Development of a risk assessment for BSE in the aquatic environment

Journal of Applied Microbiology 1998, 84, 467–477

A REVIEW

Development of a risk assessment for BSE in the aquatic environment

P. Gale, C. Young, G. Stanfield and D. OakesWRc plc, Medmenham, Marlow, UK

6500/11/97: received 24 November 1997, revised 21 January 1998 and accepted 26 January 1998

1. Summary, 467 6.4 The biochemical evidence for a threshold2. Introduction, 468 dose, 472

2.1 Problems in developing a risk assessment 7. Inputs of BSE infectivity to the aquaticapproach for a novel pathogenic agent, 468 environment, 473

3. The biological nature of the BSE agent, 468 7.1 Landfills, 4734. Assumptions about the infectivity of BSE- 7.2 Abattoirs, 473

infected bovine brain and spinal cord to humans, 7.3 Rendering plants, 473000 8. Barriers to BSE infectivity in the environment,4.1 The cow-to-man species barrier, 469 4734.2 Each bovine with clinical symptoms 8.1 Dilution as a barrier in the aquatic

contributes 1000 human oral ID50 units, 470 environment, 4735. The nature of BSE agent in the aquatic 8.2 Degradation of BSE infectivity in the aquatic

environment, 470 environment, 4745.1 BSE agent will be bound to particulates and 8.3 Resistance of infectivity to inactivation, 474

suspended solids in the aquatic 9. A quantitative risk assessment for a renderingenvironment, 470 plant disposing of treated effluent to a sub-

5.2 BSE agent will be dispersed and diluted to a surface irrigation system above a chalk aquifer,large degree in the aquatic environment, 470 474

6. The risks from exposures to sub-fractions of an 9.1 Concentrations of BSE infectivity dischargedID50 through drinking water, 471 to the soakaway in the effluent, 4746.1 The number of PrPBSE molecules comprising 9.2 Concentrations of BSE infectivity reaching

a human oral ID50, 471 the aquifer, 4746.2 A dose-response curve for BSE infection, 471 9.3 Risks to consumers’ drinking water from the6.3 Low dose extrapolation suggests that even a aquifer, 475

single BSE prion molecule has a small but 10. Discussion, 475finite probability of initiating infection, 11. Disclaimer, 476472 12. References, 476

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1. SUMMARY

Bovine spongiform encephalopathy (BSE) is believed to betransmitted by the ingestion of proteinaceous agents calledprions which accumulate in the brain and spinal cord ofinfected bovines. Concern has been expressed about the risks

Correspondence to: Dr Paul Gale, WRc plc, Henley Road, Medmenham,Marlow, Buckinghamshire SL7 2HD, UK.

© 1998 The Society for Applied Microbiology

of transmission of BSE to humans through BSE prions dis-charged to the aquatic environment from rendering plants,abattoirs and landfills. The disease-related form of the prionprotein is relatively resistant to degradation, and infectivitydecays rather slowly in the environment. Levels of dis-infection used for drinking water treatment would have littleeffect. This paper presents the assumptions which were usedto model the risks from a rendering plant disposing of cull

468 P. GALE ET AL.

cattle carcasses in the catchment of a chalk aquifer which isused for a drinking water abstraction. The risk assessmentapproach focused on identifying the hydrogeological andphysical barriers which would contribute to preventing BSEinfectivity gaining entry to the aquifer. These barriersincluded inactivation of BSE agent by the rendering process,removal from the effluent by treatment at the plant, filtrationand adsorption in the clay and chalk, and dilution in theground water. The importance in environmental risk assess-ment of the cow-to-man species barrier is considered. Twokey conclusions about the environmental behaviour of theBSE agent are that prion proteins are ‘sticky’ and bind toparticulates, and that the millions of BSE prion moleculescomprising a human oral ID50 are subject to some degree ofdispersion and hence dilution in the environment. Assumingthe rendering plant processes 2000 cull cattle carcasses perweek, the risks to drinking water consumers were estimatedto be remote. Indeed, even using worst case assumptions anindividual would have to consume 2 l d−1 of tap water for 45million years to have a 50% chance of infection throughdrinking water drawn from the aquifer.

2. INTRODUCTION

On 20 March 1996, the UK Government announced thatthere was a possible link between a new and distinct variantof Creutzfeldt-Jakob disease (vCJD) in humans and exposureto the agent that has caused the epidemic of bovine spongi-form encephalopathy (BSE) in cattle. Twenty cases of vCJDhave been recognized in unusually young people in the UK,and a further case has been reported in France. Bruce et al.(1997) showed that both vCJD and BSE are almost certainlycaused by the same agent. The most likely source of exposure(although this has not yet been proved) was through eatingbeef products that included infected offal (brain and spinalcord) before it was banned from human food in late 1989.

To restore consumer confidence in beef and beef-productsat home and abroad, the UK Government introduced in 1996a scheme to cull cattle over the age of 30 months. In 1996, itwas estimated that some 800 000 cattle would be culled in theUK. Although only a small proportion of the national herdwas infected with BSE in 1996 (Anderson et al. 1996), con-cerns were expressed about the potential hazards from BSEagent entering the aquatic environment, e.g. from abattoirs,rendering plants and landfills receiving culled cattle carcasses.

