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1 Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
2 National Veterinary Research and Quarantine Service, Anyang, Republic of Korea
3 National Veterinary Institute (SVA), SE-75189 Uppsala, Sweden
Correspondence
G. A. H. Wells
g.a.h.wells{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Oak Farm, Harpsden Bottom, Henley-on-Thames, Oxon RG9 4HY, UK. 
Present address: NSPAC, Defra, Whittington Road, Worcester WR5 2SU, UK.
Present address: Defra, 1a Page Street, London SWIP 4PQ, UK, and Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Outram, 1976; Prusiner et al., 2004).
While in rodent models of scrapie it has long been established that the oral route of inoculation is less efficient than parenteral routes (Kimberlin & Wilesmith, 1994), the relative efficiency of these routes is variable in different models (Taylor et al., 2001). The implication of the dietary constituent, meat and bone meal (MBM), as the vehicle of infection of cattle with the bovine spongiform encephalopathy (BSE) agent (Wilesmith et al., 1988) established that for BSE the oral route is the primary route of exposure.
Although the infective dose of the ingested scrapie-like pathogen necessary to produce the perceived attack rate in the BSE epidemic could not previously be determined directly, epidemiological studies have examined the dynamics of the exposure. In Great Britain (GB) an increase in incidence of BSE occurred in July 1989 which, given no change in the ascertainment rate at that time, is consistent with a real change in the exposure of the cattle population to the BSE pathogen in 1984 (Wilesmith, 1991). However, the increased incidence of BSE at this time was not reflected in within-herd incidence, but rather in a geographically proportional increase in the number of affected herds throughout GB. It is suggested that this phenomenon appeared because of a geographically uniform increase in the frequency with which batches of feed contained infective material, but not an increase in titre of infectivity within infected batches. Epidemiological monitoring, to determine the attack rate in affected herds, indicates that the average exposure to infection in the feed has been at a very low level, analogous to limiting dilutions in bioassays of scrapie infectivity, and might have been as low as 14 oral LD50 per tonne of an infected batch of feed (Kimberlin & Wilesmith, 1994).
The modelling of exposure dynamics in the BSE epidemic requires experimental determinations of the cumulative incidence of disease (attack rate) and dose–response characteristics after oral exposure. An oral titration protocol determines a value for the cattle oral ID50 and an IP distribution according to dose. The studies described here have been conducted in two phases. The initial study had three main aims: to determine the attack rate and IP of BSE in cattle exposed orally to four different dose levels of brain homogenate from affected cattle; to determine whether there is a dose–response effect on the IP and to determine whether, in the event of the failure of single doses to produce disease, multiple exposures were effective. This study was also intended to inform on the validity of using a 100 g dose of brainstem homogenate in concurrent studies of the pathogenesis of BSE in cattle after oral exposure, as a dose (given an equivalent titre of inoculum) that would produce a 100 % attack rate in calves (Wells et al., 1996, 1998). In a second experiment, consequent to the failure of the initial study to indicate a limiting exposure dose, a further range of lower exposure doses were examined using the same inoculum. This study extended the existing data toward an understanding of what constitutes effective exposure to BSE in the field. Data from the two experiments were combined to determine attack rate, ID50, a dose–response curve and, as far as possible, a minimum infective dose, after oral exposure.
The combined data are therefore relevant to studies of modelling the BSE epidemic, risk analyses for exposure patterns and future decisions on BSE surveillance and control.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Inoculum and estimation of inoculation dose.
A pool of undiluted homogenized brainstems (brain pool code BBP1/92) was prepared from 71 naturally occurring, histopathologically confirmed cases of BSE, killed in 1991. An end-point titration of infectivity in the pool was conducted according to a standard protocol in RIII mice, using simultaneous intracerebral (i.c.) and intraperitoneal (i.p.) inoculation of six groups of 20 mice and a dilution range of 10–1 to 10–6. The combined parenteral routes of inoculation were used to replicate the previous most efficient assays of the BSE agent across a species barrier in RIII mice (Bruce et al., 1994; Buschmann & Groschup, 2005). IPs in mice were calculated by using established criteria (Dickinson et al., 1968).
