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SID 5 Research Project Final Report

SID 5 (Rev. 07/10) Page 1 of 17

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 SE1953

2. Project title

A study of the pathogenesis of experimental scrapie in sheep

3. Contractororganisation(s)

Veterinary Laboratories agencyAddlestone RoadWeybridgeSurrey     

54. Total Defra project costs £      (agreed fixed price)

5. Project: start date................ 31/03/2007

end date................. 31/03/2011

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so......................................................................................YES x 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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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 project Se1953 was set up to support a flock of sheep in which scrapie infection could be continuously maintained over time (the so called “dirty-flock”). High levels of infection were initiated by oral challenge of sheep of different PRNP genotypes and subsequently by breeding to maintain supply of susceptible PRNP alleles. In agreement with studies performed elsewhere these data have shown that sheep of VRQ/VRQ genotype are the most susceptible to infection with a scrapie brain pool comprised mainly of VRQ/VRQ genotype sheep. ARR/ARR sheep are entirely resistant to experimental oral challenge with the scrapie brain pool used in this study. Similarly, the experiment has continuously maintained scrapie in sheep of known susceptible genotypes but despite the high levels of infection within the flock and in particular within VRQ/VRQ genotype sheep, lateral transmission has not been demonstrated in sheep of ARR/ARR or ARR/ARQ genotypes.

The early onset of peripheral PrPd amplification has been shown in VRQ/VRQ sheep from 3 months post exposure and transmission by conjuctival instillation has also been demonstrated. Studies have also shown that the dynamics of peripheral amplification of infectivity are influenced by genotype. However, the infection was initiated by challenge with a pool of brain homogenate made up of brain tissue from individual sheep of different genotypes obtained from different farms and it has not so far been determined whether infection was predominantly maintained by a single source or isolate, by a mixture of isolates or whether more than one of these sources or putative strains was serially propagated in different individual sheep over time.

Material from this project was supplied to the Defra-funded Archive and materials from the Archive have been used to support further VLA and 3rd party projects.

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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

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).

Objectives: Data relating to individual key objectives of SE1953 will be summarised in series:

Objective 1: Preparation and strain typing of a pool of natural scrapie brain for inoculation:

The RBP inocula.

Two inocula were used to experimentally challenge groups of sheep. These inocula were RBP1 (Ripley brain pool 1) and RBP2. RBP1 consisted of a homogenate created from brains obtained from 17 scrapie-affected sheep. Eight of these sheep were homebred from Ripley farm and were of Swaledale (VRQ/VRQ n=5), Welsh mountain (VRQ/VRQ, n=1), white faced Dartmoor (ARQ/VRQ, n=1) and a mixed breed sheep (VRQ/ARQ (n=1). These sheep were between 528 and 1039 days of age when killed with clinical scrapie. The remaining 9 sheep derived initially from 5 farms. All except one of these sheep which died 11 days after importation, survived for over a year at Ripley before succumbing to scrapie: 3 were Vendeen breed sheep (all ARQ/ARQ genotype), 4 were Swaledale sheep (all ARQ/VRQ genotype), 1 was a white faced Dartmoor sheep (ARQ/ARQ genotype) and the final sheep was a Welsh mountain sheep (VRQ/ARR genotype). Thus the RBP1 pool consisted of mixed genotypes and potentially mixed sources or isolates of scrapie. The RBP1 inoculum was titrated in C57bl, RIII and VM mice. No titre was established in VM mice due to the low attack rate. Titres in C57bl and RIII mice were 104.17 and 103.15 mouse i.c/i.p LD50/g respectively.

The RBP2 inoculum was created from 32 brains: 20 brains from home bred Ripley sheep of Swaledale (n=13), Welsh-mountain (n=4), Cheviot (n=1) and cross breed sheep (n=2). The remaining 12 sheep derived originally from 3 farms but all sheep survived for a minimum of 933 days on Ripley farm. One negative brain was inadvertently included in the homogenate. The genotypes represented in the homogenate were 10 VRQ/VRQ, 20 VRQ/ARQ and one ARQ/ARQ. The RBP2 pool has not been titrated.

RBP1 and RBP 2 were analysed by SDS-PAGE electrophoresis and Western blotting using anti-PrP monoclonal antibodies (mAb, 6H4 [VLA Hybrid] and the BioRad Western blot techniques). Each brain pool gave a positive result showing a classical scrapie profile of PrPres with each method and the Western blot images are shown below in Figure 1. Note that there is an error on the Figure and RBP11 should be RBP2. Also note that the strain/isolate complexity of these inocula cannot be inferred from this simple biochemical analysis, but it does give a crude, pre-challenge indication of their potency which was later confirmed by titration in wild-type mice.

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Figure 1 showing Western blots of RBP1 pool revealed with 6H4 and SHA31.

Objective 2: Establishment of infected flocks of sheep

The flock was initially established in 2001 by oral challenge of ninety-seven TSE free sheep with RBP1. Forty-six sheep were challenged with 1g and fifty-one sheep were challenged with 5g. All sheep were challenged at 14 days of age and were of the VRQ/VRQ PRNP genotype.