The lack of experimental data on the behaviour of BSEagent in the aquatic environment presented a considerablechallenge for the development of the risk assessmentapproach. This paper describes how a risk assessmentapproach can be developed for a novel pathogenic agent. Italso considers how accurate a risk assessment has to be forpractical use. The principles of the approach used here could

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 467–477

be applied to assess the ‘emergence potential’ of other little-studied pathogens of relevance to the UK water industry.

2.1 Problems in developing a risk assessmentapproach for a novel pathogenic agent

The main problem in developing a risk assessment approachfor a novel pathogenic agent is the lack of specific information.In the case of BSE agent, there were:1 no dose–response data for humans (indeed, the link

between BSE and vCJD has only recently been established)and no dose–response curve or information on the risksfrom ingestion of small fractions of an ID50;

2 no information on the behaviour of the BSE agent in theaquatic environment;

3 no information on the effectiveness of different barriers inattenuating the movement of the BSE agent into drinkingwater supplies;

4 limited information on the decay of BSE infectivity in theenvironment and some suggestions that prion proteins areimmortal.It was therefore necessary to derive information on each

of these for the purposes of developing a quantitative riskassessment.

3. THE BIOLOGICAL NATURE OF THE BSEAGENT

There is growing evidence to support the ‘protein-only’hypothesis that the transmission of mammalian transmissiblespongiform encephalopathies (BSE in cattle, scrapie in sheep,CJD and kuru in humans) depends on a protein agent calledthe prion protein or PrP (Prusiner 1991; Prusiner 1996) ratherthan a nucleic acid-containing structure. During progressionof the disease, the disease-related form of the prion proteinaccumulates in the brain and spinal cord of the infectedanimal. The nomenclature for the disease-related form of theprion reflects the species of origin, i.e. PrPSc for scrapie insheep, PrPBSE for BSE in cattle, and PrPCJD for CJD inhumans. The disease-related forms are also referred to asPrP-res to indicate their resistance to proteases. Accordingto the ‘protein-only’ hypothesis (Weissmann 1995; Mastersand Beyreuther 1997), initiation and progression of infectionare mediated by PrP-res which converts the cellular form ofthe prion protein (called PrPC (C for cellular) or PrP-sen(sensitive to proteases)) in the tissues into more PrP-res.This transformation involves a shift in the three-dimensionalstructure of the protein (Masters and Beyreuther 1997) witha change in the relative proportions of a-helix and b-sheet(Prusiner 1996). Since the structure of a protein determinesits function, it is not surprising that PrP-res and PrPC havevery different properties. The PrPC conformer is protease-sensitive and will be rapidly degraded in the environment

BSE RISK ASSESSMENT 469

before it can be converted into more PrP-res. Therefore, it isconcluded that the possibility of the BSE agent replicatingfurther in the aquatic environment is remote. PrP-res,however, is relatively resistant to degradation by proteolyticenzymes, and scrapie infectivity has been shown to persistfor several years in the environment (Brown and Gajdusek1991).

The three-dimensional protein structure of PrPC has beendetermined (Riek et al. 1996). However, there is very littlestructural information for PrP-res itself. There is evidence tosuggest that PrPSc exists in scrapie-infected hamster brains asan integral membrane protein within the cellular membranes(Meyer et al. 1986; Gabizon et al. 1987). However, it appearsto be able to exist in several different forms. In particular,PrPSc can form long rods under certain extraction conditions(Prusiner et al. 1983). Large plaques containing aggregatedPrP-res are rare in brains of BSE-infected bovines (Wells andWilesmith 1995), although they are a feature of some priondiseases including vCJD in humans (Will et al. 1996; Almondand Pattison 1997).

4. ASSUMPTIONS ABOUT THE INFECTIVITYOF BSE-INFECTED BOVINE BRAIN ANDSPINAL CORD TO HUMANS

Infectivity titres in bovine tissues are measured by titrationin mice after intracerebral or intraperitoneal injection (Kim-berlin 1996). Brain stem from confirmed cases of BSE hasthe highest level of infectivity with 5·2 log10 ID50 units g−1

(by mouse assay). Infectivity levels in whole brain extractsfrom confirmed BSE cases are lower, measuring between 2·7and 3·6 log10 ID50 units g−1 (Kimberlin 1996) by mouse brainbioassay. The actual levels of infectivity in bovine brain willbe underestimated by mouse bioassays because of the cattle-to-mouse species barrier effect. Indeed, Kimberlin (1996)writes that if bioassays were carried out intracerebrally incattle, then the BSE titre in the bovine brain would be about6·0 log10 ID50 units g−1, suggesting that the cattle-to-mousespecies barrier is a factor of about 1000.

The oral/intragastric route of infection is less efficient thanthe intracerebral and intraperitoneal routes (Kimberlin 1996).The magnitude of the intracerebral/oral difference is about100 000 for transmission of BSE to mice. Experiments inwhich mice were fed BSE-infected bovine brain have shownthe oral ID50 to be 6·3 g (Kimberlin 1996). In experimentswith sheep of a certain PrP genotype, an oral challenge of0·5 g of BSE-infected bovine brain homogenate infected oneout of the six sheep, with an incubation period of about twoyears (Foster et al. 1996). It should be noted that hamstersand at least one PrP genotype of sheep appear to be resistantto BSE (see Raymond et al. 1997).