Estimations of the initial range of doses to which the calves were exposed were made from limited data on the results of a parenteral inoculation of calves with the BSE agent. Experimental transmission of BSE in cattle had established that 100 mg brain i.c. plus 500 mg intravenously is an infectious dose (Dawson et al., 1990). Since, in a cattle-to-cattle transmission, there are no species-barrier effects, it was considered likely that 100 mg i.c. would have been sufficient to produce disease. Using these data, together with analogy with experimental rodent TSE, and by extrapolation from the estimates of the exposure via feed in the natural epidemic of BSE, it was proposed that, by the oral route of inoculation, 10 g infected brain would probably cause clinical disease, and that 100 g would be certain to induce clinical disease (R.H. Kimberlin, personal communication).
Animals.
For the first phase of the study, 40 castrated male calves (19 Friesian/Holstein, 20 Friesian crossbred and one Aberdeen Angus X Jersey) were assembled, in 1991, from 12 GB herds with no history of BSE and no history of feeding proprietary concentrates at the time of sourcing and, on subsequent investigation, no BSE case in animals born in the same year as the sourced animals. The Ministry of Agriculture, Fisheries and Food (MAFF)/Veterinary Laboratories Agency (VLA) main BSE database, and any other information available at the time, were used to assess the current BSE status of the original source herds and, if necessary, to identify possible new source herds for BSE-unexposed animals. For the second experiment, 60 castrated male Friesian/Holstein calves were similarly acquired from 13 GB herds, during 1997.
Polymorphisms of the PrP gene are determinants of susceptibility and IP in most species affected by TSE but, in cattle in GB, until very recently (Juling et al., 2006), no such associations were established for BSE. It has long been established that an octapeptide-repeat polymorphism in cattle occurs as either five or six copies, with genotypes expressed as 5 : 5, 6 : 5 or 6 : 6 (Goldmann et al., 1991). While this polymorphism has not been associated with any differential susceptibility to BSE infection in Britain (Hunter et al., 1994), cattle with six octapeptide-repeats predominate in the population and among BSE cases. The PrP genotype of the calves, with respect to the octapeptide-repeat polymorphism (Goldmann et al., 1991), was therefore determined from DNA extracted from EDTA blood samples (Wells et al., 2005). In the initial experiment 29 calves were of the 6 : 6 genotype and 11 were of the 6 : 5 genotype and in the second experiment 55 calves were of the 6 : 6 genotype and five were of the 6 : 5 genotype. The different genotypes were allocated to provide, as far as possible, a mix within test (Tables 1 and 2) and control groups.
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To reduce environmental contamination resulting from faecal excretion of inoculum in the immediate period after dosing, a strict post-dosing husbandry regime was implemented as previously described (Wells et al., 1996). In the first experiment the groups remained segregated in housing until 14 weeks post-dosing, when they were all turned out to double-fenced pasture and allowed to mix. They were held at pasture for a further 32 weeks and then rehoused, ungrouped in covered yards for the duration of the study. For the second experiment the cattle were housed together after the initial 14-week period of segregation.
Experimental design.
For the first experiment, prior to dosing, calves were randomly assigned to four groups of ten animals each and dosed at approximately 4–6 months of age, within the estimated period of calfhood exposure in the epidemic (Wilesmith et al., 1992b). Each group of calves was dosed orally with the brainstem homogenate according to one of four different dosing regimes: single doses of 1, 10 or 100 g respectively, or 3x100 g on successive days. The means (and ranges) of ages at dosing, according to dose group, are given in Table 1
. The 3x100 g dosing strategy was used not to investigate the effect of multiple dosing per se, but simply to provide a contingency in the event that single exposures were ineffective as, at the time of initiation of the experiment, the form of an effective exposure to induce BSE by the oral route was not known. Each dose was administered using sterile syringes; 100 g doses were administered using 2x50 ml syringes, the 10 g dose by using a 10 ml syringe and the 1 g dose by using a 1 ml syringe. Inoculum was administered as pure brain homogenate as previously described, directly on to the base of the tongue, followed by enforced closing of the jaw to encourage swallowing and prevent possible loss of inoculum (Wells & Hawkins, 2004).
