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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code SE1439

2. Project title

The relative transmissibility of sheep and cattle BSE to humans.

3. Contractororganisation(s)

Institute for Animal HealthNeuropathogenesis UnitOgston BuildingWest Mains RoadEdinburghEH9 3JF

54. Total Defra project costs £ 1,153,100.00(agreed fixed price)

5. Project: start date................ 01 October 2001

end date................. 30 September 2006

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

The transmissible spongiform encephalopathy (TSE) diseases are fatal, infectious neurodegenerative diseases of animals, including humans. The infectious nature of these diseases makes them different from other neurodegenerative disorders, and presents a risk of transmission between individuals of the same or different species by oral exposure in foodstuffs, or by surgical intervention. Such cross species transmissions are thought to have already occurred where BSE has been transmitted from cows to man, but no evidence has yet emerged to demonstrate that scrapie has been transmitted from sheep to man. Recent and ongoing monitoring has found no evidence for the presence of BSE in European sheep. However if BSE had passed from cows to sheep, would it retain the strain characteristics of BSE, thus allowing transmission from sheep to man, or would it adopt the strain characteristics associated with sheep scrapie which does not appear to transmit to man? This project (SE1439) was designed to model the transmission between species (including humans) of different natural TSEs. Transgenic mice were produced that express the PrP gene of cow or human instead of the endogenous mouse gene. Two mouse lines were produced that expressed the bovine Prnp gene containing either five (B5) or six (B6) octapeptide repeats (present in the UK cattle population), and two lines expressing the human PRNP gene with either methionine (HuMM) or valine (HuVV) at the codon 129 polymorphism site. These two human transgenic lines were bred together to create the HuMV line thus providing representative lines of the three different genetic combinations observed in the human population. These mouse lines mice were Cross species transmission of TSE disease often leads to increased incubation times and a decreased susceptibility to disease. The nature of this species barrier is not clear. It has been proposed to be due to differences in PrP protein sequence and/or structure between the host and donor of infectivity. In producing mice expressing bovine PrP, we hypothesized that the incubation time would decrease compared to the wild type controls. We, in fact, observed the opposite; the mean incubation times of the B5 and B6 mouse lines were significantly longer (637±7 days and 551±12 days respectively) compared to the wild type 129Ola mice (447±27 days). However the bovine transgenic mice did appear to be more susceptible to BSE than the wild type mice, with 100% of bovine transgenic mice showing clinical or pathological signs of TSE disease, compared to only 80% of wild type mice. The BSE strain passaged through sheep prior to inoculation into the mice retained similar mean incubation times (B5 652±18 days, B6 564±8 days and 129Ola 474±22 days). None of the transgenic mice developed clinical disease with scrapie. Similarly, none of the human transgenic mice developed clinical disease following infection with any of the TSE isolates. These lines were, however, susceptible to infection by a number of different human isolates (vCJD and sCJD) when infected during a parallel project. These experiments demonstrate the PrP of the host has an important influence on the incubation time of the disease but increasing the

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identity between host and donor PrP does not always lead to a shortening of incubation time. Moreover this experiment demonstrates that a lengthening of incubation time can correlate with an apparent increase in susceptibility and thus incubation time differences are not always an accurate guide to host susceptibility

Pathological analysis of all animals revealed a number of bovine transgenic mice which showed vacuolar pathology in the absence of clinical disease following infection with BSE and experimental sheep BSE. Additionally, a small number of mice showed only PrPSc deposition in the absence of both vacuolar pathology and clinical signs of TSE. Although no signs of BSE transmission were observed in human transgenic mice, a single HuMM brain infected with sheep-passaged BSE showed a very limited focus of PrP deposition which requires to be more fully assessed through additional experimentation. The possible extent of subclinical BSE in cattle and the potential for subclinical disease on transmission of BSE in humans warrant further investigation. The use of larger groups of animals in further experiments may allow these risks to be more fully assessed.

When PrPSc is extracted from infected tissue and examined using immunoblotting techniques, three distinct protein bands are observed representing its three different glycoform states. PrPSc of different TSE isolates can be characterized in this way since differences in size can be easily discerned. The characteristics of PrPSc extracted from the brains of infected mice was assessed by immunoblot. PrPSc

from BSE-infected and experimental sheep BSE-infected mice showed similar characteristics to that of cattle BSE in that a heavy diglycosylated band was observed with the monoglycosylated and unglycosylated bands being far less intense. Some PrPSc could be detected in the brains of mice that had shown no clinical signs of disease and only some of which had evidence of vacuolar pathology. This supported the pathological evidence described above and reasserts the need to determine the extent of subclinical disease or carrier status in both animal and human populations.

In summary, cattle BSE and experimental sheep BSE were shown to infect transgenic mice expressing bovine PrP with an almost 100% attack rate. Neither the bovine nor the human transgenic mice were susceptible to natural scrapie. Transgenic mice expressing human PrP were not susceptible to BSE of cattle or sheep. Although the bovine transgenic mice had pathological evidence of subclinical disease, no evidence was found in the brains of the human transgenic mice infected with BSE (although further experimentation is required to fully assess sheep BSE in this respect). We, therefore, suggest that our mouse model demonstrates that there is a low risk of transmission of BSE and scrapie to humans. However, individuals who have succumbed to vCJD may have done so through multiple exposures to BSE. The model we have described here would allow us to examine this possibility and to assess whether multiple exposure does indeed increase the likelihood of BSE transmission.

In light of the current need to assess the risk to humans of transmission of atypical scrapie, a new Defra project has been initiated (SE1441) which will infect the human transgenic and B6 mouse lines using six isolates of atypical scrapie from the Veterinary Laboratories Agency (VLA). The same analytical techniques will be applied to that work and we will be able to compare the data with that which we obtained during the work of SE1439. Ultimately we will be able to determine the risk to humans from atypical scrapie and identify whether any specific genotype is most at risk. We concluded, therefore, that there is a significant species barrier between cattle BSE and humans which has limited the number of clinical vCJD cases in the human population but subclinical disease or could be a significant issue in both bovine and human populations.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

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details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Project Aim.The aim of project SE1439 was to determine the relative transmissibility of sheep and cattle BSE to humans.

