Bovine Spongiform Encephalopathy (BSE) – Mad Cow Disease

Review

Med Principles Pract 1998;7:172–186

Bovine SpongiformEncephalopathy (BSE) –Mad Cow Disease

Peter N. Campbell

Department of Biochemistry andMolecular Biology, UniversityCollege London, UK

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Key WordsPrionScrapieBovine spongiform

encephalopathyMad cow diseaseCreutzfeldt-Jakob disease

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AbstractThe background to the epidemic of bovine spongiform en-cephalopathy (BSE) among cattle in the United Kingdom isdescribed as the possible origin of the disease and its relation-ship to scrapie in sheep. Although the epidemic in cattle isvirtually over there is evidence of the transmission of theinfectious agent to humans to produce a new variant ofCreutzfeldt-Jakob disease. The current status of our under-standing of the molecular biology of the infectious agent isdescribed as is the evidence in support of the protein-only,prion, hypothesis. Study of the glycoforms of the prions sup-ports the view that BSE has been transmitted to humans.OOOOOOOOOOOOOOOOO

Received: March 29, 1998

Dr. Peter CampbellDepartment of Biochemistry and Molecular BiologyUniversity College LondonGower StreetLondon, WC1E 6BT (UK)

ABCFax + 41 61 306 12 34E-Mail [email protected]

© 1998 S. Karger AG, Basel1011–7571/98/0073–0172$15.00/0

Accessible online at:http://BioMedNet.com/karger

Introduction

Until 1985 there were probably not manybiochemists in the UK who were more thanvaguely aware that sheep tended to sufferfrom a degenerative disease known as ‘scra-pie’. Moreover, since the disease had existedin the UK for some 200 years and no one hadsuggested that it could be transmitted to hu-mans the subject was hardly a prominent onefor research. Nevertheless, the British Gov-ernment did continue to finance research at amodest level in order to unravel the nature ofthe infective agent, often described as a ‘slow

virus’, and similar work was pursued in othercountries, especially the USA.

All this changed following the report in1985 from a farm in Kent, that they had a cowsuffering from what is now called ‘mad cowdisease’. After a delay of about a year beforethe full significance of the finding becameapparent, the situation rapidly changed. Wehave witnessed a biological problem of mam-moth proportions which has many implica-tions in terms of the aetiology of the diseaseand the nutrition of animals and humans.Although most of the troubles have beencentred in the UK there have been a small

Sheep

Bovine Spongiform Encephalopathy(BSE) – Mad Cow Disease

Med Principles Pract 1998;7:172–186 173

Table 1. The occurrence oftransmissible dementias in about1985

Species Name of dementia

scrapieHumans kuru in New Guinea

Creutzfeldt-Jakob disease (CJD)Gerstmann-Straussler-Scheinker disease (GSS)fatal familial insomnia

Cows bovine spongiform encephalopathy (BSE)

number of cases in Switzerland, but the im-portant point is that the story has implicationson a world-wide basis. As I will show, theresearch effort to understand the problem hasbeen international.

I will start by describing the epidemiologyof ‘bovine spongiform encephalopathy’ (BSE)in the UK and then survey the state ofresearch aimed at understanding the aetiologyof the disease and the evolution of possibletherapies. I cannot provide in the limitedspace available a comprehensive list of ref-erences. Two books [1, 2] give a useful back-ground, an update by the Royal Society [3]which is authoritative, and a review providesolder references [4]; in addition I have pro-vided some references to key findings.

Epidemiology

The Incidence of TransmissibleEncephalopathiesThe situation concerning the occurrence of

the transmissible encephalopathies in the ear-ly 1980s is summarised in table 1. Scrapie insheep is characterized by an irritation of theskin caused by damage to the neuronal cells,which causes the animals to rub against afence or wall in the later stages of the clinicalcondition, and hence its name. While thereare sporadic outbreaks of scrapie in manycountries, including Europe and North Amer-

