Strain characterization of natural sheep scrapie and comparison with BSE

Moira E. Bruce1, Aileen Boyle1, Simon Cousens2, Irene McConnell1, James Foster1, Wilfred Goldmann1 and Hugh Fraser1

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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Scrapie was transmitted to mice from ten sheep, collected in the UK between 1985 and 1994. As in previous natural scrapie transmissions, the results varied between scrapie sources in terms of the incidence of disease, incubation periods and neuropathology in challenged mice. This contrasted with the uniformity seen in transmissions of BSE to mice. The scrapie and BSE isolates were characterized further by serial passage in mice. Different TSE strains were isolated from each source according to the Sinc or PrP genotype of the mouse used for passage. The same two mouse-passaged strains, 301C and 301V, were isolated from each of three BSE sources. Despite the variation seen in the primary transmissions of scrapie, relatively few mouse-passaged scrapie strains were isolated and these were distinct from the BSE-derived strains. The ME7 scrapie strain, which has often been isolated from independent sheep sources in the past, was identified in isolates from four of the sheep. However, a new distinct strain, 221C, was derived from a further four scrapie sheep. These results suggest that there is agent strain variation in natural scrapie in sheep and that the spectrum of strains present may have changed over the last 20 years. The tested sample is too small to come to any conclusions about whether the BSE strain is present in sheep, but the study provides a framework for further more extensive studies.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The agents causing transmissible spongiform encephalopathies (TSEs) exhibit clear evidence of strain variation (Bruce, 1993 ). Studies of mouse-passaged TSE isolates have established that the phenotype of the disease depends on an interaction between the strain of TSE agent and genetic factors in the host (Bruce et al., 1991 ; Dickinson & Meikle, 1971 ). These observations led, many years ago, to the development of formal TSE strain typing methods that depend primarily on the measurement of incubation periods in panels of defined mouse strains. In particular, incubation periods are measured in mouse strains carrying different alleles of the Sinc gene. The discovery of the mouse Sinc gene was based on its dramatic effect on TSE incubation period (Dickinson et al., 1968 ), but the gene was later shown to encode the prion protein, PrP (Hunter et al., 1992 ; Moore et al., 1998 ). The two known alleles of Sinc, designated s7 and p7, encode PrP proteins that differ by two amino acids (Westaway et al., 1987 ). TSE strain discrimination also depends on an assessment of the neuropathological changes seen in infected mice. The severity and distribution of vacuolar degeneration in the brains of these mice are plotted to produce ‘lesion profiles’ (Fraser & Dickinson, 1968 ) which, together with the incubation period data, provide a ‘signature’ of the TSE strain. Using these methods, numerous distinct laboratory strains of TSE agents have been identified, with signatures that remain stable over many serial mouse-to-mouse passages (Bruce et al., 1991 ). It is clear from these studies that TSE agents carry strain-specific information that is independent of the host, but the molecular basis of this information is unresolved.

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Primary transmissions.
Transmissions to mice were set up from eight BSE Holstein–Friesian or Friesian cattle, collected at intervals between 1987 and 1992 from widely separated locations in the UK. The results for the full series have been reported elsewhere (Bruce et al., 1994 , 1997 ); here we present data from only the first three BSE transmissions. In parallel, transmissions to mice were set up from ten sheep with natural scrapie, collected from 1985 onwards, of various breeds and PrP genotypes (Table 1). The four Cheviot sheep were collected from the same farm in southern Scotland. The other sheep were all from different unrelated farms in southern Scotland and northern England. At post-mortem, brain samples from the sheep and cattle were dissected using rigorous aseptic procedures.


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Table 1. Details of natural sheep scrapie sources used for transmission

 
For each TSE source, a 10% homogenate of brain in physiological saline was injected into groups of 20–24 mice of each of the following inbred strains: RIII, C57BL (both Sincs7 or PrP-a), VM (Sincp7 or PrP-b) and the C57BLxVM F1 cross. Each mouse was injected by a combination of the intracerebral (0·02 ml) and intraperitoneal (0·1 ml) routes.

{blacksquare} 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 10–12 RIII, C57BL, VM and C57BLxVM mice were injected by the intracerebral route with 0·02 ml of 1% brain homogenates in physiological saline.

{blacksquare} 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’.