Background.While there is significant evidence to support the theory that BSE had been transmitted from cows to man1-3, there is to date no evidence to suggest that sheep scrapie is infectious to man. Questions, however, remain unanswered as to whether or not BSE has been transmitted from cattle to sheep. Experimental transmissions of BSE between cattle and sheep have been achieved, but it is unknown whether such transmissions have occurred naturally. However BSE has recently been identified in goats in Europe 4 suggesting that transmission between ruminant species is possible. Although the BSE strain appears to be highly stable, on transmission to sheep it may either retain BSE strain characteristics which would allow transmission from sheep to man, or adopt strain characteristics associated with sheep scrapie which does not appear to transmit to man (which would also be more difficult to identify). Although epidemiological monitoring of the British flock has revealed no evidence of BSE in sheep5 this is still an area of concern.

Previous studies at the NPU and elsewhere have shown that a species barrier exists whereby cross species transmission of TSE disease often leads to increased incubation times and decreased susceptibility to disease. The nature of this species barrier is not clear. It has been proposed to be due to differences in PrP protein sequence and/or structure between the host and donor of infectivity1, 6, 7. There are a number of polymorphisms in the PrP sequence of mice, sheep, cows and human that influence the incubation time of disease within each species8-12 although it seems likely that additional factors other than the PrP coding sequence may also influence the course of disease. In order to model the transmission of different natural TSEs between species (including to humans), gene targeted transgenic mice were produced in which the endogenous murine Prnpa gene was replaced with the bovine or human equivalent. Since these transgenic mice were produced using homologous recombination, expression of the foreign gene occurs under control of the endogenous promoter, ensuring equivalent cell specific expression in all transgenic lines thus enabling like-for-like comparison of all mice. To account for the species specific variation in Prnp alleles a number of different transgenic lines were produced:

B5 - bovine Prnp gene containing five octapeptide repeats B6 - bovine Prnp gene containing six octapeptide repeats HuMM - human PRNP gene with methionine at codon 129 HuVV - human PRNP gene with valine at codon 129

The two human transgenic lines (HuMM and HuVV) are bred together to create the heterozygous line HuMV to model the true genetic variation present in the human population. In addition to these mouse lines, the background strain, 129/Ola, was employed as a control in all experiments. An ovine transgenic line was also to be used in this project. Due to problems during the production of these mice, delays were incurred that meant that insufficient time remained to include them in this work. These mice were to be included as positive controls in the scrapie transmission experiment but we do not believe the absence of any data relating to this line of mice will have a detrimental effect on the interpretation of the data produced within the remit of this work.

ObjectivesThe objectives of this project will be referred to throughout this Project Report. They were laid out in the original application as follows:

1. To obtain and aliquot all inocula - cattle BSE, sheep passaged BSE and natural scrapie.

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2. Breed lines of transgenic mice to assemble groups for inoculation.3. To set up transmission experiments.4. To determine incubation times and assess the pathology in each line in terms of vacuolation and PrP

deposition. To assess PrPSc by immunoblot analysis for each transmission.5. To analyse data, produce dose response curves.6. To provide an estimate of infectivity for each inoculum in each transgenic line.

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MethodsSamples of cattle or sheep TSE-infected brain tissue were obtained (objective 1). From these samples, homogenate was prepared at a 10-1 dilution from 500 mg of each of the three inocula (objective 1): cattle BSE (CVL BBP12/92), sheep-passaged BSE (NPU J2501 (ARQ/ARQ)) or NPU natural scrapie (NPU P1019 (VRQ/VRQ)). Groups of 24 of each mouse line (B5, B6, HuMM, HuMV, HuVV and 129Ola) were bred containing aged matched individuals where possible and equal numbers of males and females (objective 2). The mice were intracerebrally (i.c.) inoculated in separate experiments with each inoculum; 20 µl of inoculum per mouse (objective 3). All the inoculations in each separate experiment were performed on the same day. Following the inoculations, the mice were monitored daily then scored once a week for signs of clinical disease using standard procedures. Mice showing signs of TSE disease were culled at a pre-defined clinical endpoint. Incubation time of disease was calculated as time from inoculation to cull with terminal disease (objective 4). The brain was removed for pathological study (objective 4). One half of the brain was fixed in formal saline, further trimmed to expose a number of different regions of the brain (frontal cortex, cortex, hippocampus, thalamus, cerebellum and brain stem) then wax embedded to allow 6 µm sections to be cut for use in pathological analysis of the tissue. The second half of each brain and the entire spleen were flash frozen in liquid nitrogen and stored at -80 C for biochemical analyses (objective 4).

Analysis of vacuolar pathology6 µm sections were cut from each mouse brain and stained using haematoxylin and eosin. Nine areas of grey matter (medulla, cerebellum, superior colliculus, hypothalamus, thalamus, hippocampus, septum and cerebral cortex) and three areas of white matter (cerebellar white matter, midbrain white matter and cerebral peduncle) were examined for vacuolation and scored on a scale of 0 (no vacuolation) to 5 (severe vacuolation) for the presence and severity of vacuolation. The scoring was performed blind by trained individuals and logged on the animal database. Average vacuolation scores for each mouse group in each experiment were calculated and a lesion profile compiled.

Immunocytochemical analysis of PrPSc depositionTwo methods of immunocytochemical (ICC) analysis were employed in this project. Initial studies were performed using ABC Elite kit from Vectastain (PK 4000). 6 µm sections were pretreated by alcohol dehydration and hydrolytic autoclaving prior to incubation with monoclonal antibody 6H4 (Prionics) at room temperature overnight (1µg/ml). Following several washes in PBS/BSA, biotinylated secondary rabbit anti-mouse antibody (Jackson Immuno Research Laboratories, UK) was added (2.5µg/ml) and incubated at room temperature for 1 h. After further washes, PrPSc detection was performed using the Vectastain ABC kit, with hydrogen peroxidise-activated diaminobenzidine (DAB) substrate. PrPSc deposition was detected as a brown precipitate using light microscopy.

When low or undetectable levels of PrPSc were observed with the ABC Elite kit, repeat sections were analysed using the DAKO Catalysed Signal Amplification (CSA) kit (product code K1500). This high sensitivity ICC kit works using the same principals as the method described above, but includes an additional amplification step involving a streptavidin-biotin-peroxidase complex and biotinylated phenol resulting in an amplification of the number of biotin molecules available for binding to the streptavidin-peroxidase. As with the first technique, staining is completed using DAB/hydrogen peroxide. When the DAKO CSA kit was used, the 6H4 concentration was reduced to 50ng/ml to reduce the levels of background staining. The secondary antibody was supplied in the kit.