4.1 The cow-to-man species barrier

It is only very recently that scientific evidence has beenobtained to prove that BSE in cattle and vCJD in humansare caused by the same agent (Bruce et al. 1997; Almond andPattison 1997). Not surprisingly, there are no dose–responsedata available for BSE in humans. A critical factor in deter-mining the risk that the BSE agent presents to man is themagnitude of the cow-to-man species barrier. Experimentsin which the human PrP gene has been expressed in micesuggest that some humans, at least, are more resistant to BSEagent than mice and that some humans may even be totallyresistant (Hope 1995; Collinge et al. 1995). Indeed, whiletransgenic mice expressing human PrPC were rapidly infectedby intracerebral challenge of CJD-infected human brain, noneof those mice to date (Hope, personal communication) hasbeen infected by challenge with BSE-infected bovine brain.Wild type mice expressing murine PrPC were infected byintracerebral challenge with BSE-infected bovine brain.Results of in vitro experiments in which human PrPC waschallenged with BSE prions, PrPBSE, are also consistent witha high cow-to-man species barrier (Raymond et al. 1997). Aworst case therefore would be to assume that the cattle-to-man species barrier is no higher than the barrier betweencattle and mice (which is a factor of about 1000), and thatman is no more resistant than mice to BSE infection (Kim-berlin 1996). This would mean that as a worst case, the humanoral ID50 would also be about 6 g of infected brain from abovine with clinical symptoms.

An additional factor to consider when defining the mag-nitude of the cow-to-man species barrier arises from dif-ferences in genetic susceptibility between individual humans.Polymorphisms at codon 129 of the human prion gene appearto be important in this respect. Human (hu) PrP has twocommon allelic forms that encode either methionine (huPrP-met) or valine (huPrP-val) at codon 129. Human individualshomozygous for huPrP-val are the most susceptible genotypefollowing exposure to human prions (Collinge et al. 1995),for example, from administration of growth hormoneextracted from human pituitary glands (Collinge et al. 1991).In contrast, evidence would suggest that humans homozygousfor huPrP-met are the most sensitive genotype to BSE prions.Thus, Will et al. (1996) reported that all eight cases of the newvariant CJD investigated were homozygous for methionine atcodon 129. Furthermore, Raymond et al. (1997) have shown,using cell-free systems, that huPrP-met is converted by BSEprions, PrPBSE, into PrP-res approximately threefold moreefficiently than huPrP-val.

The human PrP transgene used in the mice challengedwith BSE-infected bovine brain in the experiments of Col-linge et al. (1995) encoded valine at residue 129 and may not,therefore, give a worst-case representation of the infectivityof BSE to humans. However, to assume there is no species

470 P. GALE ET AL.

barrier for humans homozygous for methionine at codon129 is almost certainly too pessimistic because huPrP-met isconverted by PrPBSE into PrP-res about 10-fold less efficientlythan bovine PrP-sen (Raymond et al. 1997). Collinge et al.(1995) write that it will be important to repeat the studieswith mice expressing huPrP-met, and also with transgenicheterozygotes, to determine whether heterozygosity at codon129 protects against the transmission of BSE, as it does againstthe development or rate of progression of some types ofhuman prion diseases. A comparison of the percentage con-version efficiencies for PrP-sen into PrP-res by PrPBSE (Ray-mond et al. 1997) suggests that murine PrP-sen is moreefficiently converted than both huPrP-met and huPrP-val.This is consistent with humans homozygous for methionineat codon 129 being at least as resistant to BSE as mice, i.e.using a cow-to-man species barrier of 1000 is indeed a worstcase assumption. Furthermore, data from Raymond et al.(1997) show that the conversion efficiencies of hu-PrP-metand huPrP-val into PrP-res by BSE prions are no greaterthan by PrPSc from scrapie-infected sheep, which is a sourceof no measurable risk to the human population. Indeed, Ray-mond et al. (1997) conclude that BSE is no more inherentlytransmissible to humans than sheep scrapie.

4.2 Each bovine with clinical symptoms contributes1000 human oral ID 50 units

For the purposes of the risk assessment here, a mass of 1 g ofBSE-infected bovine brain/spinal cord was used as thehuman oral ID50. Bovine brain and spinal cord weigh about750 g in total, although a more conservative value of 1000 gwas assumed for the risk assessment. It was concluded, there-fore, that each bovine infected with BSE contributes 1000human oral ID50 units. This figure applies to cattle with fullclinical symptoms of BSE but was also used in the riskassessment here for infected cattle not exhibiting full clinicalsymptoms. Those cattle have much lower levels of infectivityand this infectivity figure would therefore represent anextreme worst case.

5. THE NATURE OF BSE AGENT IN THEAQUATIC ENVIRONMENT

The aggregation state of the PrPSc molecules in the aquaticenvironment is an important consideration in risk assessmentbecause it defines whether the molecules are freely dispersed,bound to particulates, or associated with each other.