ica, it is no longer present in Australia orUSA, some breeds being very resistant. Scra-pie could be transmitted to other sheep byintracranial injection of infected brains butmore significant was the transmission to otherspecies such as goats. After the first passage toa goat there was a long incubation period andthis became shorter on further transmission toother goats. This so-called ‘species barrier’,whereby the clinical symptoms of the diseasetake longer to emerge when the recipient is ofa different species from that of the donor, isan important characteristic of the disease.The length of the incubation period is relatedto the evolutionary gap between the donorand recipient. In 1961 it was shown that thedisease could be transmitted to mice with amuch shorter incubation period and more cer-tain outcome and they, therefore, became afavourite test animal. The Syrian golden ham-ster has also proved to be a useful experimen-tal animal particularly in the hands of Prusi-ner in San Francisco. In humans Zigas andGajdusek discovered a disease named ‘kuru’among the Fore tribe in New Guinea. Theyshowed that this was spread as a result of acannibalistic feast involving ritual consump-tion of their dead relatives. The custom hasdied out but even today, some 30 years afterthe practice of cannibalism was suppressed,about 10 people a year die from kuru. Theexplanation of kuru was that by chance some-one suffering from Creutzfeldt-Jakob disease

174 Med Principles Pract 1998;7:172–186 Campbell

Table 2. The chronology of the BSE epidemic

1985 (Apr) BSE first observed clinically1986 (Nov) Disease identified as spongiform encephalopathy1987 (Apr) Initial epidemiological studies started1987 (Dec) Initial epidemiological studies incriminate ruminant-derived MBM as a

cause of BSE1988 (Apr) Southwood Committee set up

(July) Ruminant feed ban introduced for MBM(Aug) All cattle with symptoms of BSE to be slaughtered

1989 (Feb) Southwood report; risk to humans ‘remote’; estimates expected number ofcattle with BSE to reach 17,000–25,000

(Nov) Ban on use of certain bovine offals for human consumption1991 (Mar) EU bans export of British cattle over 6 months old

(Apr) EU bans export of British offal(May) First case of ‘BSE’ in cat(Sept) Offal banned from animal feed

1992 BSE epidemic peaks at 36,681 cases in a year1993 (July) 100,000th confirmed case of BSE1994 (Nov) Thymus and intestines added to offal ban; all mammalian protein banned

from cattle and sheep feed1995 (Nov) First 3 deaths of younger humans1996 (Mar) Suspected link between BSE and nvCJD; EU bans all British beef exports

(July) Controls on slaughter of sheep1997 (Dec) Ban on sale of beef on the bone because of infectivity in spinal cord and

possibly in bone marrow

(CJD), which as I will explain is a nervous dis-ease occurring sporadically all over the world,appeared among the Fore people. When theaffected person died the surviving relativesbecame infected. In view of research associat-ed with scrapie, Gajdusek inoculated chim-panzees and other primate species with sus-pensions of kuru brains. The chimpanzeessuccumbed after about 1.5 years. Gajdusekwas awarded the Nobel prize in 1976.

In addition to CJD, which I will refer toagain later, there are two other diseases inhumans with a rather similar pathology,namely Gerstmann-Straussler-Scheinker dis-ease (GSS) and fatal familial insomnia. It wasin 1985 that the first reports were received ofa dementia in cows from farms in England.Although there was some delay in clearlyrecognizing the disease it became known as

‘bovine spongiform encephalopathy’ (BSE) inview of the spongy appearance of sections ofthe brains of the dead animals.

The Chronology of the Epidemic of BSEThe number of cases of BSE rose dramati-

cally and investigations of the cause were setup. The critical dates in the unravelling ofthe epidemic are given in table 2. Suspicioncentred on a high protein dietary supplementprepared from meat and bone meal (MBM)from sheep and cattle that were not suitablefor human consumption. MBM was particu-larly used for feeding to high milk-yield dairycows. This is a common practice in manycountries. It transpired that for various rea-sons the method of preparation of MBM inthe UK had been changed around 1980 inthat the hydrocarbon extraction of fat had



been reduced and the additional steam treat-ment that was used to remove the solvents forreuse was omitted. A ban on the use of MBMas a feed for cattle and sheep was rapidly in-stituted (1988). In 1988 a working party un-der Prof. Southwood was set up. They madesome interim recommendations during 1988and issued a final report in 1989. The sameyear they confirmed the suspicion concerningMBM, and recommended that all affectedcattle be destroyed. At that time it was sus-pected that the trouble was caused by scrapiefrom infected sheep being transmitted to cat-tle via MBM but as I will show this now seemsunlikely. (Moreover, it has not been clearlyshown that scrapie can be transmitted to cat-tle.) Since scrapie had been known for some200 years and had never been shown to betransmitted to humans, the Southwood Com-mittee reported that although transmission ofBSE to humans was possible and could not beruled out, the chances of it happening were‘remote’. The British Government took upthis advice which was a relief to the farmers,and the general picture was that humans weresafe from BSE especially since measures hadbeen taken to ban the use of MBM for cattleand sheep although not for pigs and chickens(this did not come until 1996; the delay proba-bly caused some problems in that the manu-facturers of MBM did not realise how impor-tant it was to disinfect their machinery andsome of the still suspect MBM meant for poul-try probably was fed to cattle). Soon after, thehuman consumption of brain, spinal cord andother offal was banned and slaughterhouseswere told to remove all spinal cords. Becausethe chance of transmission to humans wasregarded as remote it is clear that some of therecommended precautions were regardedmerely as ‘window-dressing’ and were not ful-ly implemented.