Immunoblot analysis of PrPSc

In order to maximise sensitivity of detection, two different methods of sample preparation for immunoblot analysis were employed. In the first method 10% brain homogenates were prepared in NP40 buffer (0.5% NP40, 0.5% sodium deoxycholate, 150mM NaCl, 50mM Tris/HCl pH 7.5) and clarified by low speed centrifugation (10,000g/15min). The clarified supernatant was treated with 20 μg/ml Proteinase K for 1 hour at 37 C. In the second method to increase sensitivity of detection, PrPSc was selectively precipitated out of the clarified supernatant using Sodium Phosphotungstic Acid (NaPTA). Samples produced by both methods, were denatured and loaded onto a 12% Novex Tris/Glycine gel (Invitrogen, UK). After electrophoresis proteins were transferred onto PVDF membrane by electroblotting. PrP bands were detected using the SuperSignal West Dura chemiluminescence detection kit (Pierce) with primary antibody 8H4 (Dr Man-Sun Sy, Ohio, USA) at 50ng/ml and an anti-mouse IgG peroxidase-linked secondary antibody (Jackson Immuno Research Laboratories, UK) at 100ng/ml. Images were captured on x-ray film and by a Kodak 440CF Digital Imager.

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ResultsInfection of bovine transgenic mice using cattle BSE.Given the hypothesis that the TSE transmission barrier, or species barrier, is caused by differences in PrP sequence between host and donor of infectivity, we predicted that expression of the bovine PrP gene in transgenic mice would lead to reduced incubation times and greater susceptibility to BSE, with respect to wild type mice. However, following i.c. inoculation with BSE the incubation time of disease in B5 (637±7 days) and B6 (551±12 days) transgenic mice was significantly longer than in the 129Ola mice (447±27 days) (table 1). These data clearly indicate that factors in addition to sequence similarity play an important role in determining the incubation time of disease. Also the incubation time of the B5 group was significantly longer than that of the B6 group, thus indicating a role of the octapeptide repeat length in determining the incubation time of disease. The numbers of mice in each group exhibiting clinical signs of disease were 46%, 68% and 75% of the B5, B6 and 129Ola groups respectively (table 1). This indicated a lack of full penetrance of BSE in these mouse lines.

It is possible that increased incubation times with BSE were due to incompatibility between other bovine and murine factors. In order to address this possibility, we performed sub-passage of a BSE-infected B5 brain into further groups of B5, B6 and 129Ola mice (i.c.) to see if the incubation time would decrease on adaptation of the agent to mouse. However no decrease in incubation time was observed. Indeed slight increases of 59, 37 and 12 days were observed in the B5, B6 and 129Ola mice respectively (table 1). This was somewhat unexpected and we assume this result is due to a lower titre of infectivity in the B5 brain than in the original cattle brain. Unfortunately there was insufficient material remaining following the subpass to perform a full titration which would be required to determine if this was indeed the explanation.

Brain tissue from all BSE inoculated transgenic and 129Ola mice was analysed for the presence of TSE associated vacuolar pathology and PrPSc deposition. All bovine transgenic mice (B5 and B6) inoculated with BSE showed positive TSE disease pathology, whereas only 80% of the 129Ola mice showed either vacuolation or PrPSc deposition. The average vacuolation scores for each experimental group were compiled as described (see methods) and plotted against brain area to produce a lesion profile (figure 1). The profiles of the B5 and B6 mice were very similar but differed significantly from that of the 129Ola controls. Whereas the greatest amounts of vacuolation in the 129Ola mice were located in the medulla, hypothalamus and septum, the bovine transgenic mice showed less vacuolation in the hypothalamus; rather it was observed in the superior colliculus, thalamus, hippocampus and cerebral cortex. This indicates that the regional targeting of BSE in the brain was altered when bovine PrP was expressed in place of the endogenous murine protein. Similarly, when the PrPSc deposition was studied in the brains of these mice (using ICC analysis) it was noted that PrPSc was found in different regions of the bovine transgenic brains compared to the 129Ola brains. In the B5 and B6 brains significant levels of PrP Sc

were observed in the thalamus, medulla, dorsal motor nuclear vagus and hippocampus (figure 2). Some PrPSc

deposition was located in the molecular layer of the cerebellum, but at lower levels. In the 129Ola mice, however, PrPSc deposition was specifically targeted to the CA2 region of the hippocampus, although the thalamic deposition was similar to that of the bovine transgenic mice. Overall, PrPSc deposition was present as medium to large punctuate deposits in the brain. No amyloid deposits were detected following staining using thioflavin.

The presence of vacuolar pathology and PrPSc deposition in all bovine transgenic mice inoculated with BSE indicated that expression of the bovine PrP gene did appear to increase susceptibility to BSE (from 80% to 100%), but also resulted in the extension of the incubation time, possibly beyond the lifespan of the mouse. 9 out of 13 clinically negative B5 and 4 out of 7 clinically negative B6 mice showed vacuolar pathology. Further to this, the remaining clinically negative mice in both groups showed PrPSc deposition despite having no clinical or vacuolar signs of disease (figure 3). While this PrPSc deposition was not as evident as that seen in terminally ill animals, it was clearly identifiable by ICC. These clinically negative mice were culled for intercurrent disease between 330-500 days post injection and it is likely that in due course they would have developed symptoms of disease, but we cannot say this definitively. The finding is, however, very important to note since it implies that there is a risk that BSE infection could remain undetected and it underlies the need for reliable diagnostic tests especially for older cattle entering the food chain.

Biochemical analysis of the PrPSc in BSE infected brain tissue was performed by immunoblot of PK-treated brain homogenate. The blots were performed as described (see methods) and showed that PK resistant PrPSc was present in the brains of B5, B6 and 129Ola mice. In all brains analysed the characteristic BSE glycoform (prominant diglycosylated band) was observed (Figure 4). The PrPSc produced by B5 and B6 mice was of slightly higher molecular weight than that of the 129Ola PrPSc, but this not unexpected given the additional octapeptide repeats present in the transgenic PrP, and has been observed previously on characterisation of PrP C in uninfected transgenic mice.

