Volume 2: Science
3. The nature and cause of BSE
Dose
Dose-related experiments (including the attack rate experiment)
Summary
Dose-related experiments (including the attack rate experiment)
3.162 The epidemiological studies described above identified that the amount of infective material required to infect cattle was small. They could not, however, determine the actual quantity of material required. Until the preliminary results of the attack rate experiment in 1994 revealed the small amount of material needed to infect cattle with BSE, many MAFF scientists and officials assumed that much larger amounts would be necessary.
3.163 It was recognised as far back as 1987 that estimates of the minimum dose of the agent required for transmitting BSE should be sought by appropriate experimentation. Mr Raymond Bradley, head of the research programme at the CVL, had, indeed, laid this out in his paper 'The logical approach to research', which he sent to his Director, Dr Watson, in December of that year. 1 However, estimating the minimum infective dose was complicated. As there was no biochemical method to measure the amount of PrPSc in biological material, the titre of activity had to be measured by biological assay using known quantities of brain samples from terminally affected animals. Again, because of the high cost and long length of time required to carry out the experiment in calves, the mouse bioassay was used, with the inherent difficulty of the species barrier effect discussed above. However, this was not the only complication, and several other factors also had to be considered:
Genetic factors in the recipient animal - such factors were known to affect susceptibility and resistance to infection. Different strains of mice, for example, had been shown to be more or less susceptible depending on their genotype at the sinc locus.
The route of infection - was known to have a considerable effect on the infectious dose required to produce disease. A general scientific consensus existed, based on the evidence surrounding experiments with scrapie, that it was very difficult to transmit infection by the oral route. Oral or intragastric infection of scrapie had been found to be about 50,000 to 100,000 times less effective than the intracerebral route in mice, 2 with a difference as high as 109 reported in hamsters. 3 However, for any given route the dose-response curve had been shown to be consistent, ie, the higher the dose, the shorter the incubation period. 4
The amount of agent administered - the effective dose depended both on the mass or volume of the material given, and on its infectivity titre. 5
3.164 These factors, and the assumptions surrounding them at the time, were critical in the interpretation of the standard assay in experimental mice, and hence the conclusions reached regarding dose. The question of minimum infective dose of BSE agent was not in the end addressed directly, but was discovered as a result of the attack rate experiment. However, before we describe this experiment, we turn to a study examining transmission of BSE to sheep, the results of which had significant implications for the design of the attack rate study.
Transmission of BSE and scrapie to sheep and goats by intracerebral and oral routes
3.165 Initiated in June 1988 at the NPU, this experiment set out to compare transmission of both BSE and scrapie to two breeds of sheep, one susceptible, the other resistant to scrapie infection, and to goats which are rarely susceptible to natural scrapie. Its aim was to determine, firstly, if BSE behaved differently from scrapie in these animals and, secondly, if BSE transmission was influenced by the route of challenge.
3.166 Sheep of both breeds and goats were inoculated either intracerebrally with 0.05 g of infected BSE brain material, or orally with 0.5 g of infected material. The size of the intracerebral inoculations was based on that used for standard scrapie transmissions and was effectively limited by the volume of material that could be introduced into the brain. 6 Amounts used for oral administration were limited by the physical quantity of bovine material available, and so, in order to inoculate several animals, it was necessary to use a smaller quantity of inoculum (0.5 g) than if the available material had been administered to one animal.
3.167 BSE was found to transmit to both scrapie-resistant and susceptible sheep breeds as well as to goats following intracerebral inoculation. This result was significant in that it showed that BSE transmitted to animals which were not susceptible to scrapie infection. BSE was also found to transmit orally to these animals, showing that the amount of infectivity contained in 0.5 g of brain material was sufficient to cause disease. The results also showed that the two inoculation routes were similarly efficient in transmitting disease, when it had been commonly accepted that the intracerebral route was much more efficient than the oral route.
3.168 Results of positive oral transmission to scrapie-susceptible sheep were known to scientists conducting the experiment by November 1990, 7 although it was not clear if the sheep had succumbed to infection with natural scrapie or BSE. This was more than a year before the start of the attack rate experiment. Oral transmission of BSE to negative-line sheep was not recorded until October 1991. 8
3.169 It is not clear when MAFF officials became aware of the oral transmission of BSE to sheep, let alone the amount of material that was sufficient to cause disease. Results were supplied periodically by the NPU to the CVL in the form of a data sheet which contained updates of all NPU experiments. It is not clear who received these reports, though it appears that they were distributed prior to the NPU/CVL R&D meetings. 9 The quantity of material administered orally to the animals was not recorded on these data sheets, so those not familiar with the design of the experiment would not necessarily have appreciated the significance of the oral transmission. Interim results of this experiment were published in the Veterinary Record in 1993. 10
3.170 These results showed that a small amount of infectious material administered orally was sufficient to cause BSE, even across a species barrier. This information could have usefully guided decisions made on the design of the attack rate experiment in cattle.