The number of cases of BSE rose dramati-cally as shown in figure 1 [5], the final total

Fig. 1. Number of cases by year of clinical onset ofBSE. a From 1986 to 1997, the latter number (3,592)involving some estimates. b The predictions of casesby year for 1997–2001 assuming no selective cull isimplemented. A further cull would be expected toreduce the numbers further.


being about 180,000. It is estimated thatabout 1 million cattle have been infected inthe UK with several hundred cases in Switzer-land and a few elsewhere. In addition to theslaughter of animals showing symptoms therehas been the slaughter of older but apparentlyhealthy animals in affected herds so that in allsome two million cattle will have been slaugh-tered. The British Government has had to paya compensation to the farmers which has costmore than a billion pounds sterling. It is esti-mated that about 54,000 infected animalshave entered the human food chain. This cal-culation takes into account the large numberof infected animals even after the implemen-tation of the ban on MBM. The measures tak-en to control BSE in cattle have been success-ful and it is slowly dying out. In 1998 it is pre-dicted that there will be 1,714 cases, with 641in 1999 and 235 in the year 2000. Such esti-mates in the past have had a good record ofaccuracy. There is some evidence of maternaltransmission when birth is close to the onsetof BSE in the mother but the level of its occur-rence is unlikely to be able to sustain the epi-demic.

Transmission of BSE to Other SpeciesIncluding ManAfter 1987, cases of BSE appeared in var-

ious other species of animals, particularlyamong the various ungulates in the zoos andalso a few cases among cats (from 1991). Theincidence in cats caused particular concernfor it was the first case of transmission to ameat-eating animal rather than a herbivore.This strengthened the concern about the pos-sible transmission to humans. ConcerningCJD, apart from kuru three different kindscan be discerned: First, there is the ‘sporadic’which is the most common and affects indi-viduals, usually in later life, for no known rea-son. Those affected reside world-wide andthere has been no evidence of a recent epi-

demic in the UK. Second, the ‘familial’, this isan autosomal dominant disease which is rath-er rare. A good deal of genetic work has beendone to understand this. Third, the ‘iatrogen-ic’ which has been caused by the administra-tion of growth hormone preparations madefrom a pool of 3,000 cadaver brains in theUSA to children with problems of growth.There has been the emergence of CJD in chil-dren as a result of such treatment in severalcountries. In the UK, 1,908 children weretreated and there have been 10 cases of CJD.The incidence of the various kinds of CJD inthe UK is shown in figure 2. We can concludethat until 1995 the incidence of CJD in theUK was comprehended in terms of the inter-national scenario.

Later a different kind of CJD was toemerge. In August 1995 the first young per-son, then aged 19, died of CJD followed byseveral others who were aged about 29. Thebrains of these patients had a pathology whichclosely resembled that of cattle with BSE andwas quite different from those who had diedof sporadic CJD. In March 1996 when 10young people had died of CJD the BritishGovernment took the advice of its scientificadvisory committee that we were witnessingthe emergence of a ‘new variant’ of CJD(nvCJD) and that it seemed likely that thishad arisen by the transmission of BSE as aresult of the consumption of infected beef.There was also the worry that farm workerswere particularly susceptible when the deathsof 3 farmers from CJD were reported. It wassubsequently shown that the incidence amongfarmers was no higher in the UK than else-where and subsequent scientific work has dis-counted this particular concern, for the farm-ers had not died of nvCJD. In view of the longincubation period of 5–10 years for nvCJD,based largely on the assumption that thegreatest chance of people eating infected beefwas between 1980 and 1988 when the ban on



Fig. 2. Deaths from CJD in England and Wales 1st January 1970–30th April 1995.g = Sporadic; i = familial; W = iatrogenic.