Overall we concluded that expression of the B5 and B6 alleles in transgenic knockin mice led to an increase in disease susceptibility, and a change in incubation time of disease and targeting in the CNS compared to the wild type controls. Additionally, there was evidence of sub- or preclinical disease as shown by the presence of vacuolation and PrPSc deposition in the mouse brains despite the absence of clinical signs of disease.

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Infection of bovine transgenic mice using sheep-passaged BSE.In order to assess the capacity for BSE to retain infectivity and strain characteristics following passage through sheep, a homogenate prepared from the brain of an experimentally BSE-infected sheep was used to infect 24 each of B5, B6 and 129Ola mice. The results obtained demonstrated that the sheep-passaged BSE produced the same disease profile in these mice as had been observed following cattle BSE infection, indicating that the strain specific characteristics of BSE had been retained on passage through sheep. The incubation times in the B5, B6 and 129Ola mice were 652±18 days, 564±8 days and 474±22 days respectively. The proportion of each group showing clinical signs of disease were 58% (B5), 70% (B6) and 59% (129Ola). As observed in the BSE-infected mice, the total number of affected animals increased when the vacuolar pathology and PrPSc deposition data were included such that 95% of B5, 90% of B6 and 65 % of 129Ola mice showed signs of TSE disease following infection with sheep-passaged BSE. The lesion profiles (figure 1), patterns of PrPSc deposition (figure 5) and glycoprofiles (figure 4) mirrored those observed in the BSE infected animals. Overall there was no discernable difference between like-for-like groups infected using cattle or sheep-passaged BSE.

As with the bovine transgenic mice infected using cattle BSE, the sheep-passaged BSE infected mice showed degrees of sub clinical infection. 5 clinically negative B5 mice and 3 clinically negative B6 mice showed vacuolar pathology with a further one mouse in each group showing PrPSc deposition in the absence of any clinical or pathological signs. This again indicates the potential that BSE infection could remain undetected in animals unless full post mortem diagnostic techniques were applied.

Intraperitoneal infection of bovine transgenic mice using cattle BSE.TSEs are thought to be acquired mainly by the oral route, and although i.c. inoculation provides an indication of susceptibility, it does not examine the influence of peripheral processing on disease progression. In order to examine the effect of bovine PrP expression in the periphery, the same BSE inoculum as used in the i.c. infection of bovine transgenic mice was employed in the intraperitoneal (i.p.) inoculation of additional groups of the same mouse lines. While 84% of the 129Ola mice showed clinical signs following i.p. injection of BSE, only 31% of the B6 mice and none of the B5 mice showed clinical signs of disease. The vacuolation scores were very low (≤1.2) in the B6 mice and no vacuolation, only PrPSc deposition, was observed in the B5 mice. We, therefore, conclude that the i.p. route of BSE infection is not as efficient in these transgenic models as i.c. infection. As the bovine transgenic mice were produced by gene targeting, expression of PrP in the periphery should be identical to that of wild type mice, and it is unclear why the peripheral route of infection is less efficient in the presence of the bovine gene. Further analysis of the transgenic mice and peripheral tissues from the infected animals will be required to address this.

Infection of bovine transgenic mice using natural scrapie.The brain of a naturally scrapie-infected sheep (from the NPU farm) was homogenized and used to infect B5, B6 and 129Ola mice. The mice were allowed to age to >800 days and after that time only one, a 129Ola, showed any clinical signs of disease (incubation period 594 days). A further 129Ola mouse showed evidence of subclinical PrPSc deposition by ICC but no evidence of disease was observed by any methods in any of the other animals, transgenic or wild type. This result suggests an extremely low transmissibility of scrapie to cattle consistent with the presence of a species barrier.

Infection of human transgenic mice using cattle or sheep-passaged BSE and natural scrapie.In order to model the susceptibility of humans to ruminant TSE isolates, the same BSE and natural scrapie inocula used to infect the B5 and B6 mice were inoculated into human transgenic mouse lines HuMM, HuMV and HuVV (infections performed at the same time as the bovine transgenic inoculations). Despite the high BSE susceptibility rates observed in the B5 and B6 mice, no definitive evidence of TSE disease was observed in the human transgenic mice . Additionally, no transmission of natural sheep scrapie to human transgenic mice was observed (table 1). Brain sections from all mice were analysed by ICC using the sensitive DAKO CSA kit (figure 2, 5 and 6), but no PrPSc was detected. Furthermore PrPSc was not detected by immunoblot, even following centrifugal purification (SAF preparation) of detergent insoluble PrP. In a single HuMM mouse infected using sheep-passaged BSE, a small foci of PrPSc deposition was detected using the DAKO CSA kit (figure 5). Given the location, amount and appearance of the deposition it was difficult to confirm whether or not it was true staining or whether it was an artefact. Unfortunately as this animal showed no clinical signs of disease and was culled for welfare reasons, the brain was not removed aseptically so subpassage of the material to further mice was not possible to assess the presence of infectivity (This observation is being further investigated by repeat transmission of experimental sheep BSE to human transgenic mice in a new DEFRA contract).

Based on the evidence obtained in these transmissions, we conclude that humans have a low susceptibility to BSE either from cattle or passaged through sheep, and natural sheep scrapie. However, failure to transmit disease to the human transgenic mice could also reflect a transgene construction problem which prevents TSE infection. As a control for ruminant TSE transmission, a parallel project (Dept. Health 007/0085) has studied the susceptibility of the human transgenic lines to different isolates of human TSE disease, namely vCJD and sCJD. Two of 17 HuMM transgenic mice showed clinical symptoms following inoculation with vCJD. However, of the 17 HuMM mice 11 showed subclinical signs of PrPSc deposition and vacuolation. The HuMV transgenic mice also

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showed low susceptibility, with one clinical case and 11 pathological cases out of 16 mice. The HuVV mice showed the lowest susceptibility with only one animal showing subclinical disease out of 16. Different susceptibilities were noted following infection using a number of different sCJD isolates but the overall evidence allows us to dismiss any possibility that the human transgenic mice are not expressing PrP (thereby leading to resistance to TSE infection). Rather the evidence from this project (SE1439) supports the ongoing epidemiological and surveillance data that indicates a low incidence rate of transmission of BSE from cattle to humans.