The attack rate study
3.171 As already noted, the question of minimum infective dose was not directly addressed by any particular project, and was in fact answered as a result of the attack rate experiment entitled 'Effect of Oral Inoculum Dose on Attack Rate and Incubation Period of BSE in Cattle'. This experiment started at the CVL in January 1992, and sought to investigate how many cattle in a group would be affected by oral inoculation with different doses of affected material, as well as to understand how incubation period might be related to dose. 11 It also aimed to look at whether attack rate and incubation period differed for single and multiple exposures. In theory, the study of multiple exposure could have been of relevance to the cumulative ingestion of infected tissues, although it is not clear whether this was specifically under consideration. Groups of calves received oral doses of BSE-affected cattle brain homogenate (1 g, 10 g, 100 g, or 3 x 100 g on successive days), and were monitored clinically through to the point at which they succumbed to the disease. 12
3.172 The original idea for the cattle attack rate experiment appears to have been stimulated by the pathogenesis study, which started in December 1991. 13 The earliest reference to this in the SEAC minutes is at its sixth meeting on 1 November 1990, when it was stated that:
The Committee had 'reservations' about the proposed BSE pathogenesis studies . . . there was a risk that not enough animals might be infected to enable pathogenesis to be properly examined. 14
3.173 Much discussion ensued over the design of the pathogenesis study, especially over the need to test different dose levels, 15 and it was finally agreed that two separate experiments would take place - the pathogenesis study and the attack rate study. 16
3.174 However, Mr Gerald Wells, the project leader at the CVL, suggested in both written and oral evidence that the attack rate study was initially driven by the epidemiological need for a dose-response curve for cattle to be used in field modelling studies. 17 The experiment thus appears to have had several objectives.
3.175 The experimental design for both this study and the pathogenesis study was based on previous observations of scrapie and TME, which showed that the oral route of inoculation was less efficient than parenteral routes. Furthermore, studies had revealed the amount of infective material sufficient to cause infection by intravenous and intracerebral (i.c.) inoculation in calves. By analogy with mouse studies showing that the oral route was about 100,000 times less efficient than the i.c. route, it was possible to estimate a quantity of material that would very likely cause disease. 18
3.176 Dr Kimberlin, who provided the original calculations, explained his rationale, 19 using data laid out in a 1996 publication. 20 The amount of infectivity from brains of confirmed cases of BSE was found to be about 1 million i.c. ID50 units per gram; therefore only a millionth of a gram of brain would need to be injected intracerebrally to give a 50 per cent risk of causing disease, and 1 g of diluted brain could in theory infect half a million animals by the i.c. route.
3.177 Dr Kimberlin relied upon data from studies of mouse-adapted scrapie in mice, assuming that this meant there was no species barrier. As mentioned above, the results of these studies suggested that the oral route was 100,000 times less efficient than the i.c. route. 21 It thus followed that a dose of 100,000 i.c. ID50 units would be required to give an effective exposure of 1 oral LD50 unit, and that if the i.c. injection of only a millionth of a gram of BSE-infected brain gave a 50 per cent risk of causing disease, then 0.1 g would be required by the oral route to achieve the same risk.
3.178 On this basis, Dr Kimberlin estimated that 100 g and 10 g doses would be certain and likely respectively to cause disease if given orally. It was also considered that a 1 g oral dose of BSE-infected brain would 'possibly' cause disease. 22
3.179 Although the experiment was not yet complete, by September 1994 it was clear that animals in all groups were succumbing to disease, even those given the 1 g dose. It was reported at a meeting of MAFF officials with UKASTA representatives at which both the Assistant Chief Veterinary Officer and Mr Wilesmith were present, that following inoculation of animals with single doses of infective material of 100 g, 10 g and 1 g, it had become apparent that 'a very low dose was sufficient to cause BSE'. The implications of this for potential cross-contamination at feedmills and effective enforcement of the feed ban were discussed, especially with regard to BABs. 23
3.180 These results were greeted with surprise by many. Dr Watson (by now a member of SEAC) stated that both he and Mr Bradley were surprised at the small amount of CNS that did transmit orally to cattle. 24 Mr Wells also expressed surprise that a single gram of brain homogenate proved to be an effective dose. The finding helped to explain why the ruminant feed ban had been less effective than intended, 25 and led to the introduction of the Specified Bovine Offal Order 1995 in August of that year. This prohibited the practice of removing the brain from the skull and required the entire skull, after removal of the head meat, to be disposed of as if it were SBO.
3.181 The finding of disease in animals in the 1 g group was finally confirmed by histopathological examination in February 1996, 26 and various periodic updates ensued. While references to the results of this study have been made in various publications, 27 they have not been published in their own right.