The second experiment replicated the approach used in the first experiment but with a range of single exposure doses of 1, 10 and 100 mg and 1 g of the same pooled brainstem homogenate (BBP1/92) used in the first phase. In the second experiment, the exposure doses were considered to be more closely representative of the exposure of the majority of commercial cattle via contaminated MBM in the course of the BSE epidemic in GB. Calves were allocated to treatment groups and dosed orally, as in the initial experiment, but three groups of 15 calves received 1, 10 or 100 mg respectively and one group of five calves received 1 g of the brainstem homogenate. Ages at dosing are given in Table 2. In contrast to the initial experiment, because of the low mass of the inoculum dose range, the volume of inoculum administered was adjusted to a minimum of 10 ml by dilution with saline. Ten unchallenged calves served as environmental/cohort controls, which after the initial 14-week post-exposure (p.e.) period of separation were in contact with exposed animals.
Clinical monitoring.
Pre-exposure clinical examinations were carried out on each animal to establish a baseline assessment. Clinical observations were maintained throughout the studies by methods described previously (Wells et al., 1996; Austin et al., 1997; Wells & Hawkins, 2004). In the initial experiment, behaviour studies were made over 24 h periods (Austin et al., 1994) at approximately three month intervals from 12 months post-dosing.
For the second study, clinical examination methods were subject to some modifications due to differing operational circumstances in the two phases of the study. Clinical examinations were conducted monthly and comprised assessment of the animal's behaviour (replacing periodic 24 h monitoring periods), reactivity and gait. Every third month, this was supplemented with a more detailed neurological examination (Konold et al., 2004). In both experiments, the assessments were made without knowledge of the inoculation status of the animals and were categorized as possible, probable or definite signs of BSE (Wells & Hawkins, 2004).
Individual animals were killed after they had been judged to have a clinical classification of definite BSE. In the initial phase of the study, cattle that did not progress to this clinical stage were retained until 110 months p.e. and were then euthanized. This end point to the study was based on survival data beyond the IP of the animal with the longest IP (72 months) and estimation that the end-point of the oral titration had been exceeded. The second phase of the study is as yet incomplete and will be reviewed at 110 months p.e.
Post-mortem examinations and sampling.
Post-mortem examination procedures were similar in both phases of the study. In the first experiment, the whole brain and spinal cord were removed, fixed in formal saline and then processed routinely through to paraffin wax. Additionally, in the second experiment, fresh tissue was taken from the caudal medulla for freezing at –80 °C and subsequent biochemical analysis for PrPSc detection, prior to fixation of the remaining brain tissue. Additional tissues were sampled from animals which succumbed to intercurrent disease problems for diagnostic examinations.
Histopathology, immunohistochemistry and Western immunoblotting.
Sections of brain and spinal cord were prepared and stained with haematoxylin and eosin (HE) and examined for evidence of vacuolar changes. The brain and spinal cord of any animal that had to be killed due to intercurrent disease or welfare reasons were examined similarly.
Profiling of vacuolar changes was carried out on HE-stained sections of the brains of the affected animals by scoring 17 neuroanatomically distinct sites representative of major brain regions, according to a method previously detailed by Simmons et al. (1996). The sites scored were considered to be representative of a range of different intensities of vacuolar pathology in naturally occurring BSE. The mean score for each neuroanatomical area was calculated and plotted against the area ‘code number’ to produce a lesion profile. Equivalent data from natural field cases of BSE studied over three annual periods (Simmons et al., 1996) were compared graphically with those of the orally challenged cattle.