Estimation of infectivity for each inoculum in each transgenic lineWhen designing this programme of work, it was anticipated that bovine transgenic mice would provide a model of BSE transmission with short incubation times which would allow us to titrate infectivity levels in different BSE isolates and construct dose response curves for the analysis of further samples. The cattle BSE inoculum was serially diluted to concentrations of 10-1-10-7 then injected i.c. into groups of B5, B6 and 129Ola mice. Titres of infectivity of 105.52, 105.18 and 104.65 were calculated for the B5, B6 and 129Ola mice respectively. However, as described previously the numbers of affected mice were low and the ID50 calculation was, as a result, difficult to make due to the long incubation periods of disease observed in these mice. As detailed in the original proposal subsequent titrations were to be set up on the basis of successful transmissions. Due to the long incubation periods of some of the inocula in some of the mouse lines, and given the absence of transmission to the human transgenic lines or following scrapie infection, we considered that further titration experiments were inappropriate and would constitute an unethical use of a large number of mice. This was agreed by Defra.

SummaryTo summarize the results of this project:

• PrP sequence has a major influence over incubation time of TSE disease • Although sequence identity between the host and the donor of infectivity can increase disease incidence,

it does not always lead to shorter incubation times.• The disease profiles of cattle BSE and sheep BSE are indistinguishable from each other in the bovine

transgenic and 129/Ola lines.• In the bovine transgenic and 129/Ola lines a proportion of the total affected cases showed no clinical

disease, only vacuolation or PrPSc deposition suggesting that there is a risk of sub clinical disease being present in some individuals.

• No definitive signs of TSE disease were detected in the human transgenic lines suggesting that humans have a low susceptibility to BSE of cattle or passaged through sheep.

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DiscussionThe true nature of the species barrier observed in TSE disease transmission is unknown. Efficient disease transmission is seen to occur between individuals with identical PrP sequence, and it was therefore proposed that PrP sequence identity was the key to the species barrier. We anticipated that the introduction of a foreign PrP sequence into transgenic mice by gene targeting would remove the barrier to transmission to mice and allow transmission of disease from another species with high susceptibility and short incubation times. Although this has been shown in mice overexpressing foreign PrP sequences 13 it is unclear from these experiments whether shortened incubation times are due to the PrP sequence or the degree of overexpression of the PrP gene. In the experiments described here using gene targeted transgenic mice with physiological levels of PrP expression, it is clear that the expression of bovine PrP does indeed result in high levels of susceptibility to BSE in mice, but with incubation times which are longer than those observed in wild type mice. While BSE transmits efficiently to transgenic mice expressing bovine PrP, it does not transmit to mice expressing human PrP, indicating the presence of a significant species barrier for BSE infection of humans. This result applies whether the BSE isolate is from cattle or following passage through sheep. These data imply that it is a combination of PrP sequence and strain of agent which determines both incubation time of disease and degree of susceptibility.

There has been concern that BSE may have been transmitted to sheep, and it is unknown whether the resulting disease would be of similar or increased risk to humans. In addition it was unknown whether the phenotype of such BSE transmission in sheep would be recognised. Transmission of experimental sheep BSE to bovine transgenic mice produced identical clinical and pathological profiles to cattle BSE and, as such, indicates a high degree of stability of the BSE strain. Although no definitive transmission of experimental sheep BSE to human transgenic mice was observed, we have to assume that the agent, if it were to infect sheep naturally, would be of similar risk to humans as cattle BSE. This risk should be explored by further experimental transmission studies (see later).

Although both 129Ola mice and bovine transgenic mice are susceptible to BSE and experimental sheep BSE, the replacement of the murine gene with the bovine gene resulted in significant changes in disease pathology. No lesion profile or PrPSc deposition differences were observed between the B5 and B6 lines. However, when compared to the 129Ola control line significant differences in both targeting of vacuolation and PrPSc deposition were observed. This indicates that PrP sequence has an effect on the localization of PrPSc deposition and the degree of vacuolar pathology in different regions of the brain. Whether this is due to alternative folding patterns of the PrPSc or due to different interactions of PrPSc with the mouse cellular proteins we can not predict from these results. These results do, however, raise some interesting possibilities for future in vitro studies using primary cell cultures derived from the transgenic lines. Protein-protein interactions could be studied in order to identify any differences in interactions between cellular proteins and bovine transgenic PrP or wild type (129Ola) PrP. In vitro conversion of the transgenic versus wild type PrP could also be studied using techniques as described in Kirby et al.14 2006.

It will also be interesting to assess protein interactions and conformational stability based on the extra octapeptide repeat in the B6 transgenic line since this extra repeat had an apparent effect on incubation time of disease. The work of Castilla et al.15 has previously demonstrated that the presence of an additional octapeptide repeat leads to a reduction in incubation time of disease and a reduced survival rate. In that work, transgenic mice overexpressing PrP with six or seven octapeptide repeats were used and mice expressing the 7 octapeptide PrP showed significant reductions in incubation time. The fact that the B5 and B6 mice are single copy with fewer octapeptide repeats, yet a significant difference in incubation times can still be discerned, demonstrates a critical role of PrP sequence in the control of TSE incubation time.

The lack of clinical disease and vacuolar pathology in human transgenic lines inoculated with BSE suggests that the expression of human PrP has introduced a significant species barrier which confers on these three mouse lines a high degree of resistance to BSE. However it is also possible that genetic manipulation may have for some reason rendered the human transgenic mice resistant to all TSE infection. Through the work of Bishop et al. we have demonstrated that the human transgenic lines are susceptible to vCJD10 and sCJD (unpublished). We are, therefore, satisfied that the results we have obtained here indicate the presence of a species barrier and are not due to an error in transgenic mouse production. If such a significant species barrier exists between cattle and humans, the question is raised as to how 162 individuals in the UK became infected and developed vCJD. Possible causes which are not addressed in this work are multiple dosing of the agent through repeated exposure in food, or a specific age related susceptibility which is indicated by the young age of onset of vCJD. Additionally other specific genetic factors may be involved in the small number of clinical cases observed, however the risk then exists of significant levels of subclinical disease in the general population. This has also been indicated by the identification of two PrP positive appendix tissues in a retrospective survey of archived tonsil and appendix tissue 16, 17, and possibly by the small focus of PrP deposition observed here in one HuMM mouse following inoculation with experimental sheep BSE. Of major importance to this report is the finding that many of the bovine transgenic mice not displaying clinical symptoms of disease did however show vacuolar pathology and/or PrPSc deposition. This indicates that

SID 5 (Rev. 3/06) of 22

subclinical infection could be present in the bovine population which would be more difficult to detect and may be missed altogether if screening was abandoned. Stringent active surveillance measures remain in place to prevent BSE-infected meat entering the food chain. However, these results highlight the need for accurate diagnostic tests and continuing surveillance. The infectious status and level of infectivity in such subclinical disease could be assessed by subpassage of such cases in bovine and human transgenic mice. Similar transmissions from non-clinical BSE inoculated human transgenic mice should also be performed to determine whether any subclinical infection or carriage of infectivity exists in these animals.