{blacksquare} 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.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Primary transmissions of scrapie from sheep
Table 2 shows the results of the ten primary transmissions of scrapie from sheep to mice, compared with results for our first three transmissions of BSE from cattle to mice. The three BSE transmissions, which were typical of the full series of eight that has been reported elsewhere (Bruce et al., 1994 , 1997 ), illustrate the uniformity of the transmission characteristics of BSE from different cattle. In contrast, no consistent pattern was seen amongst the ten sheep scrapie transmissions and none resembled BSE, in terms of either incubation periods (Table 2) or lesion profiles (data not shown) (Bruce et al., 1997 ). Only one scrapie source (SCR1) produced disease and/or neuropathology in 100% of mice of all genotypes (Table 2). Seven other sources (SCR3, 4, 5, 8, 9, 10, 11) produced clinical disease in some mouse strains after prolonged incubation periods, usually with only a partial ‘take’. The remaining two sources (SCR2, 6) produced no clinical disease in mice, although vacuolar pathology, diagnostic for TSE disease, was seen in the brain of one mouse in each transmission, 700 and 763 days after injection.


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Table 2. Results of BSE and scrapie transmissions

 
Serial mouse-to-mouse passage of BSE and scrapie isolates
Serially mouse-to-mouse passaged isolates have been established from the three BSE sources and as many of the scrapie sources as possible. Agent strain characteristics, based on incubation periods and lesion profiles, were tested in the mouse strain panel after up to three serial mouse-to-mouse passages. Figs 1 and 3 show the latest incubation period data available for each isolate. The same two distinct mouse-passaged strains, designated ‘301C’ and ‘301V’, were isolated from each BSE source: 301C, by serial passage in either C57BL or RIII mice (Fig. 1) and 301V by serial passage in VM mice (Fig. 3). These two BSE-derived strains had distinct lesion profiles (illustrated in Figs 2a and 4a), which also differed from the characteristic profiles seen in primary transmissions of BSE to mice (Fraser et al., 1992 ). 301C and 301V do not resemble any previously characterized mouse-passaged scrapie strains, in either their incubation periods or lesion profiles. It is notable that 301V in VM mice has the shortest incubation period we have ever observed for TSE disease in non-transgenic mice (around 120 days).



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Fig. 1. Mean incubation periods (±SEM) in mice infected with Sincs7 mouse-passaged isolates derived from BSE and sheep scrapie sources. Each isolate was tested in the following mouse groups: RIII (blue diamonds), C57BL (red circles), VM (yellow triangles) and C57BLxVM (grey squares). Data are given for C57BL-passaged isolates (C) and RIII-passaged isolates (R), at either the first or third mouse-to-mouse passage, as indicated. The results are compared with archive data for strains (ME7, 87A) previously isolated from natural scrapie.

 


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Fig. 2. (a) Examples of lesion profiles in mice infected with Sincs7 mouse-passaged isolates derived from BSE and sheep scrapie sources. Profiles are shown for C57BL-passaged (dashed line) and RIII-passaged (solid line) isolates from each source. The profiles are derived from the same experiments as are shown in Fig. 1. Examples are given of ME7-like (SCR1) and 221C-like (SCR4) scrapie profiles and these are compared with profiles from a BSE isolate (BSE1). The SCR8 source gave a 221C-like profile when passaged in RIII mice and a different profile when passaged in C57BL mice. (b) Dendrogram showing the hierarchical clustering of lesion profiles in mice infected with Sincs7 mouse-passaged isolates derived from BSE and sheep scrapie sources. The profiles are derived from the same experiments as are shown in Fig. 1. The analysis shows that the BSE-derived isolates (BSE1, 2, 3) are distinct from the scrapie isolates, and that the ME7-like (SCR1, 3, 9, 11) and 221C-like isolates (SCR4, 5, 10) form separate clusters. The analysis places the RIII-passaged isolate from SCR8 with the 221C-like cluster, but separates it from the C57BL-passaged isolate from the same source.

 
Serial passage lines have been set up in C57BL or RIII mice from eight of the scrapie sources (SCR1, 3, 4, 5, 8, 9, 10, 11) (Fig. 1). Four of the sources (SCR1, 3, 9, 11) have given mouse-passaged isolates with incubation periods closely resembling those of ME7, a strain that was isolated from most natural scrapie sources studied in this laboratory the past (Bruce & Dickinson, 1987 ; Bruce et al., 1991 ). In the case of SCR1, the same strain was isolated by passage in both C57BL and RIII mice. Three other sources (SCR4, 5, 10) have yielded mouse-passaged isolates that are closely similar to each other but unlike any mouse-passaged strain derived from natural scrapie in this laboratory in the past. This uniformity strongly suggests that the same scrapie strain has been isolated from each of these sources; this has been designated ‘221C’. For SCR4 and SCR5, the 221C strain has been isolated by passage in both C57BL and RIII mice. The remaining scrapie source (SCR8) gave a 221C incubation period pattern for the RIII-passaged isolate, but another distinct pattern, unlike either of the above, following a single passage in C57BL mice. At this stage the incubation periods of the C57BL-passaged isolate from SCR8 resemble those of 87A, a strain commonly isolated from natural scrapie cases in the past (Bruce & Dickinson, 1987 ).