5.1 BSE agent will be bound to particulates andsuspended solids in the aquatic environment

Infectivity is associated with biological membranes and infec-tious prion particles are insoluble (Prusiner 1991). This is


responsible for many of the difficulties which have beenencountered during attempts to purify and characterize theinfectious agent in scrapie. Indeed, infectivity is sometimesdescribed as ‘sticky’. The reasons for this ‘stickiness’ and theinsolubility of PrPSc are explained by the biochemistry andbiophysical properties of the PrPSc molecule and, in particu-lar, its hydrophobicity. The PrPSc molecule is amphipathic(Meyer et al. 1986). This means that both hydrophilic (water-loving) and hydrophobic (water-hating) regions exist on eachand every PrPSc molecule. Its amphipathic properties accountfor both its binding to cellular membranes and its aggregationinto rods (Meyer et al. 1986). In view of their hydrophobicmoieties, molecules such as prions cannot exist free in water.Indeed, through the maximization of hydrophobic inter-actions, amphipathic molecules attach with great tenacity toother molecules to the exclusion of water. It is thereforebelieved that any BSE agent in the aquatic environment willbe bound to solids and particles. In whole cattle carcasses,and rendered cattle carcasses, those solids and particles towhich PrPBSE attaches will be proteinaceous and carbohydratein nature. Through decay of those components in the aquaticenvironment, the bound prions will be released and hencedispersed (molecular dispersion), immediately attaching toother particles. Thus, prions will be subject to progressivedispersion and dilution in the aquatic environment whileremaining bound to particulates at all times.

5.2 BSE agent will be dispersed and diluted to a largedegree in the aquatic environment

Amyloid plaques containing large aggregates of PrP-res fea-ture prominently in the brains of some animal species infectedwith transmissible spongiform encephalopathy. However,such plaques are not a feature of all species (Prusiner 1996).Indeed, such plaques are rarely observed in the brains ofBSE-infected bovines (Wells and Wilesmith 1995). The prob-ability of ingesting a dose of BSE prions approaching thehuman oral ID50 in a single exposure through the aquaticenvironment is remote because of molecular dispersion anddilution of the prions. There is no obvious mechanismwhereby all the prions in the millions of neurones in 1 g ofbovine brain stem could aggregate to form a single particlecomprising a human oral ID50 unit. PrPSc under certainextraction conditions has been observed to aggregate intolong rods which become entangled to form clumps. Thelargest clump reported in scrapie-infected hamster brainextracts contained some 100 000 PrPSc molecules (Prusineret al. 1983), which is small compared with the estimated 1013

BSE prions in a human oral ID50 (see below).It is interesting to note that dispersion of the PrPSc from

those rods into lipid complexes results in a 10- to 100-foldincrease in scrapie infectivity (Gabizon et al. 1987). Whetherthe lipid component is responsible for the increase in infec-


tivity is not clear. However, in intact bovine brains, PrPBSE

will be associated with membrane lipids (Meyer et al. 1986)and during decay of the bovine carcass, those membranelipids will be rapidly degraded. This may lead to a decreasein the infectivity of BSE agent in the aquatic environmentand could account for some of the loss of infectivity in scrapieagent in the soil environment reported by Brown andGajdusek (1991).

6. THE RISKS FROM EXPOSURES TO SUB-FRACTIONS OF AN ID 50 THROUGHDRINKING WATER

Risk assessment approaches for bovine products model theprobability of exposure to a whole ID50 unit and assume thatthe ID50 unit is indivisible, i.e. the particle/infectivity ratiois one (Kimberlin 1996). In the aquatic environment, thedilution and dispersion effects on the infected bovine material(and hence, the component PrPBSE molecules) will eliminatethe chance of exposure to a whole ID50 in a single challenge(for example, through a glass of drinking water). Therefore,environmental risk assessment approaches should considerthe risks from ingestion of sub-fractions of an oral ID50. Thedose is the total number of PrPBSE molecules ingested.

6.1 The number of PrP BSE molecules comprising ahuman oral ID 50

About 100 000 PrPSc molecules make up an intracerebral ID50

in experimental scrapie models which are species adapted(McKinley et al. 1983). Assuming a cow-to-man species bar-rier of 1000, and that the oral route is 105-fold less efficientthan intracerebral challenge (Kimberlin 1996), the humanoral ID50 would be 1013 PrPBSE molecules. Thus, accordingto this calculation, 1 g of brain from a confirmed bovine wouldcontain in the region of 1013 BSE prions. Some assurancethat this conclusion is reasonable comes from reports that 1 gof scrapie-infected hamster brain contains about 10mg ofPrPSc (Meyer et al. 1986). Using a molecular weight for PrPSc

of 33 kDa (Prusiner 1996) and a value of 6·022× 1023 mol−1

for the Avogadro constant (Atkins 1978), it may be calculatedthat 10mg of PrPSc is equivalent to 1014 PrPSc molecules.

Compared with conventional water-borne pathogens, thetransmission of BSE to humans requires a huge number ofparticles. For comparison, the ID50 for calf Cryptosporidiumparvum in humans is about 150 oocysts (calculated from thedose–response curve of Haas et al. (1996) applied to thehuman infectivity data of DuPont et al. (1995)) and forGiardia lamblia, the ID50 is about 35 cysts (using thedose–response curve from Rose and Gerba 1991).


6.2 A dose–response curve for BSE infection

To quantify the risks from minute sub-fractions of an oralID50 requires a dose–response curve. Titration data for BSE-infected bovine brain into mice (intracerebral challenge) showthat the probability of infection falls off rapidly at doses belowthe ID50 (Fig. 1). Indeed, while a 10−2 dilution of bovinebrain homogenate infected nine out of 14 mice, a 10−4 dilutioninfected 0 out of 13 mice (Taylor et al. 1995). However, itwould be complacent to assume that doses below an ID50

present zero risk in quantitative risk assessment. Indeed, bycalculating binomial confidence limits, the risk of infectionby the 10−4 dilution, for which 0/13 mice were observed tobe infected, has a 5% chance of being higher than 0·206.

