Institute for Animal Health, Neuropathogenesis Unit, Ogston Building,West Mains Road, Edinburgh EH9 3JF, UK1
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK2
Author for correspondence: Moira Bruce. e-mail moira.bruce{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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Previous studies have established that the BSE strain of agent can be identified from its incubation period and lesion profile signature in mice experimentally infected directly from cattle (Bruce et al., 1994 , 1997
; Fraser et al., 1992
). The BSE signature has been recognized also in transmissions of novel TSEs of other animal species, providing evidence that these species have been infected accidentally with BSE (Bruce et al., 1994
; Fraser et al., 1994
). More recently, transmissions of variant CJD (vCJD) to mice have also given the BSE strain signature, providing compelling evidence of a link with the cattle disease (Bruce et al., 1997
). As BSE can be transmitted experimentally to certain PrP genotypes of sheep by oral exposure (Foster et al., 1993
, 2001
; Goldmann et al., 1994
), it is theoretically possible that BSE was transmitted accidentally to sheep that were fed concentrates containing meat and bone meal during the BSE epidemic. It is also possible that the BSE strain was present in sheep before the start of the BSE epidemic in cattle. Transmissions to mice from the tissues of experimentally BSE infected sheep give the BSE strain signature (Foster et al., 1996
). Therefore, if BSE has spread accidentally to sheep, or if it has always been present in the sheep population, we predict that it would be detectable by strain typing in mice.
During the late 1960s and early 1970s, transmissions to mice were attempted in this laboratory from about 20 sheep with natural scrapie from the UK (Dickinson, 1976 ). The results of these transmissions were variable in terms of the proportion of mice developing disease and the incubation periods and neuropathology of disease in the infected mice. None of the transmissions gave the BSE signature. Twelve of the scrapie isolates were characterized further by serial mouse-to-mouse passage (M. E. Bruce, unpublished observations). Typically, the strain typing signature stabilized over the first two or three mouse-to-mouse passages, although some mouse-passaged isolates took longer to reach stability. These changes in the early mouse-to-mouse passages were consistent with the selection of scrapie strains with shorter incubation periods from an original mixed population of strains. Passage in mouse strains carrying different alleles of the Sinc gene resulted in the isolation of different strains from the same sheep source, again in a manner that was consistent with the selection of strains with shorter incubation periods in the particular mouse strain used for passage. However, despite the variation seen in the primary transmissions of scrapie to mice, only four mouse-passaged strains were isolated from the natural scrapie sources: 87A or ME7 by passage in Sincs7 mice (Bruce & Dickinson, 1987
) and 87V or 111A by passage in Sincp7 mice. In at least six of the Sincs7 mouse-passaged isolates from different individual sheep, the 87A strain was isolated initially and was maintained on serial mouse passage at low dose. However, when passaged at high dose, the shorter incubation period ME7 strain often emerged from these isolates, in a manner consistent with the generation of a variant strain derived from 87A (Bruce & Dickinson, 1987
).
We have now completed ten further transmissions from individual sheep with natural scrapie, collected since the start of the BSE epidemic. This sample of ten scrapie sources is far too small to test whether or not BSE has been present in the national sheep flock. Rather, the aim of this study is to explore strain variation in natural scrapie. The results of most of these primary scrapie transmissions have been reported briefly elsewhere (Bruce et al., 1994 , 1997
; Fraser et al., 1992
). Here, we document the full series of transmissions in more detail. We also report further characterization of the scrapie isolates by mouse-to-mouse passage and compare the strains isolated in mice with those isolated from BSE cattle.
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Methods |
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Serial mouse passage.
Further characterization of the isolates was performed by serial passage of the infection in the RIII, C57BL and VM mouse strains. For each experiment, inoculum was prepared from a brain sample from an individual mouse of the appropriate strain, showing clinical and neuropathological signs of TSE disease. Groups of 1012 RIII, C57BL, VM and C57BLxVM mice were injected by the intracerebral route with 0·02 ml of 1% brain homogenates in physiological saline.
Incubation period measurement and histopathological assessment.