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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Fig. 3. Mean incubation periods (±SEM) in mice infected with Sincp7 mouse-passaged isolates derived from BSE and sheep scrapie sources. Each isolate was tested in the following mouse groups: RIII (blue diamonds), C57BL (red circles), VM (yellow triangles) and C57BLxVM (grey squares). Data are given for VM-passaged isolates at either the first or third mouse-to-mouse passage, as indicated. For SCR8, clinical disease was seen only in a small number of C57BL mice. The results are compared with archive data for strains (87V, 111A) previously isolated from natural scrapie sources.

 


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Fig. 4. (a) Examples of lesion profiles in mice infected with Sincp7 (VM) mouse-passaged isolates derived from BSE and sheep scrapie sources. The profiles are derived from the same experiments as are shown in Fig. 3. (b) Dendrogram showing the hierarchical clustering of lesion profiles in mice infected with Sincp7 mouse-passaged isolates derived from BSE and sheep scrapie sources. The profiles are derived from the same experiments as are shown in Fig. 3. The analysis shows that the profiles for BSE-derived isolates (BSE1, 2, 3) are distinct from those for the scrapie isolates (SCR1, 9, 10, 11).

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The remarkable uniformity in the results of primary BSE transmissions to mice, presented here and in previous publications (Bruce et al., 1994 , 1997 ), suggests that each bovine source tested contained the same major strain of agent. This conclusion is supported by the isolation, reported here, of the same mouse-passaged TSE strains from each of three BSE sources tested. Two distinct strains, 301C and 301V, were isolated, depending on which of the two Sinc (or PrP) genotypes of mouse was used for passage. A striking feature of primary BSE transmissions is a 100 day difference in mean incubation period between the two Sincs7 mouse strains, RIII and C57BL. This has been shown, by quantitative trait loci (QTL) analysis, to be a multigenic, PrP-independent effect (Manolakou et al., 2001 ). We have shown here that the same BSE strain, 301C, is isolated by passage in both RIII and C57BL mice. Therefore the incubation period difference in primary transmissions of BSE does not reflect the isolation of different TSE strains. As RIII and C57BL mice are equally susceptible to BSE (Fraser et al., 1992 ), it is likely that the genetic difference between them results in differences in the details of pathogenesis. RIII mice had shorter incubation periods than C57BL mice for most of the mouse-passaged BSE and scrapie isolates described here, suggesting a general effect on TSE pathogenesis, which is strongest in the primary BSE transmissions.

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 5000–10000 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.


   Acknowledgments
 
We would like to thank the staff of the Neuropathogenesis Unit, particularly those concerned with care and monitoring of the experimental animals and the preparation and assessment of histopathological sections. This study was supported by MAFF.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Belt, P. B. G. M., Muileman, I. H., Schreuder, B. E. C., Bos-de-Ruijter, J., Giielkens, A. L. J. & Smits, M. A. (1995). Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. Journal of General Virology 76, 509-517.[Abstract]

Bruce, M. E. (1993). Scrapie strain variation and mutation. British Medical Bulletin 49, 822-838.[Abstract]

Bruce, M. E. & Dickinson, A. G. (1987). Biological evidence that scrapie agent has an independent genome. Journal of General Virology 68, 79-89.[Abstract]

Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. Journal of General Virology 72, 595-603.[Abstract]

Bruce, M. E., Fraser, H., McBride, P. A., Scott, J. R. & Dickinson, A. G. (1992). The basis of strain variation in scrapie. In Prion Diseases of Humans and Animals , pp. 497-508. Edited by S. B. Prusiner, J. Collinge, J. Powell & B. Anderton. Chichester:Ellis Horwood.

Bruce, M., Chree, A., McConnell, I., Foster, J., Pearson, G. & Fraser, H. (1994). Transmission of bovine spongiform encephalopathy and scrapie to mice – strain variation and the species barrier. Philosophical Transactions of the Royal Society of London B Biological Sciences 343, 405-411.

Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., Cousens, S., Fraser, H. & Bostock, C. J. (1997). Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389, 498-501.[Medline]

Carp, R. I. & Callahan, S. M. (1991). Variation in the characteristics of 10 mouse-passaged scrapie lines derived from five scrapie-positive sheep. Journal of General Virology 72, 293-298.[Abstract]

Dickinson, A. G. (1976). Scrapie in sheep and goats. In Slow Virus Diseases of Animals and Man , pp. 209-241. Edited by R. H. Kimberlin. Amsterdam:North-Holland.