These data from transmissions in human and bovine transgenic mice show that the risk to human health from ruminant TSEs appears to be low, mirroring the fact that only 162 clinical cases of vCJD have occurred from the millions of UK individuals who were undoubtedly exposed to the BSE agent during the epidemic. Why these cases occurred is not known, and it is clear that our knowledge of BSE and scrapie is far from complete. Increased surveillance has recently identified new atypical cases of both scrapie and BSE 18-20 and the risk of transmission to humans of these newly discovered diseases remains unclear. We have recently initiated a new study (SE1441) in which the relative transmissibility of different atypical scrapie cases to humans will be assessed using the human transgenic mouse lines described in this report. In separate projects we will also assess the risk of transmission of CWD and BASE to humans. Continued monitoring of stock animals and patients will enable us to prevent spread of TSE disease both to and within the human population, as well as allowing us to determine the risk of future transmissions.

ImplicationsThe major implications of this work are that subclinical BSE infection may exist that is reaching the food chain and, therefore, creating a risk to human health. With the age limits that are imposed in beef production this risk is vastly reduced, but as the original precautionary measures are lifted, it may prove to be prudent to maintain stricter testing of older carcasses before they enter the food chain. Effective diagnostic tests will need to be developed, and refined where they already exist. If the human transgenic mice infected using BSE in this project were harbouring undetectable levels of PrPSc then it is possible that a risk of human-to-human risk of infection may exists and we would like to test this possibility (see Future Work).

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Future workAs evidence of disease pathology was present in clinically unaffected bovine transgenic mice inoculated with BSE, the risk of transmission from such cases should be assessed. It is possible that such cases may have been culled during a pre-clinical phase, but this may mirror the status of some apparently healthy cattle presented for slaughter. In order to establish the levels of infectivity present in the brains of the clinically negative/pathologically positive bovine transgenic mice a sub pass could be performed into a new panel of bovine transgenic and wild type mice. With current surveillance measures in place one would predict that such animals would be identified, but this work could certainly provide new information on associated levels of infectivity and risk of transmissibility.

Although no clinical disease or vacuolar pathology was observed in the human transgenic mice inoculated with BSE, one HuMM mouse which received experimental sheep BSE did show an extremely small focus of PrP deposition in the thalamus by ICC. Although we have been unable to prove whether this represents infection and propagation of the agent, such deposition may correspond to subclinical infection or early stages of disease, and as such merits further investigation by repeat transmission studies (inoculation planned Jan 2007). The apparent lack of BSE transmission in the human transgenic mice, while providing some reassurance, also leaves some questions unanswered. Only approximately 162 individuals of the susceptible PrP codon 129MM genotype have succumbed to vCJD, even though around 40 % of the UK population which was exposed to BSE share that genotype. It is possible that the mouse numbers used in this project (24 mice per genotype) were so low that a low transmissibility rate was indefinable. Further BSE transmissions involving larger groups (up to 100) of each human transgenic mouse line may produce a more accurate assessment of the transmissibility of BSE to humans. We would also propose to perform multiple challenges of HuMM mice by both i.p. and oral routes to determine whether continued exposure to the agent increases the risk of disease. Although no evidence of clinical disease, or TSE disease related vacuolation was observed in BSE infected human transgenic mice, it is possible that subclinical infection may occur, as suggested recently by the identification of PrPSc in lymphoid tissues of 129MV and 129VV individuals 17, 21. We would therefore propose to perform subpassage from the oldest surviving human transgenic mice following inoculation with BSE into human and bovine transgenic mice, 129Ola and also Tg20 indicator mice to determine whether low levels of infectivity are present in these animals. This would provide important information on possible levels of carriage of infectivity in the population and subsequent risk of human-to-human transmission by, e.g. blood transfusion or surgery.

We have recently initiated a new Defra project (SE1441) which follows the work described here and is designed to assess the risk to humans of transmission of atypical scrapie. This new project will infect the human transgenic and B6 mouse lines using six isolates of atypical scrapie from the Veterinary Laboratories Agency (VLA). The same analytical techniques as were used in SE1439 will be applied to the new project and we will be able to compare the data with that which we obtained during the work of the project described in this report. In this way we will be able to determine the risk to humans from atypical scrapie and identify whether any specific genotype is more at risk. In addition, Defra has requested that a second sample of sheep-passaged BSE is also passed through the human and B6 transgenic mouse lines to assess further the risk of transmission of sheep BSE to humans in the light of the identification of the small focus of PrPSc deposition in one HuMM mouse brain. While this may simply have been an artefact, we feel it merits further assessment.

Potential for intellectual property arising from this work.No direct commercialisation opportunities have arisen from this project. However, pilot studies are examining the potential for use of the human and bovine transgenic mice in the production of primary cell cultures. These could be used in in vitro studies of TSE disease as well as providing a potential source of human or bovine PrP. Further experimental work is required to assess such future potential for commercialisation of these primary cultures. The transgenic lines themselves are under consideration for commercial exploitation and we have engaged business development organisation Genecom to assess the marketability of these mice.

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References1. Scott, M. R. et al. Compelling transgenetic evidence for transmission of bovine spongiform

encephalopathy prions to humans. Proceedings of the National Academy of Sciences of the United States of America 96, 15137-15142 (1999).

2. Hill, A. F. et al. The same prion strain causes vCJD and BSE. Nature 389, 448-50, 526 (1997).3. Bruce, M. E. et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent.