MBM was instituted, no sound estimate couldbe given of the likely scale of the epidemic ofnvCJD, but the possibility was mentionedthat it could run into thousands of cases. For-tunately, so far the worst predictions have notbeen fulfilled since the number of new cases isabout 1 per month with a total to date of 23.All the cases except 1 are in the UK, theexception being in France.

The Possible Origin of BSEI have indicated above that initially the

cause of BSE was thought to be the transfer ofscrapie to cattle. More recently it has beenshown that BSE can be experimentally trans-ferred to sheep who then show symptomsmore akin to those of BSE than scrapie. Thusscrapie and BSE are two different diseases.BSE in cattle and nvCJD in humans bear thehallmarks of BSE rather than scrapie and Iwill mention scientific evidence to supportthis view. Thus it now seems more likely thatBSE arose as a result of an unusual event – the

mutation in a cow which developed BSE andthat this cow was used for the manufacture ofMBM which was fed back to cattle. The aeti-ology seems similar to that ascribed to kuru.

More Recent PrecautionsAlthough no wild sheep has been found

with BSE the possibility exists that some willbe found so that in the meantime sheep brainsand if possible spinal cord, which is difficultto dissect, have been banned for human con-sumption. Although the neuropathologies donot have an immunological pathogenesisthere is evidence that the immune system isimportant for transporting infection from theperiphery to the brain [6]. Mice that are genet-ically immunodeficient (either SCID mice orRAG knockout mice) cannot be infected byperipheral injection with the infective agentbut can be so infected by injection directlyinto the brain. The prospect has been men-tioned of removing white cells from bloodbefore administration but this would be ex-


tremely difficult and expensive. Blood takenfrom donors will not in the future be pooled.Pooled blood has been used as a source ofserum albumin used in various vaccines. Forthis reason serum obtained from pooled bloodin Britain is no longer being used for the prep-aration of medical products. In the meantimethe message is that blood must be considered‘dirty’ unless it is your own.

Experiments to Discover the Nature ofthe Infective Agent

Some Characteristics of the AgentFollowing the discovery in 1961 that the

scrapie agent could be transmitted to mice,rapid progress was made in determining thecharacteristics of the infective agent. Theagent was resistant to formalin and seemed tohave the size of a small virus, e.g. the picor-navirus. However, the results of irradiationindicated that the scrapie agent was muchsmaller than any known virus and corre-sponded more closely to plant viroids whichonly contain RNA but unlike them the agentwas resistant to nucleases. These were majorfindings and to the present day no one hasbeen able to demonstrate that a nucleic acid isassociated with the infectivity of the scrapieagent. Another important characteristic of thescrapie agent was its resistance to heat. Thusinfectivity was retained even if the brain wasboiled but infectivity was destroyed at tem-peratures above 121°C. No doubt this findingis relevant to the changes in the method ofpreparation of MBM.

Prusiner [7] in California worked with Syr-ian golden hamster-adapted scrapie where theincubation period was shorter than in themouse and the amount of scrapie agent inclinical brain was at least tenfold greater. Hecame to the conclusion that the main compo-nent of the scrapie agent was a hydrophobic

protein which polymerized easily. He coinedthe name ‘prion’ (proteinaceous infective par-ticle) [7] and subsequently isolated proteinfrom scrapie brain which he claimed was themajor prion protein. Thus emerged the so-called ‘protein-only hypothesis’ for whichPrusiner was awarded the Nobel prize in1997.

Another characteristic of the scrapie agenthas been the claim that it is partially resistantto breakdown to amino acids by proteolyticenzymes with no loss of infectivity being in-volved. It is true that it is largely resistant tothe proteinase K which is a fungal enzymemuch favoured by protein chemists and refer-ence will be made to this later. This findinghas been linked to the fact that in the trans-mission of BSE the active agent is not de-stroyed in the gastro-intestinal tract and ru-men of sheep and cattle, and that the agent isresistant to the proteolytic enzymes therein.Against this is the fact that sheep and cattleare herbivores and may not be able to breakdown animal protein in the same way as car-nivores, so this conclusion seems to be un-proven. If so, it may be fortunate that man isan omnivore and it is possible that we mayusually be able to break down the prion pro-tein.