For the first experiment, in the absence of vacuolar changes, PrP immunohistochemistry (IHC) using the polyclonal antibody R486 (Cooley et al., 2001) was applied to the brainstem at the level of the obex, the rostral medulla at the level of the cerebellar peduncles and the mesencephalon at the level of the rostral colliculi. For the second experiment, histopathological and immunohistochemical examinations were applied to brainstem at the level of the obex, rostral medulla and mesencephalon. For the immunohistochemical examinations, the monoclonal antibody R145 was used (Terry et al., 2003). This antibody is an anti-PrP rat mAb raised against the same bovine peptide sequence as R486, and to which it is comparable in performance. Lesion profiling was performed as previously described on the brains from terminally affected cattle (Simmons et al., 1996).
Western immunoblotting (Wb) for PrPres was carried out using the Prionics-Check WESTERN technique (Prionics Ag), as previously described (Stack, 2004).
Statistical analyses.
For single-dose results, data on the IP for each animal and on the survival of the non-affected animals (Tables 1 and 2) were used to estimate the dependence of both the IP distribution and attack rate on the dose, via maximum likelihood.
The IP was assumed to follow a log-normal distribution, with the mean depending linearly on the log dose, d, as found for scrapie in hamsters (Prusiner et al., 1982; Robinson et al., 1990). The normal distribution was also fitted to the IP data but produced an inferior fit compared to the log-normal. Heterogeneity of variance with respect to dose was investigated using Levene's F-test. The attack rate was assumed to follow a logistic regression curve, i.e. the probability of infection given the dose, S(d), could be expressed as:
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, 
were parameters to be determined from the statistical analysis. To perform the maximum-likelihood estimation, the probability that an animal reached clinical onset and would thus have a recorded IP was assumed to be given by S(d)f(t|d), where f(t|d) denotes the dose-dependent IP distribution. For the purposes of the statistical model it was assumed that infection would ultimately result in disease should the animal live long enough. Therefore, the probability that the animal did not develop clinical signs was given by:
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The above probabilities are then used to obtain maximum-likelihood estimates of the parameters for the IP distribution and probability of survival given the dose; since the titre of the initial inoculum was known only in terms of mouse i.c./i.p. ID50 units, the dose was expressed in terms of mouse i.c./i.p. ID50. The 95 % confidence interval for the ID50 was obtained by Fieller's method (Fieller, 1940).
There were insufficient data to provide interpretable analysis of either 3x100 g results with respect to possible multiple dose effects, or the possible effects of PrP genotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Clinical diagnosis
The times of first onset of the defined clinical stages and the times of euthanasia of individual exposed animals in each phase of the study are detailed in Tables 1 and 2. The clinical signs in all confirmed cases were similar to those observed and reported in naturally infected cattle. Behavioural and/or sensory changes were usually the earliest clinical signs and abnormalities in muscle activity, locomotion and posture occurred later in the clinical course.
The three different clinical stages could be identified in most, but not in all, animals that succumbed to the experimental exposure. Individual animals did not all show evidence of a steady progression from one stage to the next. Some animals abruptly entered a stage without earlier signs of a preceding stage. In other cases, there was remission of signs and therefore regression into a preceding stage.
The mean period from exposure to onset of the definite clinical stage of the different dosage groups varied according to dose (Table 3), but there was also considerable variation in this period within dose groups (Tables 1 and 2).
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and 2. Two control cattle were killed due to bone fractures; one (CM908), culled at 39 months after test group exposures, had a fracture of the second lumbar vertebra, the other (CM925), culled at 59 months, a femoral fracture. A further three control animals (CM918, CM856 and CM858) were killed at 97, 101 and 103 months, due to a pleural mesothelioma, septic arthritis and muscle injury respectively. None displayed definite signs of BSE but CM858 and CM856 displayed probable signs of BSE at 74 and 75 months after test group exposures, respectively. Currently (December 2006), there are surviving animals from all test groups in the second experiment (1 g, 1/5; 100 mg, 6/15; 10 mg, 11/15; 1 mg, 11/15) at 106 months post-dosing. There are also five surviving uninoculated in-contact control animals.
