brain homogenates from cattle with BSE caused similar lesions in inbred or transgenic mice, and that the PrP in these lesions as in the donor cattle was similar or identical to that in  new variant (nv)CJD. This could mean that a malformed PrP is itself the common infectious agent, or that it is produced by the same infectious agent and modified by genetic processes, or that BSE and  nvCJD are independent phenotypic expressions of similar modifications of Prions  It does not mean that a transmissible agent from BSE in beef from British cattle is a cause of  nvCJD although, on zero-risk criteria, the possibility still cannot be excluded.  Epidemiologically, the selective distribution of BSE, the occurrence of nvCJD in persons at low or zero risk and the entire absence to date of any cases in persons with maximal and continuous exposure exclude a directly infectious causal agent unless it is one which becomes pathogenic only by interacting with genetic or other intermediate factors. If insistence on zero risk is excluded, the scientific evidence to date, after intensive and continuing research, is insufficient to establish a causal connection between transmissible BSE in cattle and  nv- or any other form of  CJD in humans.

The occurrence and investigation of BSE has nevertheless revealed serious defects in our understanding and management of actual as well as potential transmissible diseases in cattle and in the food chain. If there had been a real risk in 1985 - 88, conventional practice in feeding and  slaughtering cattle, and for marketing meat products would not have controlled it. Reforms in abattoirs such as separating nervous tissue from viscera, or prohibiting the sale of beef-on-the-bone would not have overcome cross-contamination, as exemplified in simultaneous occurrences of outbreaks of infections with E coli 0157. Removal in 1974 of responsibility for testing from local to central authorities lowered standards in abattoirs, wholesale markets, shops and catering establishments. Management of the problem centrally by MAFF and QUANGOS with virtual elimination of all branches of the public health service from participation led to ill-advised precipitate action motivated by unrealistic scientific arguments and then by political panic. Writing from Weybridge in 1991, Bradley (19) concluded that there was no evidence that either BSE or scrapie were hazards to humans. Despite the wealth of original research since then, this could still be the underlying truth.

 It is usual to regard organised science, bureaucracy and government as agencies for the prevention of disasters like epidemics but, in the aetiology and spread of BSE, their reverse role can hardly be overlooked. even with the saving grace that their response to the problem which they created might lead indirectly and eventually to reassurance, albeit at enormous and unnecessary cost. To protect the public purse as well as the public health in these matters, it is time to revive the pre-1974  function wherein local authorities had an independent, executive role in the detection and control of all forms of potentially-communicable disease, and in the safety of food, water, atmospheres and other hazards arising in their localities. It is a matter of record that the fallibility of the public health function in these matters was predicted before the 1974-75 reorganisation, and has been apparent on many occasions since then. 
 

*Gordon T. Stewart,    Present address:   Glenavon,
Emeritus Professor of Public Health,              Clifton Down,
University of Glasgow,               Bristol, BS 8 3HT, UK
Glasgow, UK.    Tel/fax 0117 973 6532 <g stewart@gifford.co.uk>

        29th July, 1998. 
G T Stewart. 

   BOVINE SPONGIFORM ENCEPHALOPATHY:      REFERENCES.

1 Bovine Spongiform Encephalopathy: A Prigress Report, December 1997. Ministry of Agriculture, Food and Fisheries, London: HMSO.
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See also: 

Diringer H. Lancet 1995; 346; 1208.
Wyatt JM et al Vet Rec 1991; 129; 233.
Schreuder BEC. Vet Q 1993; 15; 167.
Guarda F  Schweizer Arch fur Tierheilkunde 1995; 137; 101.
Wells GA, Wilesmith JW.  Brain Pathology 1995; 5; 91.
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Nathanson N et al. Amer J Epid 1997; 145; 959.
Donnelly CA et al Phil Trans R Soc Lond B 1997; 352; 781.
Schreuder BE et al. Vet Rec 1997; 141; 187.
Poser SR  Deutsche Med Wochenschrift 1997; 122; 1099.
Zeidler M et al Lancet 1997; 350; 903.
Ibid  Lancet 1997; 350; 908.
Wilesmith JW et al. Vet Rec 1997; 141; 239.
MAFF. BSE Enforcement Bulletins, monthly.
MAFF  BSE in Great Britain. Progress Report, June 1998.

SUBIR