The Structure of PrionsThe primary structure of the biologically

active prion was determined and is given infigure 3. This includes a signal peptide at theN-terminus which is lacking in the matureprotein. At the C-terminus there is a glycosyl-phosphatidylinositol (GPI) anchor which isused for attachment to the plasma membrane.The active prion which has an Mr of 33–35 kis degraded by proteinase K to a protein withan Mr of 27–30 k, as a result of the removal ofsome 60 amino acid residues. An oligonucleo-tide probe was synthesized to correspond tothe N-terminus of this protein. This probe



Fig. 3. Schematic representa-tion of the 230-amino acid back-bone of the human PrP proteinshowing sites of mutation associat-ed with the occurrence of GSS atcodons 102, 105, 117, 198 and 217.The signal peptide at the N-termin-us is not present in the mature pro-tein. At the C-terminus a 23-aminoacid residue is replaced by a GPImembrane anchor labelled glyco-lipid. The octapeptide repeat re-gion is shown with the normal fiverepeats. The different symbols in-volved the prediction of secondarystructure before this was deter-mined experimentally. From ref.24.

was used on the cDNA from hamster brainand an oligonucleotide for a 27–30 k proteindetected. Southern blotting gave a single genewith the same restriction patterns in normaland scrapie-infected brain DNA. Similar re-sults were obtained in mice and humanswhere the gene concerned is located on theshort arm of chromosome 20. PrP-relatedmRNA at similar levels was found in normalscrapie hamster brain and in many other nor-

mal tissues. By the use of an antiserum againstPrP 27–30 k, a PrP-related protein was de-tected in crude extracts of infected brain andto a lesser extent in normal brain. ProteinaseK treatment yielded PrP 27–30 k in infectedbrain extracts, but the protein was completelydegraded in normal brain. This establishedthat there was a normal protein in many tis-sues which had the same properties as the bio-logically active prion except that it had no


Fig. 4. Schemes for the conversion of PrPCC to PrPSc. a One hypothesis predicts that anabnormal protein PrPSc contacts its normal twin PrPC and changes it to an abnormal form.b An alternative hypothesis. In this a ‘seed’ is needed so that infection by PrPSc seeds rapidpolymerization of the molecules leading to self-replication of the infectious agent. After Mes-tel [25].

ba



scrapie activity and was completely degradedby proteinase K. The normal protein was des-ignated PrPC while the scrapie form was des-ignated PrPSc. I will use this nomenclature forthe proteins involved in both scrapie andBSE.

The Origin of the Infective PrionAs a result of the above findings the con-

cept arose that the active prion arose from aconversion of the normal PrPC protein. Thiswas confirmed when ‘knockout’ mice wereproduced that lacked the normal protein.These could not be infected with scrapie butinfectivity was restored when PrPC was rein-troduced, the infectivity being greater in ho-mozygotes than heterozygotes with respect toPrPC [8]. Two schemes for the conversion ofPrP have been put forward as shown in fig-ure 4. In the first it is conjectured that there issome event which causes PrPSc to bind toPrPC, and this sets off a cascade of conversionwhich generates lots of PrPSc. In the second,PrPC and PrPSc are in reversible equilibriumfollowed by an event, a seed, which involvesPrPSc binding to PrPC and a cascade results asin the process of crystallization. In analysingthe conversion of PrPC to PrPSc use is made ofthe increased resistance of PrPSc to the actionof proteinase K.

The Tertiary Structure of the PrionsThere has been an intense interest in the

comparison of the structure of PrPC andPrPSc. Although they both have at least twoglycosylation sites this does not account forthe difference in resistance to proteinase Kalthough there are some interesting aspects towhich I will refer later. The carbohydrate moi-ety may play an important part in plaque for-mation in the brain. There is no difference inthe primary structure of the two proteins soefforts have been directed to a comparison ofthe tertiary structures. Neither protein has

Fig. 5. Models for the tertiary structure of PrPSc onthe left and PrPSc on the right. After Mestel [25].

been crystallized so this work has centred onNMR and physical measurements such asFourier-transform infrared and circular di-chroism. A model of the structure of PrPC isshown in figure 5. It is difficult to produce amodel of PrPSc for the protein is insoluble andalthough it can be denatured it then loses itsinfectivity. However, it is possible to estimatethe content of ·-helix and ß-sheet in the twoforms. PrPC has 43% ·-helix and 3% ß-sheetcompared with 34% ·-helix and 43% ß-sheetfor PrPSc. Based on these results a model forPrPSc has been produced and is shown in fig-ure 5. It is conjectured that the extra contentof ß-sheet is in accord with its greater resis-tance to proteinase K. The complete tertiarystructure of PrPC has been determined byNMR and is shown in figure 6 [9]. This is inclose accord with the model and shows thatthere is a large random coil chain at the N-terminus. It is possible that it is this randomcoil that is converted into a ß-sheet in PrPSc.