In addition, further VRQ/VRQ sheep were challenged to provide a temporal series of pre-clinical cases, 30 VRQ/VRQ sheep were dosed with control brain inoculum and 4 VRQ/VRQ sheep were left as environmental controls. These sheep will be discussed below.

In 2002 a further 12 VRQ/VRQ sheep, 26 ARQ/ARQ sheep, 31 VRQ/ARQ and 27 VRQ/ARR sheep were challenged at 2 weeks of age with 1g RBP1. These groups developed clinical disease with incubation periods of 184-208; 495-511, 247-268 and 349-397days.

The incubation periods in both the ARQ/ARQ and VRQ/ARR sheep are significantly different from those obtained with a corresponding dose in VRQ/VRQ sheep indicating relative resistance to the RBP1. However, when the incubation periods of RBP1 challenged ARQ/ARQ sheep are compared with the incubation period of ARQ/ARQ sheep challenged with a different inoculum derived from VRQ/VRQ sheep, (data from Se1950) the incubation periods of RBP1 ARQ/ARQ sheep are significantly shorter. This suggests that the RBP1 pool has unusually high titre or is composed of different strains from that used in Se1950.

In addition 9 VRQ/VRQ environmental controls and additional ARQ/ARQ and VRQ/ARQ sheep were dosed to provide study of pre-clinical disease (see below).

Objective 3 : Monitoring of clinical and metabolic status of the flock.

Clinical examinations were carried out according to a standard protocol to assess changes in behaviour, sensation and movement. This included the assessment of the sheep’s response to scratching of the dorsum (scratch test). The response was classified as “positive” (repeatable stereotypical response, such as head movements and/ or nibbling movements/licking of the lips), “inconsistent” (response not always repeatable), “inconclusive” (some equivocal head or lip movements) and “negative” (no response). As the testing required animals to remain calm, the scratch test response could not be evaluated in those sheep that were too restless during the procedure (“unable to test”). The body condition was scored on a scale from 0-5 to separate sheep with a poor condition (<2.5) from those with good body condition.

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A separate assessment of the sheep’s pruritic behaviour consisted of scratch testing only, which was done at regular (usually monthly) intervals.

Weekly observations for behavioural and sensory changes were conducted in 32 sheep: six VRQ/VRQ sheep inoculated with 200 μl BP1 intraocular for 5 days, 19 sheep orally dosed with 1 g BP1 (three VRQ/VRQ, five ARQ/VRQ, six ARR/VRQ and five ARQ/ARQ sheep) and seven ARR/ARQ sheep orally dosed with 5x 1 g BP1 for five days.

Animals were culled at pre-determined time points (timed culls) or when they reached clinical end-stage [display of pruritus (positive scratch test or excessive rubbing/ scratching resulting in alopecia) in combination with tremor or gait abnormalities that are likely to cause recumbency].

Results and conclusionsPre-cull examinations were available for 174 (48%) sheep; the scratch test response prior to cull was evaluated in 234 (65%) sheep, eight of which did not have a postmortem diagnosis. Table 1 summarises the scratch test findings by inoculation group and postmortem test result.

Table 1. Scratch test results by inoculation groups

Inoculum Genotype N Scratch test result/brain positive1 drop RBP1 skin VRQ/VRQ 4 Positive 4/41g RBP1 orally (single dose) ARQ/ARQ 17 Positive

InconsistentInconclusiveNegativeUnable to assess

5/52/21/0*5/4*4/1

VRQ/ARQ 22 PositiveNegative

13/139/9

VRQ/ARR 18 PositiveNegativeUnable to assess

14/143/11/1

VRQ/VRQ 25 PositiveNegative

17A /173/3

1g RBP2 orally (single dose) VRQ/VRQ 3 Positive 3/3200μl RBP1 intraocular for 5 days VRQ/VRQ 22 Positive

InconsistentInconclusiveNegative

10/101/0*4/47/7

5g RBP1 orally (single dose) VRQ/VRQ 22 PositiveNegative

19 B/192/2

5g RBP1 (1g orally for 5 d) ARR/ARQ 24 PositiveInconclusiveNegativeUnable to assess

2/12/0*14B/5**5/2

ARR/ARR 33 PositiveInconclusiveNegativeUnable to assess

1/02/028/02/0

Control brain VRQ/VRQ 10 PositiveInconsistentNegative

4/43/33/0*

Control brain, then RBP1 1g orally at 250 d

VRQ/VRQ 5 Positive 5/5

Undosed (environmental control) ARR/ARQ 7 NegativeUnable to assess

6/01/0

ARR/ARR 3 Negative 2B/0VRQ/ARR 9 Negative

Unable to assess8/01/0

VRQ/VRQ 4 PositiveNegative

3/31/1

Undosed and kept in isolation VRQ/VRQ 6 InconclusiveNegative

1/05/0

SID 5 (Rev. 07/10) Page 6 of 17

*Sheep with detectable PrPsc in lymphoid tissue but not in the brain (number of asterisks = number of sheep). Excluded are Afive sheep / Bone sheep without postmortem diagnosis.