Neuropathological diagnosis and observations
The diagnostic outcome of examinations of the brains of all exposed cattle killed to date from the two experiments is given in Tables 1 and 2. In none of the animals killed or found dead due to intercurrent disease problems, or those (in the first phase of the study) surviving to the termination of the experiment, was there any pathological evidence of BSE. The lesion profile for animals of the seven dose groups compared to that generated from naturally infected field cases of BSE was plotted (Fig. 1). Where the data for groups were the averages of multiple individual profiles, there is overlap suggestive of a common profile.
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and 2) were used to estimate the parameters of the dose-dependent attack rate and IP distributions. For the attack rate, the parameters of the logit dose–response model were =–3.50 and =1.26, resulting in an ID50 estimate of 102.8 mouse i.c./i.p. ID50 g–1 (with a 95 % confidence interval of 102.1–103.5). This ID50 estimate is equivalent to 0.20 g of the brain homogenate used in the experiment, with 95 % confidence intervals of 0.04–1.00 g [Fig. 2, produced by plotting S(d) from equation (1) for the estimated values of , ]. Levene's F-test detected no difference in variance between the single dose groups.
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=0.21, where dose is the titre of brain homogenate in terms of mouse i.c./i.p. ID50 g–1. The relationship between mean IP [given by exp(µ+0.52) for the log-normal distribution] and the dose of brain material used in the study is given in Fig. 3.
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There was representation of both 6 : 6 and 6 : 5 octapeptide-repeat PrP genotypes among BSE-affected and -unaffected animals in the two experiments (Tables 1 and 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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100.4 RIII mouse i.c./i.p. ID50 units) from clinically affected field cases of BSE and that the limiting dose for infection of calves is lower than this exposure. It has also demonstrated that one cattle oral ID50=102.8 mouse i.c./i.p. ID50. In a separate titration study (VLA brain pool: SE1809 : BBP) of the species difference between cattle i.c. and mouse i.c/i.p., it has been shown that one mouse i.c./i.p. ID50=102.7 cattle i.c. ID50 (Hawkins et al., 2000). From these results it follows that one cattle oral ID50=105.5 cattle i.c. ID50. A parallel titration of the same cattle brainstem pool (SE1809 : BBP/BBP12/92) in RIII mice and in transgenic mice overexpressing bovine PrPC (Tgbov XV) obtained titres of 103.3 and 107.7 i.c./i.p. ID50 g–1 respectively (Buschmann & Groschup, 2005), the Tg mouse assay providing therefore an approximately 4.4 log improved sensitivity compared to RIII mice and more than 1 log than that of the i.c. assay in cattle. Since cattle oral exposure is 2.8 logs less sensitive than the RIII mouse i.c./i.p. assay, then cattle oral exposure is approximately 7 logs less sensitive than the Tgbov XV mouse assay.
A dose–response effect on IP is evident but with large IP ranges for a given dose (Table 3).
The experimental infection produced a clinical disease comparable to that of natural cases of BSE (Braun et al., 1998; Konold et al., 2004; Wilesmith et al., 1992a), with the definite stage of the disease in the experimentally infected animals comparable with a stage of disease that would have been recognized in field cases. All infected animals also displayed clinical signs suggestive of early BSE (possible or probable signs) several months before the onset of definite signs, which is also comparable to the field situation (Wilesmith et al., 1988).
In the absence of any ante-mortem confirmatory BSE test it is impossible to correlate clinical onset with onset of neuropathological changes. Current post-mortem tests, approved for statutory diagnosis, may diagnose BSE several months before the onset of definite clinical signs (Grassi et al., 2001), but early behavioural and sensory signs of BSE (possible or probable signs) may have been present several months earlier as demonstrated here. Early behavioural changes usually precede locomotor deficits in mice infected with scrapie or BSE (Dell'Omo et al., 2002) and, in variant Creutzfeldt–Jakob disease (CJD), psychiatric and sensory symptoms dominate in the early phase of the disease (Henry & Knight, 2002). These early signs are certainly less reliable as BSE-specific clinical markers in cattle and, as the definition of the possible stage implies, may overlap with expression of normal behaviour or with behaviour displayed by animals with other diseases.