The very small risks from exposure to minute fractions ofan ID50 necessitate trials involving prohibitively large num-bers of animals. The same problem prevents practicablemeasurements of risks from ingestion of doses of single water-borne pathogens in human volunteer experiments (Gale et al.1997). However, current risk assessment models for water-borne pathogens predict exposures to doses of just one patho-gen person−1 d−1 (Gale 1996). To overcome this problem,dose–response curves for water-borne pathogens are extrapo-lated to lower doses than experimental data are currentlyavailable for (Haas 1983; Haas et al. 1993). Here, a similarapproach is used to estimate the risks from exposures to lowdoses of BSE infectivity.

Dose–response curves for water-borne pathogens are typi-cally sigmoidal in shape (Rose and Gerba 1991; Haas et al.1993). This, however, is not necessarily evidence of a thresh-old because the dose-axis is plotted on a log scale. Themagnitudes of the risks of infection presented by oral dosesof water-borne pathogens comprising sub-fractions of an ID50

depend on the shape of the sigmoidal curve. In Fig. 1, dose–response curves modelled using parameters reported by Rose

Fig. 1 Comparison of dose–response curves for two water-bornepathogens (Rose and Gerba 1991) with dose–response data for BSEinfectivity in mice (Ž). BSE data from Taylor et al. (1995)

472 P. GALE ET AL.

and Gerba (1991) for Salmonella typhi (b-Poisson model) andG. lamblia (exponential model) are superimposed on the datafor BSE infectivity titrations in mice (Taylor et al. 1995).It should be noted that the two curves have been shiftedhorizontally so that their ID50s match that of the BSE dataset. This aids visual comparison of the shapes of the curvesand in particular, how the risk of infection drops off withexposure to 10-fold dilutions of the ID50. In terms of theshape of the curves, the BSE data appear to fit the G. lambliacurve better than the curve for Salm. typhi. This wouldsuggest that the negative exponential dose–response curvefor G. lamblia could serve as a model to test assumptionsabout the risks from exposures to small sub-fractions of anoral ID50 of BSE agent.

The mathematical form of the negative exponential model(Haas 1983) is written as:

P� 1− e−rN (1)

where P is the probability of infection from an exposure ofN pathogens and r is a parameter specific for each species ofpathogen and the exposed human population. For low doses,it may be shown that Equation 1 approximates to

P� rN (2)

suggesting that there is a direct linear relationship betweenthe probability of infection and the dose (Haas 1983). Com-puter simulations showed that for doses below the ID50, therisk predicted by the dose–response model for G. lamblia(r� 0·0199; Rose and Gerba 1991) approximates that cal-culated as 0·5× fraction of ID50 ingested. This suggests thatit is justifiable to calculate the risk from ingestion of minutefractions of a human oral ID50 unit as half of the fraction ofthe oral ID50 ingested.

6.3 Low dose extrapolation suggests that even asingle BSE prion molecule has a small but finiteprobability of initiating infection

Using this approach, the risks of infection from oral ingestionof fractions of an oral ID50 are calculated in Table 1. It should

Table 1 Risks of infection to humans from single exposures tofractions of an oral ID50 assuming a linear dose–response relationship—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Fraction of Number of PrPBSE Risk ofan ID50 molecules infection—–––––––––––––––––––––––––––––––––––––––––––––––––––––1 1013 0·5

10−5 108 0·5 × 10−5

10−8 100 000 0·5 × 10−8

10−13 1 0·5 × 10−13

—–––––––––––––––––––––––––––––––––––––––––––––––––––––


be noted that a minute but finite risk is associated even witha dose of just a single PrPBSE molecule. The risk to humanhealth from a dose of 105 molecules is worthwhile consideringbecause it corresponds to the largest single aggregate of PrPSc

supported by biochemical evidence (Prusiner et al. 1983). Thepredicted risk is very small. Thus, if each of 200 millionpeople ingested a clump of 105 BSE prion molecules, thenone person would be infected.

6.4 The biochemical evidence for a threshold dose

Low dose extrapolation assumes that doses of a single patho-gen, or doses of a minute sub-fraction of an oral ID50, areindeed capable of causing infection. In this respect, low doseextrapolation presents a worst case, particularly if the negativeexponential mathematical model is used for the dose–response curve instead of the log-probit model (Haas 1983).The risks presented to humans by the minute fractions of anID50 calculated in Table 1 would be smaller still if there werea requirement for a minimum number of molecules, i.e. athreshold dose. One biochemical model suggests that singlePrP-res molecules are unable to convert PrPC into PrP-res.Indeed, that model postulates that an aggregate of PrP-res isneeded to seed conversion (Bessen 1996) through a crys-tallization process involving PrP-res amyloid formation.Assuming that the cow-to-man species barrier is 1000 forBSE and that the oral route is 100 000-fold less efficient thanthe intracerebral route for transmission, this evidence of athreshold would suggest that the risks presented by oralchallenges as large as 108 PrPBSE molecules (10−5 humanoral ID50) could be considerably smaller than the value of0·5× 10−5 predicted by low dose extrapolation assuminga linear dose–response relationship (Table 1). Indeed, thepresence of a threshold raises the possibility that oral inges-tion of small numbers of BSE prions could present zero risk.