Nature 389, 498-501 (1997).4. Eloit, M. et al. BSE agent signatures in a goat. Vet Rec. 156, 523-b-524 (2005).5. Stack, M. J., Chaplin, M. J. & Clark, J. Differentiation of prion protein glycoforms from naturally occurring

sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropathologica 104, 279-286 (2002).

6. Kocisko, D. A. et al. Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc Natl Acad Sci U S A 92, 3923-7 (1995).

7. Race, R. E. et al. Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent. Neuron 15, 1183-91 (1995).

8. Barron, R. M. et al. Polymorphisms at codons 108 and 189 in murine PrP play distinct roles in the control of scrapie incubation time. J Gen Virol 86, 859-868 (2005).

9. Barron, R. M. et al. Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers. EMBO Journal 20, 5070-5078 (2001).

10. Bishop, M. T. et al. Predicting susceptibility and incubation time of human-to-human transmission of vCJD. Lancet Neurol 5, 393-8 (2006).

11. Goldmann, W., Hunter, N., Smith, G., Foster, J. & Hope, J. PrP genotype and agent effects in scrapie - change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie. J. Gen. Virol. 75, 989-995 (1994).

12. Maciulis, A. et al. Polymorphisms of a scrapie-associated fibril protein (PrP) gene and their association with susceptibility to experimentally induced scrapie in Cheviot sheep in the United States. Am J Vet Res 53, 1957-60 (1992).

13. Scott, M. et al. Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847-857 (1989).

14. Kirby, L., Goldmann, W., Houston, F., Gill, A. & Manson, J. A novel resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres. Journal of General Virology 87 (2006).

15. Castilla, J. et al. Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein. Journal of Neuroscience 24, 5063-5069 (2004).

16. Hilton, D. A. et al. Prevalence of lymphoreticular prion protein accumulation in UK tissue samples. Journal of Pathology 203, 733-739 (2004).

17. Ironside, J. et al. Variant Creutzfeldt-Jakob disease; protein protein gentype analysis of positive appendix tissue samples from a retrospective prevalence study. British Medical Journal 332, 1164-1165 (2006).

18. Buschmann, A. et al. Atypical BSE in Germany-Proof of transmissibility and biochemical characterization. Vet Microbiol 117, 103-16 (2006).

19. Konold, T. et al. Atypical scrapie cases in the UK. Vet Rec 158, 280 (2006).20. Casalone, C. et al. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular

similarities with sporadic Creutzfeldt-Jakob disease. Proceedings of the National Academy of Sciences of the United States of America 101, 3065-3070 (2004).

21. Peden, A. H., Head, M. W., Ritchie, D. L., Bell, J. E. & Ironside, J. W. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 364, 527-529 (2004).

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Transgenic Strain

TSE Agent Incubation time ± SEM

Affected1/Total2

129Ola Cattle BSE (ic) 447±27 16/20 (15)

B5 Cattle BSE (ic) 637±7 24/24 (11)

B6 Cattle BSE (ic) 551±12 22/22 (15)

Hu129MM Cattle BSE (ic) no clinical 0/18

Hu129MV Cattle BSE (ic) no clinical 0/23

Hu129VV Cattle BSE (ic) no clinical 0/23

129Ola Cattle BSE (ip) 430±16 24/25 (21)

B5 Cattle BSE (ip) no clinical 7/9 (0)

B6 Cattle BSE (ip) 694±17 10/13 (4)

Hu129MM Cattle BSE (ip) no clinical 0/19

Hu129MV Cattle BSE (ip) no clinical 0/25

Hu129VV Cattle BSE (ip) no clinical 0/24

129Ola Sheep BSE (ic) 474±22 11/17 (10)

B5 Sheep BSE (ic) 652±18 18/19 (11)

B6 Sheep BSE (ic) 564±8 18/20 (14)

Hu129MM Sheep BSE (ic) no clinical 0/24

Hu129MV Sheep BSE (ic) no clinical 0/24

Hu129VV Sheep BSE (ic) no clinical 0/23

129Ola Natural Scrapie (ic) 594 2/25 (1)

B5 Natural Scrapie (ic) no clinical 1/21 (0)

B6 Natural Scrapie (ic) no clinical 0/23 (0)

Hu129MM Natural Scrapie (ic) no clinical 0/24

Hu129MV Natural Scrapie (ic) no clinical 0/24

Hu129VV Natural Scrapie (ic) no clinical 0/24

129Ola Cattle BSE 2nd pass (ic) 514±20 11/17 (11)

B5 Cattle BSE 2nd pass (ic) 630±11 17/18 (12)

B6 Cattle BSE 2nd pass (ic) 588±14 13/15 (10)

Table 1: The incubation times of 129Ola, bovine transgenic and human transgenic mouse lines following infection using BSE, sheep-passaged BSE or scrapie via intracerebral (ic) or intraperitoneal (ip) routes (± standard error). The total number of affected animals per group (by clinical and pathological assessment) are shown with numbers of animals showing clinical signs in parentheses.

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A

0.00

1.00

2.00

3.00

4.00

1 2 3 4 5 6 7 8 9 1* 2* 3*

brain region

129/Ola

B5

B6

B

0.00

1.00

2.00

3.00

4.00

1 2 3 4 5 6 7 8 9 1* 2* 3*

brain region

Fig. 1: Lesion profiles are shown for 129 Ola, B5 and B6 mice infected with BSE (A) or sheep BSE (B). Brain regions: 1. medulla, 2. cerebellum, 3. sup. colliculus, 4. hypothalamus, 5. thalamus, 6. hippocampus, 7. septum, 8. and 9. cerebral cortex, 1*. cerebellar white matter, 2*. midbrain white matter, 3*. cerebral peduncle.

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Figure 2: Immunocytochemical staining of PrPSc in brain of B5, B6, 129Ola, HuMM, HuVV and HuMV mice following BSE infection. Area of brain shown is hippocampus and thalamus. Primary antibody used was 6H4. Scale shown for panels a-f at x4 magnification.

Figure 3: Immunocytochemical staining of PrPSc in brain of B5 and B6 mice recorded as being clinically and pathologically negative following BSE infection. Area of brain shown is hippocampus and thalamus. Primary antibody used was 6H4. Scale shown for panels a-f at x4 magnification. The B5 animal was culled at 556 days post injection and the B6 animal at 330 days post injection.