Five of the animals in the first experiment (two inoculated with a 10 g dose and three inoculated with a 1 g dose; Table 1) expressed possible or probable signs of BSE, suggestive of an early stage of BSE at around the same time as animals that later progressed to definite signs of BSE. At 110 months post-dosing, when the experiment was terminated, the animals were judged to be clinically normal. The experimental outcome in these animals is open to two main interpretations; either the contemporary clinical assessments were misleading or the animals showed remission or recovery from the early stages of the disease. With regard to the former possibility, other clinical conditions can produce signs that overlap with those of BSE, especially the early signs. This has been experienced in clinical veterinary practice throughout the epidemic and is supported by the observation of possible and probable signs of BSE in two control animals. Recovery from prion disorders is contrary to the dogma that they are considered invariably fatal neurodegenerative diseases (Prusiner, 2004).
However, mice inoculated with a low-dose inoculum may display reversible clinical signs and never progress to terminal disease (Thackray et al., 2002). Other clinical or biochemical methods are clearly desirable to improve criteria for defining possible and probable signs of BSE as is done for variant and sporadic CJD (Zerr & Poser, 2002).
The profile of vacuolar changes (Fig. 1) and the distribution pattern of PrPSc deposition in the brainstem closely resembled that observed in naturally affected cattle, providing experimental evidence that this route of exposure simulates natural infection and subsequent pathogenesis. This is entirely consistent with the view that the BSE epidemic in GB has been sustained by a single major cattle-adapted strain of a scrapie-like agent, which is stable when transmitted between cattle by experimental passage or via rendering (Bruce et al., 1994; Casalone et al., 2006; Hawkins et al., 1997; Simmons et al., 1996; Wells & Simmons, 1996; Wells & Wilesmith, 1995).
Although not recognized when the first experiment was initiated, the subsequent occurrence of variant CJD in humans, considered to have been the consequence of infection with the BSE pathogen (Will et al., 1996), led to additional requirement for quantitative risk-assessment studies of the exposure of humans to the BSE agent from ingestion of infected bovine products. For this purpose it was necessary to utilize interim experimental data on cattle ID50 from this study as a proxy for the human ID50, making the precautionary assumption that the cattle-to-human species barrier is a factor of 1 (EFSA, 2005). On the basis of interim data from the present studies, the range of cattle oral ID50s in 1 g brain from a clinically affected cow was calculated to be approximately 0.52–5 (EFSA, 2005). Using the same reasoning, the results of the present study indicate that this range should be revised to 1.0–20, although with higher titres of BSE-affected brain as starting material, the range could extend upward. With a BSE-affected brain titre of 105.2 mouse i.c./i.p. ID50 g–1 (Fraser et al., 1992), for example, the range could extend to 1000.
Epidemiological studies have indicated that the mean IP of cattle infected in the field is in the range of 5–5.5 years (Arnold & Wilesmith, 2004; Wilesmith et al., 1988). Mean IPs in this range would correspond to single doses in the range of approximately 100 mg–1 g. However, it is difficult to make any precise inference on the distribution of doses received in the field due to uncertainty over the shape of such a distribution and the large variability of IPs for a given single dose. A further complication is the possibility of infected cattle receiving multiple doses, which would also affect the observed age of onset of field cases; multiple doses have been found to affect both the IP and the probability of infection for scrapie in hamsters (Diringer et al., 1998; Gravenor et al., 2003), and these results could apply to BSE in cattle. Furthermore, little is known regarding the effect of age at exposure on attack rate or IP, but in this study this parameter had a narrow range and is recorded (Tables 1 and 2) for future reference to prospective studies of transmission of the BSE agent. Other experimental factors may also have affected the IPs and probability of infection recorded in this study, such as the method of oral dosing, how recently the animals had eaten and sex of the animals, none of which has been explored due to insufficient data. Recent work (Juling et al., 2006) suggesting that in certain cattle breeds, polymorphisms in the regulatory region of bovine PRNP are linked to BSE incidence, requires investigation in the animals in this and related experimental studies of BSE in cattle.