Prusiner (1996) does not appear to be convinced by therequirement for crystallization involving seeds or aggregatesof PrPSc for prion replication, and as evidence against this,presents the case of genetically mutated prions causingdisease. This, however, need not be inconsistent with therequirement for a minimum number of prions for initiationof infection. Thus, a single cell with a mutated PrP gene willproduce millions of copies of the mutated PrPC protein whichare localized in the intracellular membranes (Meyer et al.1986). PrPSc is also membrane bound (Meyer et al. 1986) andGabizon et al. (1987) have suggested that it may exist as anintegral membrane protein. Integral membrane proteins canaggregate with each other within the plane of the membraneas shown for bacteriorhodopsin (Sternberg et al. 1989). Itcould be speculated that a co-operative effect between themany closely packed PrP molecules within the lipid bilayermembrane is required to trigger conversion of PrPC to PrP-res in the case of mutated prion proteins. In this case, a


minimum number of molecules or a threshold would berequired for the co-operative effect to initiate conversion ofPrPC to PrP-res. Evidence that the infectivity of prion rodsis increased 10- to 100-fold by dispersion into a lipid environ-ment (Gabizon et al. 1987) is consistent with the lipid bilayercontributing to the infection mechanism.

7. INPUTS OF BSE INFECTIVITY TO THEAQUATIC ENVIRONMENT

Since 1991, all confirmed or suspected cases of BSE-infectedcattle in UK herds have been destroyed by MAFF vets bylethal injection and their carcasses incinerated. This barriergreatly reduces the input of BSE infectivity entering theenvironment because infected bovines without clinical symp-toms of BSE have much lower levels of infectivity thanthose in the later stages of the disease and exhibiting clinicalsymptoms.

7.1 Landfills

Up to 1991, some BSE-infected cattle carcasses were disposedof in landfill sites. On decay of the carcass, BSE infectivitywill bind to particulate matter. With the possible exceptionof flows in the vicinity of extraction wells, the rate of flow ofleachate through landfilled wastes is generally slow and non-turbulent, with the result that particulate material is unlikelyto be taken up into suspension. It is therefore consideredunlikely that leachate would contain much PrPBSE, mostremaining bound to solid matter and settled sediments inthe landfill. Landfill leachate contains a complex mixture ofinorganic ions and organic molecules, in particular, volatilefatty acids. It is unlikely that those chemical componentswould affect or alter the solubility properties of PrPBSE. Over-all, it is considered that the potential for release of prions tothe aquatic environment via leachate is small.

7.2 Abattoirs

Three barriers are in place at abattoirs to prevent entry ofBSE infectivity from slaughtered cattle to the sewer. First,cattle which are symptomatic on arrival are not slaughteredat abattoirs. Second, organs known to contain infectivity,including the brain and spinal cord, are removed as specifiedbovine material (SBM) and disposed of at rendering plants.Third, 4mm screens have been put across drains in abattoirsto prevent particles larger than 0·1 g entering the sewer.

7.3 Rendering plants

Rendering is a process whereby remains of animal carcassesdiscarded by abattoirs are cooked. Under the Government’sOver 30 Month Scheme (OTMS) for the cull of cattle to


eradicate BSE from the UK herd, all cattle components arerendered. The process produces three products: meat andbone meal (MBM), tallow (from the fats) and water. Ren-dering has been shown to destroy at least 98% of BSE infec-tivity (Taylor et al. 1995). Residual BSE infectivity has beenfound in the MBM but not in the tallow.

8. BARRIERS TO BSE INFECTIVITY IN THEENVIRONMENT

The effectiveness of various barriers at protecting theenvironment against BSE agent may be determined by thefundamental assumption that any BSE agent will be bound toparticulates. Therefore, any process which removes particlespresents a barrier to the transmission of BSE agent throughthe aquatic environment. Those processes which will atten-uate the movement of prions into drinking water suppliesinclude:

, treatment processes which remove suspended solids fromaqueous effluents at abattoirs and rendering plants;

, sewage treatment processes;, clay subsoils and chalk above aquifers;, drinking water treatment processes (chemical coagulation/

filtration, granular activated carbon).

8.1 Dilution as a barrier in the aquatic environment

The degree of dilution is an important factor in calculatingthe concentration of PrPBSE in, for example, a drinking watersupply. Using reported data for daily intake of tap water byconsumers, the exposures to consumers may then be calcu-lated. Dilution therefore serves as a barrier by diminishingthe net PrPBSE load ingested by the population. The impactof dilution on the societal risk across the drinking waterpopulation is not influenced by the aggregation state (degreeof molecular dispersion) of the PrPBSE, because a linear lowdose extrapolation (according to the negative exponentialmodel) is used here in this risk assessment (Equation 2).Thus, for example, if one infectious dose unit (IDU) werediluted into 100 l of water and 100 consumers each drank 1 l,then one consumer would be infected irrespective of whetherall 100 consumers were exposed to 0·01 of the IDU or justone consumer ingested the whole IDU in a 1 l sample. Thisenables the risk assessment to be simplified into calculatingthe net ID50 loading on the human population through drink-ing water consumption.