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A B

C D

Figure 4: Immunoblot analysis of PrPSc isolated from B5, B6, HuMM, HuMV, HuVV or 129 Ola (WT) brains following infection using cattle BSE (panels A and B) or sheep-passaged BSE (panels C and D). Primary antibody used was 8H4. Proteinase K (PK) use is indicated (+). Samples in panels B and D were precipitated from 10% brain homogenate with NaPTA prior to PK digestion. Bands in lanes 2, 4, & 6 in panel B were shown to be non-specific, and reacted with the secondary antibody on duplicate blots when the primary antibody was omitted.

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B5 B6 WT

30

25

PK - + - + - +

HuMM HuVV HuMV WT

PK - + - + - + - +

36

22

B5 B6 WT

36

22

PK - + - + - +

HuMM HuVV HuMV

36

22

PK - + - + - +

Figure 5: Immunocytochemical staining of PrPSc in brain of B5, B6, 129Ola, HuMM, HuVV and HuMV mice following sheep-passaged BSE infection. Area of brain shown is hippocampus and thalamus. Primary antibody used was 6H4. Panels g & h show small focus of PrP deposition in one HuMM inoculated with experimental sheep BSE. Scale shown for panels a-f and h at x4 magnification. Panel g at x20 magnification.

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Figure 6: Immunocytochemical staining of PrPSc in brain of B5, B6, 129Ola, HuMM, HuVV and HuMV mice following natural scrapie infection. Area of brain shown is hippocampus and thalamus. Primary antibody used was 6H4. Scale shown for panels a-g at x4 magnification. Panel g represents one of the few 129Ola animals to show PrPSc deposition.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Meeting abstracts:J.C. Manson. October 2006. TSE strains and role of PrP in host susceptibility. (Oral presentation) Prion 2006, Turin

J.C. Manson, E. Cancellotti, N. Tuzi, P. Hart, F. Wisemann, R.M. Barron, M.T. Bishop, J. Ironside and R. Will. Sept 2006. TSE strains and the role of PrP in host susceptibility. (Oral presentation) Joint Funders’ TSE Workshop, Warwick.

P. Hart, C. Plinston, M.T. Bishop, H. Baybutt, N. Tuzi, L. Aitchison, E. Gall, R.M. Barron, M. Bruce, N. Hunter and J.C. Manson. 2006. The relative transmissibility of sheep and cattle BSE to humans. (Poster) Joint Funders’ TSE Workshop, Warwick.

J.C. Manson (2005). The role of PrP in TSE strains and the species barrier. (Oral presentation) Prion 2005, Dusseldorf, Germany.

J.C. Manson, R.M. Barron, E. Cancellotti, P. Hart, N. Tuzi and M.T. Bishop (2005). The role of PrP in TSE disease. (Oral presentation) 2nd International Dominique Dormont Conference, Paris, France.

J.C. Manson. The transmissible spongiform encephalopathies (2005). SGM Symposium, Herriot Watt University, Edinburgh.

J.C. Manson. October 2004. Animal Models of Prion Disease. (Oral presentation) 2004 International Symposium of Prion Diseases. Sendai, Japan.

P. Hart, M. Bishop, H. Baybutt, N. Tuzi, L. Aitchison, E. Gall, R. Barron, M. Bruce, N. Hunter, J. Manson. September 2004. The relative transmissibility of sheep and cattle BSE to humans. (Poster) Joint Funders’ Meeting, York.

J.C. Manson. June 2004. How does host PrP control TSE disease? (Oral presentation) FASEB Summer Research Conferences. Protein Misfolding, Amyloid and Conformational Disease. Colorado, U.S.A.

J.C. Manson. May 2004. How does host PrP control TSE disease? (Oral presentation) Prion 2004 Conference. Pasteur Institute, Paris, France.

J. C. Manson, R. Barron, N. Tuzi, H. Baybutt, L. Aitchison, R. Moore, D. Melton, J. Ironside and R. Will. October 2003. (Oral presentation) How does host PrP control TSE disease? Prion 2003 Conference, Munich, Germany.

P. Hart, H. Baybutt, N. Tuzi, L. Aitchison, E. Gall, R. Barron, G. O'Neill, M. Bruce, N. Hunter and J. Manson. October 2003. The TSE species barrier in gene targeted mice carrying the bovine PrP gene. (Poster) Prion 2003, Munich, Germany.

J. C. Manson, R. Barron, N. Tuzi, H. Baybutt, L. Jamieson, L. Aitchison, E. Gall, B. Bradford, V. Thomson, R. Moore, D. Melton, A. Clarke J. Ironside and R. Will. April 2003. Targeting the murine Prnp gene. (Oral Presentation) Keystone Symposia – Molecular Aspects of Transmissible Spongiform Encephalopathies (Prion Diseases).

H. Baybutt, R. Barron, N. Tuzi, L. Aitchison, E. Gall, V. Thomson, P. Hart, N. Hunter and J. Manson. 2002. Relative transmissibility of different TSE agents in transgenic mice expressing the bovine, ovine or human PrP gene (Poster). Joint Funders’ Transmissible Spongiform Encephalopathies Workshop, Durham.

Publications:E. Cancellotti, R.M. Barron, M.T. Bishop, P. Hart, F. Wiseman and J.C. Manson. 2006. The role of host PrP in Transmissible Spongiform Encephalopathies. BBA Review (in press).

J. C. Manson, E. Cancellotti, P. Hart, M. T. Bishop and R. M. Barron. 2006. The Transmissible Spongiform Encephalopathies- emerging and declining epidemics. Biochemical Society Transactions, Vol 34 part 6 (in press).

M. T. Bishop, P. Hart, L. Aitchison, H. N. Baybutt, C. Plinston, V. Thomson, N. L. Tuzi, M. W. Head, J. W. Ironside, R. G. Will, J. C. Manson. 2006. Predicting susceptibility and incubation time of human-to-human transmission of vCJD. Lancet Neurology 5(5):393-8.

J. Manson, R. Barron, P. Hart, N. Tuzi and M. Bishop. 2005. The role of host PrP in control of incubation time. In ”Prions. Food and Drug Safety”, T. Kitamoto (Ed.) pp109-118.

J.C. Manson and R.M. Barron. 2005. The transmissible spongiform encephalopathies. In SGM Symposium 64 ”Molecular pathogenesis of virus infections”. P. Digard, A.A. Nash and R.E. Randall (Ed.) pp137-158

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