Fig. 6. The tertiary structure ofmature murine PrP mPrP(23–231)produced from rDNA in Escheri-chia coli as determined by NMR.The dots represent the 98 residuesof the N-terminal segment 23–120which shows features of a flexi-ble ‘random-coil-like’ polypeptide.From Riek et al. [8].

Model systems for the conversion of pro-teins with similar properties to those de-scribed above have been devised using bothanimal extracts [10] and yeast [11]. So it hasbeen said that prions are not unique to ani-mals. These may indeed be useful model sys-tems but care should be exercised in the use ofthe word prion if all that is measured is merelythe conversion to a proteinase-resistant pro-tein and biological activity is not involved.The fact that the primary structure is not thesole criterion for determining protein tertiarystructure came as something of a surprisewhen molecular chaperones were discovered.It is not surprising, therefore, that there is evi-dence that another protein, protein X in thenomenclature of Prusiner [12], is involved inthe conversion to PrPSc [13], but it is not clearas yet whether a chaperone is involved. In anyevent it is not possible to be sure that PrPSc isresponsible per se for the infectivity for therenatured protein after treatment with dena-

turation agents is not active, nor is that syn-thesized from recombinant rDNA. In summa-ry, it has not so far been possible under invitro conditions to convert PrPC to a formthat possesses infectivity. There are otherworrying aspects of the so-called protein –only hypothesis to which I will return to later.

The Effect of Mutations andPolymorphism of PrionsA lot of work has been done on the effect of

mutations in PrPC, in the incidence of CJD.There is no doubt that familial CJD is associ-ated with certain mutations and in sporadicCJD they make some difference to suscepti-bility, but space limits further discussion ofthis matter [14]. Of more significance is theeffect of polymorphisms in PrPC, particularlythat at position 129, which can be occupied byeither Met or Val. Patients can be homozy-gous for either Met or Val at this position. Allthe cases of nvCJD so far found are homozy-



gous for Met-29, and this is also the mostcommon genotype amongst sporadic cases ofCJD.

The Glycoforms of the PrionsIf PrPSc is isolated and treated with pro-

teinase K and an antiserum is used for a West-ern blot after electrophoresis then three bandsare obtained. The two major bands are glyco-sylated proteins, glycosylated to different ex-tents with sialic acid, and the fastest band isunglycosylated. The rate of migration of theproteins on the gel is also influenced by thepolymorphism at position 129 already de-scribed. Collinge et al. [15] examined theresults of such a procedure on the PrPSc ob-tained after infection of mice under variousconditions. The first results were published inJuly 1996 and were confirmed later. Figure 7summarises the results. These show that thepattern on electrophoresis (called a signature)differs between scrapie and BSE and that thenvCJD patients clearly had an infectionwhich was characteristic of BSE. This was thefirst such result to back up the conclusionsconcerning the origin of nvCJD which hadpreviously depended on the pathology of thebrain [15–17]. The work of Collinge et al. [15]implicating BSE as the cause of nvCJD wassupported by the transmission of BSE to Ma-caque monkeys which produced a neuropa-thology very similar to nvCJD [18].

Strains of Scrapie and BSEDuring the extensive work on scrapie in

sheep it became apparent that there weremany strains of the disease. These were de-fined in terms of the length of incubation, thebehaviour of the animal and the pathology ofthe brain. As many as 20 strains have been sodefined. In a similar manner the CJD surveil-lance group at Edinburgh has characterised allthe cases of BSE both in humans and animalsand has confirmed that all the parameters for

the emergence of nvCJD bear the hallmarksof BSE [19].