There are very few studies using experimental models of prion diseases in which dose/IP distribution characteristics have been studied after oral exposure; most have examined the i.c. and other parenteral routes (Kimberlin & Walker, 1988; McLean & Bostock, 2000). When mice were inoculated intragastrically with scrapie, the dynamics of pathogenesis were defined by the same equation as that applicable to five mouse scrapie models after i.p. inoculation (Kimberlin & Walker, 1989), suggesting that comparisons of IP characteristics, despite different routes of infection, are valid. In the present studies, there was considerable variance in the range of IPs observed, with no significant difference between single-dose groups. Even in the high-dose groups, a wide range of IPs was observed; IPs ranged from 31 to 60 months for the 10 animals exposed to 100 g challenge. This is entirely consistent with results from a study comparing the dose–responses of IM mice exposed to mouse-adapted BSE (301V) agent, by the i.c., intragastric and feeding routes (S. A. C. Hawkins, unpublished), with per os routes showing comparable IP ranges which were considerably greater than that of the i.c. route (data not shown). In an analysis of 117 titration experiments of scrapie in mice, albeit mainly using the i.c. route of inoculation, McLean & Bostock (2000) showed that IPs were often within a narrow range for the higher-dose groups, with increasing variance as the dose decreased. They noted that this variability in IP increased linearly with decreasing dose and resulted in overlap of IP across several doses. Although in the present study increase in IP variance at very low doses is not yet observed, because only a single animal in each of the lowest dose level groups has developed disease, overlap of IP across several doses is a feature.
There is no null effect group in this study, despite the small group size and a minimum dose group of approximately 3 logs below the ID50. This is in keeping with the findings of McLean & Bostock (2000) for scrapie in mice, in that there is no evidence of a threshold dose at which the probability of infection becomes vanishingly small. However, the rate at which the probability of infection declines with dose for BSE in cattle by the oral route of exposure appears to be much slower than for scrapie in mice that were infected predominantly by parenteral routes; in the experiments studied by McLean & Bostock (2000) it was common for there to be only a single dose group with an incomplete incidence of disease between those in which none and all the animals succumbed to scrapie. It remains an enigma of prion diseases in general as to whether there is a dilution of inoculum beyond which the concentration of infectious particles cannot establish infection or whether the infectious particles cannot be diluted out completely.
The experiment is as yet incomplete but further results are unlikely to significantly affect the ID50 and IP characteristics. Each future additional case would result in a small reduction in the ID50 estimate; an additional case in each of the three lowest dose groups would reduce the estimate from 0.20 to 0.11 g.
This study has provided an experimental determination of attack rate and dose–response characteristics consequent to oral exposure for modelling of exposure dynamics in the BSE epidemic. It has determined the attack rate and IP of BSE to cattle exposed to a range of seven different doses of brain homogenate from affected cattle and shown that there is a dose–response effect on both the attack rate and IP distributions. It has also confirmed the validity of using a 100 g dose of brainstem homogenate in experimental studies as a dose (given an equivalent titre of inoculum) that would produce a 100 % attack rate in calves. However, it has not established a minimum infective dose of BSE agent for cattle and, if the concept of a minimum infective dose is applicable to BSE, such a dose is less than 1 mg of the brainstem homogenate used, that is, equivalent to <100.5 mouse i.c./i.p. ID50.
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ACKNOWLEDGEMENTS |
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Received 27 July 2006;
accepted 18 November 2006.
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