3.182 By October 1999 the attack rate study had continued for 7 years and 8 months. All animals receiving 100 g and 3 x 100 g had succumbed to BSE, and 70 per cent of those that had received 10 g and 1 g had also contracted the disease. The incubation period in those that had received a 1 g dose (45 to 71 months) was longer than in those that had received 3 x 100 g (34 to 42 months).
3.183 Not all scientists were surprised by the results. Professor John Bourne, also of the IAH/NPU, told the Inquiry that the result of the attack rate experiment 'probably' caused him no surprise. 28 Dr Kimberlin, who designed the attack rate experiment, had thought it possible, though not probable, that 1 gram of brain tissue would suffice for oral transmission. Mr Wilesmith had held the view from the outset that very small amounts of tissues with high infective doses would suffice for oral transmission. 29
3.184 As already mentioned, the scientific consensus relied upon by MAFF officials was that the oral route of infection was very much less efficient than intracerebral injection. However, contrary evidence from other oral transmission experiments argued otherwise:
In 1961 Pattison demonstrated oral transmission of scrapie to both goats and sheep. 30 The maximum amount of infected brain that any one of the sheep could have ingested corresponded to 10 g of material, and the observed incubation period of 165 days was similar to that following intracerebral inoculation. 31 This suggested that in the absence of a species barrier large amounts were not necessary for transmission.
As described in paragraphs 3.165-3.170 above, the experiment carried out at the NPU in 1988 in sheep and goats, which compared oral dosage of 0.5 g of BSE cattle brain homogenate with intracerebral inoculation of 0.05 g of material, showed the two routes to be almost equally efficient in transmitting disease. This experiment also highlighted the ability of the BSE agent to infect orally with a small amount, even allowing for a species barrier.
A 1990 NPU experiment showed that BSE transmitted orally to mice, with each consuming between 6.25 g and 9.1 g of brain tissue and between 4.50 ml and 4.58 ml of undiluted cerebrospinal fluid. An incubation period was observed which was remarkably similar to that found with parenteral transmission. 32 Although this work gave no indication of the dose of material necessary to cause infection in mice by the oral route, it was noted that the results 'ran contrary to the doctrine that oral exposure is much less effective than parenteral exposure . . .' 33
3.185 None of these experiments involved the titration of the infective material in mice. Therefore, the precise infectivity titre is not known, making it difficult to draw conclusions or comparisons from these studies.
3.186 In February 1995 a report, produced following a review of the MAFF-funded TSE research, noted that a repeat experiment would be required to determine the dose needed to infect calves. 34 A new attack rate experiment was started in February 1998 in which four groups of calves were given oral doses of 1 g to 0.001 g, using the same pooled sample as in the previous experiment. 35 The repeat experiment did not include a group looking at the effect of cumulative dose.
Summary
3.187 We note that prior to 1994 there was misunderstanding about the minimum amount of BSE agent required to cause disease. It was shown experimentally that for comparatively small infective doses of scrapie agent of known titre, the inoculation period was inversely related to the size of the dose; small doses were associated with prolonged incubation. Theoretically, it was possible that even the smallest possible dose could initiate disease, but not within the normal lifespan of the animal. Mr Wilesmith and Dr Kimberlin believed that in the BSE epidemic there was a minimum threshold of infection which had to be breached before the animal developed clinical disease. 36 This was the most likely explanation for the low incidence of BSE within herds. However, as each birth cohort received the same batch of infected compound feed, it was not clear why some animals succumbed and others did not.
3.188 In sheep, it was well known that some breeds were more resistant to infection with scrapie than others, and it had been shown that this resistance was genetically determined. In cattle, study of DNA polymorphisms showed that there were no apparent genetic differences between affected and unaffected animals exposed to the same infected feed. Differences in genetic susceptibility and resistance were therefore ruled out as an explanation for the low within-herd incidence.
3.189 The explanation favoured by Mr Wilesmith and by SEAC 37 was that there were 'packets' of infection in the feed containing high titres of infective agent, and that it was a matter of chance which animal consumed the packet and contracted the disease. Evidence from the renderers and compounders provided to us strongly suggested that, while different batches of feed might contain different amounts of highly infected material (such as cattle brains), each batch of feed was ground and mixed to a high level of homogeneity. Particles of MBM were ground to a maximum size of less than 3 mm.
3.190 We believe that the 'packet' theory is difficult to reconcile with this evidence, and that consideration should be given to investigating other possibilities. One important possibility is that animal susceptibility to low doses of infectivity may depend on factors in the individual animal which might affect the absorption of the BSE agent. Lesions of the oropharynx and alimentary tract have been suggested (paragraph 3.159).