Mice were coded and scored for clinical signs of neurological disease according to previously described criteria (Dickinson et al., 1968 ). The incubation period for each mouse was calculated as the interval between injection and a defined clinical end-point, when the mice were showing unequivocal signs of neurological disease. For each mouse group the mean incubation period was calculated. Histopathological assessment of haematoxylin and eosin (H&E)-stained sections was carried out on all mouse brains, to confirm the clinical diagnosis. In addition, vacuolar degeneration was scored in nine standard grey matter areas from coded H&E-stained coronal brain sections (Fraser & Dickinson, 1968
). For each mouse group the mean vacuolation score in each brain area was used to construct a lesion profile.
Statistical analysis of lesion profile data.
Similarities in the lesion profiles produced by different TSE isolates were explored using a hierarchical agglomerative approach to create hierarchically related clusters of isolates. For each TSE isolate, mean lesion scores were available for nine brain areas, for each of four mouse groups (RIII, C57BL, VM and C57BLxVM), giving a total of 9x4=36 data points per isolate. This allowed each isolate to be plotted in 36-dimensional space. The N isolates were treated initially as N separate groups and then the two closest groups were combined into a single group, giving N-1 groups. This process was repeated, with N-1 becoming N-2 groups and so on, until all sources belonged to a single group. The identification of the two closest groups for merging at any step was based on the average similarity between the isolates in each group. Similarity was measured by Euclidean distance (StatCorp. 2001 Stata Statistical Software, release 7.0: http://www.stata.com) and the results of these analyses were visualized as dendrograms. All analyses were performed using Stata 7.0.
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Results |
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Lesion profile analysis for the series of Sincs7 mouse-passaged isolates depicted in Fig. 1 showed that there were consistent differences in brain pathology between ME7-like and 221C-like isolates. Space does not permit the presentation of the full series of lesion profiles, but examples of the two types of profile are illustrated in Fig. 2(a)
. In the case of SCR8, the lesion profiles confirmed the presence of 221C in the RIII-passaged isolate and the presence of another distinct strain in the C57BL-passaged isolate. However, although the incubation periods of this second strain resembled those of 87A, the lesion profiles were not 87A-like (Bruce & Dickinson, 1987
). The lesion profiles for the Sincs7 mouse-passaged scrapie isolates all differed from those of the BSE-derived strain, 301C. Cluster analysis of the lesion profiles from the full series of Sincs7 mouse-passaged isolates shown in Fig. 1
supported the classification of different scrapie strain types based on incubation period data alone and also confirmed the difference between scrapie and BSE-derived strains (Fig. 2b
).
It was possible to derive VM-passaged scrapie isolates from five scrapie sources (SCR1, 8, 9, 10, 11) (Fig. 3). For the other five sources, there were no or very few VM mice with clinical disease in the primary transmission and it was not possible to collect brain material for further passage. The incubation period data for the five VM-passaged scrapie isolates have so far shown no consistent pattern. None resemble 301V, the VM-passaged strain derived from BSE. There are also no clear reisolations of 87V, the VM-passaged strain most often derived from natural scrapie in the past. However, the VM-passaged isolate from SCR8 may resemble 111A, a strain isolated from a ColbredxCheviot sheep collected in 1969, in that there were few clinical cases within the lifespan of the mice. However, for both the SCR8 isolate and 111A, large amyloid plaques were present in the brains of very old, clinically negative VM mice, showing that infection had been transmitted. The lesion profiles for the VM-passaged scrapie isolates were also variable, but none resembled those of the BSE-derived strain, 301V (Fig. 4a
, b
).