The magnitude of the barrier presented by molecular dis-persion and dilution of the prions comprising an ID50 is likelyto be much greater than assumed in this risk assessmentbecause1 there is evidence for a threshold dose of prions (see above)

such that the societal risk to 100 consumers each ingesting

474 P. GALE ET AL.

0·01 IDU would be lower than the risk to one consumeringesting the whole IDU;

2 it is unlikely that an ID50 can be accumulated orally over aperiod of time (Kimberlin 1996).

It therefore seems likely that dilution will present a powerfulbarrier to the transmission of BSE agent to humans in theaquatic environment.

8.2 Degradation of BSE infectivity in the aquaticenvironment

The disease-related form of the prion, PrP-res, is so namedbecause it is more resistant to degradation by certain pro-teolytic enzymes than the harmless cellular form, PrPC. How-ever, PrPSc, and scrapie infectivity, are degraded by prolongedchallenge with proteolytic enzymes (McKinley et al. 1983). Itis likely, therefore, that degradation of BSE infectivity willoccur to some degree in the environment. Studies usingscrapie-infected hamster brains have shown that a proportionof scrapie infectivity can persist for periods of years in thesoil environment. Thus, Brown and Gajdusek (1991) demon-strated that some 0·1–1% of infectivity remained after threeyears of burial in a garden. This is consistent with con-siderable degradation over the three year period.

8.3 Resistance of infectivity to inactivation

There is no information on processes which inactivate theBSE agent (with the exception of studies on the effect ofrendering reported by Taylor et al. (1995)). Virucidal treat-ments reported to inactivate × 95% of the related sheepscrapie agent include heat, phenol extraction, sodium hypo-chlorite, sodium hydroxide (1 M) and certain detergents(Rohwer 1991). Compared with bacteriophages (fX 174, fd,PM 2 and l), the sheep scrapie agent is relatively resistant tothe effects of 5·25 g l−1 sodium hypochlorite (Rohwer 1991),although the experiments were performed in 10% brainhomogenate which would present a large chlorine demand.Since the chlorine concentrations typically used in drinkingwater treatment are less than 1mg l−1, it is concluded thatfinal disinfection used in drinking water treatment will prob-ably not present an effective barrier against BSE agent.

9. A QUANTITATIVE RISK ASSESSMENT FORA RENDERING PLANT DISPOSING OFTREATED EFFLUENT TO A SUB-SURFACEIRRIGATION SYSTEM ABOVE A CHALKAQUIFER

The application discussed here is the assessment of the risksposed by the disposal of an effluent from a plant which wasrendering carcasses from the cull of cattle over 30 monthsold. The effluent from the plant was treated by dissolved air


flotation and sand filtration before discharge to a soakawayconstructed in the chalk substrata some 80m above an aquifer.The questions to be answered were:1 Could the aquifer become contaminated with the BSE

agent?2 Did the level of contamination present a risk to drinking

water supplies derived from the aquifer?

9.1 Concentrations of BSE infectivity discharged tothe soakaway in the effluent

The aqueous effluent produced from the water boiled offfrom cattle carcasses during the rendering process containssmall amounts of MBM as particulate. Assuming that all theBSE infectivity stuck to MBM and other solids during therendering process, the amount of infectivity lost in the efflu-ent was modelled as the proportion of suspended solid in theeffluent. Similarly, the efficiency of dissolved air flotation andsand filtration at removing BSE infectivity from the effluentduring treatment was modelled as the rate of removal ofsuspended solid. From the total number of cattle carcassesprocessed, it was calculated that 0·004 human oral ID50 d−1

in the effluent were disposed of to soakaway.

9.2 Concentrations of BSE infectivity reaching theaquifer

Following disposal to the soakaway, the remaining barriersare the removal which filtration, hydrodynamic dispersionand adsorption in the chalk would achieve, and any degra-dation which occurs in the time it takes for the particles toreach the aquifer. On the basis of data presented by Brownand Gajdusek (1991), some 98·3–99·7% of scrapie infectivityis removed by decay over a period of three years. The absoluteminimal flow time for the rendering plant effluent to arriveat the pumping boreholes is two years, although more realisticflow times are much longer (14–40 years). Therefore, for thepurposes of risk assessment, it was assumed that degradationin the clay and chalk would reduce infectivity by 93%, whichis equivalent to two years of removal in the Brown andGajdusek (1991) study. No removal or attenuation of the BSEagent by filtration or adsorption in the clay and chalk wasallowed for, although in reality, the magnitude of those pro-cesses is likely to be considerable. Thus, available data forvirus removal demonstrate a halving of virus density for every2m depth of chalk (Baxter and Clark 1984). Application ofthis factor to the 80m (minimum) deep chalk barrier suggestsan attenuation factor of 10−12. Indeed, in the absence of shortcircuit fissure flow, it is likely that no BSE infectivity willpass through the chalk barrier and into the aquifer. Thediluting effect of hydrodynamic dispersion of BSE agent inthe chalk was also not considered.

The dilution factor (83-fold) in the borehole was calculated


as the ratio of the volume of water pumped daily from theborehole (10 000m3) to the volume of effluent dischargeddaily (120m3) to the soakaway at the top of the chalk aquifer.The concentration of BSE infectivity in the aquifer was pre-dicted to be 3·0× 10−11 human oral ID50 l

−1.