3.191 Animals exposed experimentally to BSE have received comparatively high doses of the agent, which have been associated with high levels of transmission of the disease. Experiments in mice have shown that the size of the infective dose (of known titre) necessary for transmission of this disease by the oral route is 100,000 times higher than the dose required to produce disease when injected into the brain. Very large oral doses of high titre brain material from BSE-affected animals were used to demonstrate oral transmission of the disease in mice early in the BSE epidemic (paragraph 3.8). These results led to a widespread assumption that the oral route of transmission of BSE was very inefficient - a conclusion contrary to the observations of Mr Wilesmith. 38 This assumption might have given the unfortunate impression that extreme rigour in efforts to exclude contamination of human and cattle food with very small amounts of SBO material was unnecessary, and that occasional lapses were unavoidable and acceptable.
3.192 As the number of BABs increased after 1991, it became clear that contamination of cattle feed with comparatively small quantities of MBM, either in the compounding mill or on the farm, was of major importance in extending the epidemic. This conclusion was confirmed in 1994 when the preliminary results of the attack rate study became available (paragraphs 3.171-3.186). It was shown later that as little as 1 g of high titre BSE brain material given orally was sufficient to cause disease in 70 per cent of treated animals.
3.193 Apart from cattle feed contamination, it seems likely that maternal and lateral transmission are factors which have contributed to the number of BABs. Investigation has not yet revealed the mechanism by which either might have occurred.
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1 YB87/12.20/1.1
2 Kimberlin, R. and Walker, C. (1989) Pathogenesis of Scrapie in Mice after Intragastric Infection, Virus Research, 12, 213-20; Diringer, H., Beekes, M. and Oberdieck, U. (1994) The Nature of the Scrapie Agent: The Virus Theory, Annals of the New York Academy of Science, 724, 246-58
3 Prusiner, S., Cochran, S. and Alpers, S. (1985) Transmission of Scrapie in Hamsters, Journal of Infectious Diseases, 152, 971-8
4 Kimberlin, R. and Walker, C. (1978) Pathogenesis of Mouse Scrapie: Effect of Route of Inoculation on Infectivity Titres and Dose-Response Curves, Journal of Comparative Pathology, 88, 39-47
5 SEAC (1994) Transmissible Spongiform Encephalopathies: A Summary of Present Knowledge and Research, HMSO, London (IBD2 tab10), p. 62
6 S108A Bostock para. 2
7 S108A Bostock para. 6; YB90/10.18/9.3
8 YB91/10.20/1.2
9 YB90/10.18/9.1
10 Foster, J.D., Hope, J. and Fraser, H. (1993) Transmission of Bovine Spongiform Encephalopathy to Sheep and Goats, Veterinary Record, 133, 339-41
11 YB92/02.00/2.2
12 BSE Transmission Studies as at 31 March 1992, CVL103, 147
13 T42 p. 75
14 YB90/11.01/2.5
15 YB91/10.01/3.1
16 YB91/10.18/3.1
17 T51 p. 75; S065A Wells para. 53
18 YB92/02.00/2.2; YB91/5.25/1.4
19 S95C Kimberlin
20 Kimberlin, R. (1996) Bovine Spongiform Encephalopathy and Public Health: Some Problems and Solutions in Assessing the Risks, Transmissible Subacute Spongiform Encephalopathies: Prion Diseases, edited by Court, A. and Dodet, B. (M8a tab 42)
21 Kimberlin, R. (1991) An Overview of Bovine Spongiform Encephalopathy, Developments in Biological Standardization, 75, 75-82
22 YB91/5.25/1.4
23 YB94/9.8/4.1
24 T42 p. 64
25 YB95/2.28/1.1
26 YB96/2.29/1.1
27 For example, The Report of the Chief Veterinary Officer, Animal Health 1996, London, HMSO (M24 tab 12)
28 T44 p. 108
29 T52 p. 14
30 Pattison, I. and Millson, G. (1961) Experimental Transmission of Scrapie to Goats and Sheep by the Oral Route, Journal of Comparative Pathology, 71, 171-6
31 Ibid.
32 Barlow, R. and Middleton, D. (1990) Dietary Transmission of Bovine Spongiform Encephalopathy to Mice, Veterinary Record, 126, 111-2
33 YB91/02.26/5.1/5.6
34 YB95/2.13/1.4
35 S65B Wells para. 15.2
36 Kimberlin, R. and Wilesmith, J. (1994) Bovine Spongiform Encephalopathy Epidemiology, Low Dose Exposure and Risks, Annals of the New York Academy of Sciences, 724, 210-20
37 Spongiform Encephalopathy Advisory Committee (1994) Transmissible Spongiform Encephalopathies: A Summary of Present Knowledge and Research, September 1994, section 2.2 p. 35 (IBD2 tab 9)
38 YB88/3.4/5.1; T52, 14
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