If the protein-only hypothesis for the infec-tive agent is correct then the infectivity de-pends on the transmogrification of PrPC toproduce a specific tertiary structure of PrPSc

so that it is argued that the presence of differ-ent strains of either scrapie or BSE impliesthat there must be various structures of PrPSc.It has now been shown that experimentallysheep may be infected with BSE and so thesame PrPC must be capable of transmogrifica-tion into different structures characteristic ofeither scrapie or BSE. What is more, thesespecific PrP structures are transferred to theanimal’s progeny, for the strains are genetical-ly stable. With our present knowledge of pro-tein structure and genetics it is hard to under-stand how this can be so. The results of thepolymorphisms of CJD and the fact that allcases of nvCJD are homozygous for Met-129suggests that such polymorphisms have aninfluence on the ease of conversion of PrPC toPrPSc.

The Production of a Monoclonal Antibodyto PrPSc

A more recent development is the produc-tion by the Zurich group of a monoclonal anti-body which can discriminate between the nor-mal and disease-specific forms of PrP [20].PrP-null mice were immunized with recombi-nant bovine PrP and a monoclonal antibody,15B3, which was isolated, specifically reactedwith bovine, murine or human PrPSc but notPrPC, suggesting that it recognizes an epitopecommon to prions from different species.They also mapped three polypeptide seg-ments in PrP as the 15B3 epitope. Hopefullyby the use of such an antibody ultimately itwill be possible to detect the presence ofPrPBSE in cattle before they show clinicalsymptoms of BSE. There are claims that pa-tients who are developing CJD may have a


Fig. 7. The glycoforms of prions. Western blots of different prion strains show characteris-tic patterns of PrPSc glycoforms which are inherited even when the infectious agent is passedwithin different species. The large arrows indicate successful transmissions. The major glyco-form is shown in black and the little arrows on the Western blots indicate increased mobilitycompared with type 1. The humanized transgenic mice were transgenic for the human PrPgene. From Aguzzi and Weissmann [17].

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brain protein in their serum before the diseaseis confirmed by pathology. This also would beuseful.

The FutureIt will be seen that while the actual epi-

demic of BSE in the UK is rapidly receding ithas left many footprints particularly in termsof the possible epidemic of nvCJD. Biochem-ists find it difficult to accept that prion dis-eases are caused solely by the prion proteinand many remain attracted to the conceptthat a nucleic acid must be involved. It is truethat conclusive proof for the protein-only hy-pothesis has eluded us but against this is thefact that no one has yet identified the involve-ment of a nucleic acid. Another concern isthat although mice injected with an extractfrom BSE-infected cattle brain exhibited neu-rological symptoms and neuronal death, morethan 55% had no detectable PrPBSE. Duringserial passage PrPBSE appeared after the agentadapted to the new host [21]. This supportsthe view that while prions are involved ininfectivity other agents also play a part. Thisview is supported by the fact that the ratio ofPrPSc molecules to infection units is onlyabout 1:100,000, but there is evidence thatthe mouse assay is very insensitive when com-pared with the sensitivity of calves who maysuccumb to as little as 1 g of an extract fromthe brains of BSE-infected cattle. We alsoknow little about the way in which amyloid isformed and virtually nothing about the result-ing striking pathology. Thus there is muchwork remaining concerning the fundamentalscience as has been pointed out in two recentarticles [22, 23].

In terms of animal husbandry it seemsclear that more care must in future be takenconcerning the make-up of animal feedstocks.The general public has been shocked aboutmany of the practices that have recently cometo light. It is also clear that it is unacceptable

for a country like the UK to continue to toler-ate the presence of a disease like scrapie in theanimals used for food even though it appearedto be harmless to man. In this case the fundsfor research were not available from industry,for the profit motive could not sustain suchexpenditure so such research had to dependon government support which in the eventwas parsimonious. The enormous cost of thecompensation for the slaughter of cattle in thecase of BSE is a reminder of the results of suchparsimony.

Various therapies have been suggested, notonly because of the fear of the emergence of anepidemic of nvCJD but also because it is hardto accept that prion diseases are confined totransmissible encephalopathies. One con-cerns the role of normal PrPC. If it has nophysiological role then it has been suggestedthat it might be a good idea by genetic manip-ulation to rear cattle and sheep lacking PrPC

but the cost of such a programme is dauntingeven if it were possible. Another route is toproduce drugs which would inhibit the trans-mogrification of PrPC to an infective form. Inthis respect the model systems in yeast andbacteria that are being studied are particularlyrelevant.


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Note Added in Proof

The total of nvCJD cases is now25 (June 1998).

Bovine Spongiform Encephalopathy (BSE) – Mad Cow Disease

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