9.3 Risks to consumers’ drinking water from theaquifer

Assuming consumers ingest in the region of 1–2 l person−1

d−1 (Roseberry and Burmaster 1992; Haas et al. 1993), thedaily risk to individuals from any BSE infectivity in theaquifer is remote (³10−10) on the basis of low dose extra-polations (Table 1). Indeed, if there were a threshold suchthat a minimum number of prions were required, then dailyrisks would be even more remote. The time for an individualto accumulate an ID50 through consumption of drinking waterfrom the aquifer was calculated to be 45 million years. Twoassumptions, which are almost certainly too pessimistic, weremade.1 The level of BSE in the UK cattle herd would continue at

the 1996 level. Predictions suggest BSE will be almostextinct by 2001 in the UK herd (Anderson et al. 1996).

2 Infectivity can be accumulated over a human lifetime.Feeding studies with mice provide direct evidence againstthe ‘accumulated dose’ hypothesis (Kimberlin 1996).

It is concluded that the risks to human health are remote.

10. DISCUSSION

This work demonstrates the development of an approach forassessing the risks from novel pathogenic agents in drinkingwater supplies. Although little direct information is availablefor such agents, the necessary assumptions can be derived.The approach focuses on identifying the major barriers whichprevent or attenuate the movement of the agent into thedrinking water supply. A similar approach could be appliedto other emerging water-borne pathogens for which littlewater-related research has been conducted.

A major assumption for modelling the behaviour of BSEagent in the aquatic environment is that the BSE prions arebound to particulate matter. This conclusion was derivedfrom studying the biophysical properties of the BSE agentand supports a mechanism for effective removal by drinkingwater treatment processes. This, together with the low infec-tivity of BSE to humans, eliminates BSE as a likely water-borne threat as shown by the quantitative risk assessmentpresented here for an aquifer.

Worst case assumptions which are probably too extremewere applied throughout. For example, no allowance wasmade for attenuation of the prions in the chalk barrier, thelevels of BSE infectivity in the rendered OTMS cattle arelikely to be lower than assumed here because confirmed and


suspected BSE cases are incinerated, and there may be athreshold dose. Furthermore, the cow-to-man species barriermay offer a greater protective effect than allowed for here(Kimberlin 1996). These worst case assumptions are mul-tiplied in the risk assessment, and ultimately the final pre-diction for the risk from BSE in the aquifer may be over amillion-fold too pessimistic. This raises the question of thedegree of accuracy necessary for a quantitative risk assessmentto be of practical use.

Currently, there is no epidemiological evidence to identifythe vehicle of transmission of BSE to humans. There is noevidence that BSE has been transmitted to humans throughdrinking water supplies. This state of affairs is thereforedifferent to that for known water-borne pathogens such asCryptosporidium for which water-borne outbreaks are well-documented. In effect, the risk assessment presented here isused to address whether BSE could conceivably be trans-mitted through drinking water under normal circumstances.Risk assessment approaches have been developed for severalwell-known water-borne pathogens, e.g. enteric viruses (Haaset al. 1993). Those risk assessments need to be accurate intheir prediction and it would be unacceptable for them to bea factor of a million or even just 10-fold too pessimistic. Toimprove water quality 10-fold requires a major investment,which if the risk assessment is wrong, should be directedelsewhere. Worst case scenarios therefore should be avoidedif possible in quantitative risk assessment.

One advantage, however, of using worst case scenarios isthat they are easier to defend in terms of public health protec-tion. For example, the worst case scenario for the cow-to-man species barrier is not to assume it is the same as the cow-to-mouse barrier but rather to assume, in the case of the mostsusceptible humans at least (e.g. those who are homozygousfor methionine at codon 129 in the PrP gene), that there isno barrier at all, i.e. the cow-to-man barrier is one. (Datafrom Raymond et al. (1997) suggest that this assumption isalmost certainly too pessimistic (see above).) In such a scen-ario, the oral ID50 would be as little as 1mg of BSE-infectedbovine brain, and BSE-infected SBM might be expected topresent a considerable risk to abattoir and rendering plantworkers. However, the risks through drinking water drawnfrom the aquifer (described above) would still be remote.Indeed, assuming BSE prions did indeed pass through thechalk barrier into the aquifer, the time for a human drinking2 l d−1 to accumulate an oral ID50 would be 45 000 years.Thus, while the magnitude of the cow-to-man species barrieris of critical importance in determining the safety of con-sumption of beef products, it is of much less significance inenvironmental risk assessments where hydrogeological andphysical barriers make major contributions.

The approach described here focuses on the prion proteinas being the agent of transmission. Although the ‘protein-only’ hypothesis seems to have gained general acceptance,

476 P. GALE ET AL.

some researchers still challenge its involvement in the trans-mission of BSE (Lasmezas et al. 1997). This raises the ques-tion of whether this risk assessment would be invalidatedif it were shown that spongiform encephalopathies have avirological basis rather than being directly associated withprions. The answer is that it would not be affected. This isbecause this risk assessment is based fundamentally on ID50

units as measured in mouse bioassay studies. The assump-tions about the environmental barriers would also still applybecause studies have shown the infectivity itself is ‘sticky’and difficult to purify. It would therefore bind to particulatesin the aquatic environment.

11. DISCLAIMER

The views expressed in this paper are the authors’ and donot necessarily represent the views of WRc plc.

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Development of a risk assessment for BSE in the aquatic environment