Dickinson, A. G. & Meikle, V. M. H. (1971). Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent. Molecular and General Genetics 112, 73-79.[Medline]

Dickinson, A. G., Meikle, V. M. H. & Fraser, H. (1968). Identification of a gene which controls the incubation period of some strains of scrapie agent in mice. Journal of Comparative Pathology 78, 293-299.[Medline]

Foster, J. D. & Dickinson, A. G. (1988). The unusual properties of CH1641, a sheep-passaged isolate of scrapie. Veterinary Record 123, 5-8.[Medline]

Foster, J. D., Hope, J. & Fraser, H. (1993). Transmission of bovine spongiform encephalopathy to sheep and goats. Veterinary Record 133, 339-341.[Medline]

Foster, J. D., Bruce, M., McConnell, I., Chree, A. & Fraser, H. (1996). Detection of BSE infectivity in brain and spleen of experimentally infected sheep. Veterinary Record 138, 546-548.[Medline]

Foster, J. D., Parnham, D., Chong, A., Goldmann, W. & Hunter, N. (2001). Clinical signs, histopathology and genetics of experimental transmission of BSE and scrapie to sheep and goats. Veterinary Record 148, 165-171.[Medline]

Fraser, H. & Dickinson, A. G. (1968). The sequential development of the brain lesions of scrapie in three strains of mice. Journal of Comparative Pathology 78, 301-311.[Medline]

Fraser, H., Bruce, M. E., Chree, A., McConnell, I. & Wells, G. A. H. (1992). Transmission of bovine spongiform encephalopathy and scrapie to mice. Journal of General Virology 73, 1891-1897.[Abstract]

Fraser, H., Pearson, G. R., McConnell, I., Bruce, M. E., Wyatt, J. M. & Gruffydd-Jones, T. J. (1994). Transmission of feline spongiform encephalopathy to mice. Veterinary Record 134, 449.[Medline]

Goldmann, W., Hunter, N., Martin, T., Dawson, M. & Hope, J. (1991). Different forms of the bovine PrP gene have five or six copies of a short, G–C-rich element within the protein coding exon. Journal of Geneneral Virology 72, 201-204.

Goldmann, W., Hunter, N., Smith, G., Foster, J. & Hope, J. (1994). PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie. Journal of General Virology 75, 989-995.[Abstract]

Hoinville, L., McLean, A. R., Hoek, A., Gravenor, M. B. & Wilesmith, J. (1999). Scrapie occurence in Great Britain. Veterinary Record 145, 4-5.

Horn, G., Bobrow, M., Bruce, M., Goedert, M., McLean, A. & Webster, J. (2001). Review of the Origin of BSE. Published by Department for Environment, Food & Rural Affairs: www.defra.gov.uk/animalh/bse/bseorigin.pdf.

Hunter, N., Dann, J. C., Bennett, A. D., Somerville, R. A., McConnell, I. & Hope, J. (1992). Are Sinc and the PrP gene congruent? Evidence from PrP gene analysis in Sinc congenic mice. Journal of General Virology 73, 2751-2755.[Abstract]

Hunter, N., Goldmann, W., Smith, G. & Hope, J. (1994). Frequencies of PrP gene variants in healthy cattle and cattle with BSE in Scotland. Veterinary Record 135, 400-403.[Medline]

Kimberlin, R. H., Cole, S. & Walker, C. A. (1987). Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. Journal of General Virology 68, 1875-1881.[Abstract]

Kimberlin, R. H., Walker, C. A. & Fraser, H. (1989). The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. Journal of General Virology 70, 2017-2025.[Abstract]

Manolakou, K., Beaton, J., McConnell, I., Farquhar, C., Manson, J., Hastie, N. D., Bruce, M. & Jackson, I. J. (2001). Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice. Proceedings of the National Academy of Sciences, USA 98, 7402-7407.[Abstract/Free Full Text]

Moore, R. C., Hope, J., McBride, P. A., McConnell, I., Selfridge, J., Melton, D. W. & Manson, J. C. (1998). Mice with gene targetted prion protein alterations show that Prnp, Sinc and Prni are congruent. Nature Genetics 18, 118-125.[Medline]

Westaway, D., Goodman, P. A., Mirenda, C. A., McKinley, M. P., Carlson, G. A. & Prusiner, S. B. (1987). Distinct prion proteins in short and long scrapie incubation period mice. Cell 51, 651-662.[Medline]

Received 25 September 2001; accepted 23 October 2001.