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Discussion |
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In contrast to the uniformity seen with BSE, the transmission characteristics of sheep scrapie varied between sources. Although the results for the ten primary scrapie transmissions reported here showed little consistency, serial passage in mice resulted in the isolation of only a limited number of strains. This was also the case for natural scrapie sources previously studied in this laboratory, which generated only four mouse-passaged strains (M. E. Bruce, unpublished observations). However, only four of the scrapie sources in the present series gave ME7, the strain most commonly isolated from natural scrapie in the past by serial passage in Sincs7 mice. The other sources gave a new distinct strain, 221C. Although the sample of scrapie sources tested is very small, this observation might suggest that a different spectrum of strains is present in the two series of field scrapie cases, collected about 20 years apart, either before or during the BSE epidemic. A study elsewhere of five scrapie sheep collected in the USA also showed variation in the characteristics of mouse-passaged isolates derived from different sources (Carp & Callahan, 1991 ), but none of these mouse-passaged isolates showed a clear identity with any of those reported here.
There is considerable variation in the sheep PrP gene, which has been shown to control the occurrence of both natural and experimental scrapie (Goldmann et al., 1994 ). Furthermore, there is evidence that different strains of scrapie preferentially target different PrP genotypes of sheep (Foster & Dickinson, 1988
; Foster et al., 2001
; Goldmann et al., 1994
). An important question therefore is whether the strains of agent present in cases of natural scrapie are related to the PrP genotype of the sheep. The only consistency to emerge from the primary transmissions reported here was that the two sheep sources that were homozygous for alanine at codon 136 of the PrP gene (SCR2, 6) were the ones that were most difficult to transmit to mice (Table 1
). It was not possible to derive mouse-passaged isolates from these sources. Amongst the other sources there was no correlation between the scrapie strains isolated in mice and the PrP genotype of the donor sheep. From the six sources with a VA:RR:QQ genotype (SCR1, 3, 4, 5, 8, 11), ME7 emerged from three sources (1, 3, 11) but not from the others. ME7 also emerged from SCR9, a source with a VV:RR:QQ genotype. There was also no correlation with the breed of sheep or where the sheep were farmed. For example, of the four Cheviot sheep from the same farm, three gave ME7 and one gave 221C.
Our results demonstrate the existence of agent strain variation in natural scrapie which does not depend only on the PrP genotype of the infected sheep. This diversity is likely to have arisen because scrapie is a well-established disease in a genetically diverse population of animals. There is experimental evidence that certain laboratory TSE strains generate variant strains that may be selected by changing the PrP genotype of host through which the isolate is passaged (Bruce et al., 1992 ). A similar selective process may operate when natural scrapie infections spread amongst sheep of different PrP genotypes. The five significant alleles of the PrP gene in sheep (Belt et al., 1995
) give a total of 15 possible PrP genotypes, each likely to favour the selection of different variant scrapie strains. The natural spread of scrapie within this genetically heterogeneous sheep population would therefore be expected to maintain scrapie strain diversity. In contrast to sheep, cattle show little variation in the PrP protein. The only polymorphism described in cattle is the presence of five octapeptide repeats, rather than the usual six (Goldmann et al., 1991
), but this does not appear to influence the occurrence of BSE (Goldmann et al., 1991
; Hunter et al., 1994
). The fact that BSE is a new disease that has been disseminated rapidly through a genetically homogeneous population may explain its surprising uniformity.
The origin of BSE is obscure, but one possibility is that it was derived from rendered sheep scrapie tissues included in cattle feed supplements (Horn et al., 2001 ). If BSE did originate from sheep scrapie, the BSE strain may have pre-existed in sheep and have been unchanged by passage through cattle. Alternatively, it is possible that a scrapie strain distinct from BSE transmitted to cattle, and that the BSE strain was selected from it as a result of repeated cycling through cattle; indeed, previous studies have shown that the characteristics of TSE strains may sometimes be changed permanently by passage through a new species (Kimberlin et al., 1987
, 1989
). Our results do not rule out either of the above possibilities. There are an estimated 500010000 cases of sheep scrapie a year in the UK (Hoinville et al., 1999
). Although none of the sheep scrapie isolates we have studied has shown any strain identity with BSE, the tested sample is too small and unrepresentative to come to any conclusions about whether the BSE strain is present naturally in sheep or whether BSE has spread accidentally from cattle to sheep. However, we document an approach that can be applied to these questions, if a large enough sample of scrapie cases can be tested.
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Acknowledgments |
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References |
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Received 25 September 2001;
accepted 23 October 2001.