The sensitivity and specificity of detecting a scrapie case based on the scratch test only was 67.8% and 97.6% respectively (scrapie case = sheep with PrPd in the brain, only scratch test response 3 counted as positive). If an inconclusive or inconsistent response was also considered to be indicative of scrapie, the sensitivity increased to 74% whereas the specificity fell to 89.2%.The low number of scrapie-positive animals within certain groups prevented us from comparing signs between dose groups or genotypes but the number of confirmed scrapie cases with a positive scratch test culled at clinical end-stage was usually 100% in all groups independent of inoculum, dose or genotype. Only three ARQ/ARR sheep, orally challenged with 5 g of RBP1 and culled at 2196-2685 days of age, developed clinical end-stage and only one displayed a positive scratch test although pruritic behaviour was consistently displayed on weekly behavioural observations.This suggested that scratch testing was a very useful method to detect cases of scrapie, particularly if the time for behavioural observations to detect other signs of pruritus is limited, although this test may be less reliable in sheep with more scrapie-resistant genotypes.

When the scratch test results were compared between two time points (three months prior to cull and prior to cull, see Table 2), VRQ/VRQ or ARQ/VRQ sheep usually developed a positive scratch test within the three month period irrespective of inoculum whereas an equal or higher proportion of ARQ/ARQ and VRQ/ARR sheep reacted to scratching at both time points. In these cases, the onset of movement disorders (abnormal gait, tremor), which in combination with pruritus was indicative of clinical end-stage, usually occurred later in the clinical phase than in VRQ/VRQ or ARQ/VRQ sheep.

Table 2. Scratch test result progression between two time points in the incubation period

InoculumGenotype

Genotype N Scratch test result progression (N sheep)Three months prior to cull – prior to cull

200μl RBP1 intraconjunctival for 5 d

VRQ/VRQ 6 No response – positiveInconsistent/inconclusive – positivePositive – positive

411

1g RBP1 orally (single dose)

ARQ/ARQ 5 Inconsistent/inconclusive – positivePositive – positive

14

VRQ/ARQ 5 No response – positiveInconsistent/inconclusive – positive

41

VRQ/ARR 6 No response – positivePositive – positive

33

5g RBP1 (1g orally for 5 d)

ARR/ARQ 3 No response – positiveInconsistent/inconclusive – negativeNegative – negative

111

Control brain VRQ/VRQ 5 No response – positiveNegative – inconsistent/inconclusivePositive – positive

221

Undosed (environmental control)

VRQ/VRQ 4 No response – positiveInconsistent/inconclusive – positivePositive – positive

211

As stated below, incubation period (time from infection to cull) is influenced by the prion protein genotype, with VRQ/VRQ sheep having a shorter incubation period. The clinical findings suggest sheep with a longer incubation period also have a longer clinical duration (longer time from onset to clinical signs as determined by the display of a positive scratch response to clinical end-stage in ARQ/ARQ and VRQ/ARR sheep).

Selected signs at clinical end-stage that were displayed by scrapie-affected sheep within their group are listed in Figure 1. The number of animals per group displaying a particular sign was too small for comparison by statistical methods but there was variability in the display of clinical signs even between groups of the same genotype, e.g. 35% of 20 VRQ/VRQ sheep dosed with 5 g RBP1 displayed changes in behaviour and mental status whereas it was displayed twice as often in VRQ/VRQ sheep dosed with 1 g RBP1 (71% of 17 sheep); see Figure 1.

It is well documented that the clinical signs can vary between sheep of the same genotype but it cannot be excluded that the dose had an effect on the clinical presentation; a higher number of sheep would be required to test this hypothesis.

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Figure 1. Signs in sheep at clinical end-stage separated by group

The total number of sheep per group examined is displayed on the right-hand side of the graph.

When groups were combined by genotype regardless of inoculum to increase the number of sheep per group (VRQ/VRQ – ARQ/ARQ & ARQ/VRQ – ARR/VRQ & ARR/ARQ) significant differences were found between VRQ/VRQ sheep and ARR heterozygous sheep for the display of bruxism (more frequent in VRQ/VRQ), ataxia (less frequent in VRQ/VRQ) and poor body condition (more frequent in VRQ/VRQ) (P<0.05, Chi-Square test with Bonferroni’s correction).It has been shown that the clinical picture in sheep with scrapie can be influenced by the prion protein genotype, which is supported by these findings. The main clinical sign associated with scrapie is pruritus, which is often detected in a field situation by evidence of wool loss with or without skin lesions. The finding that ataxia is more frequent in sheep carrying one ARR allele whereas the scratch test is less reliable in these sheep (see above) may be concerning with regards to the detection of clinical cases in the field. The introduction of more resistant genotypes to eradicate scrapie may shift the clinical presentation towards a more “ataxic form” of scrapie where signs of pruritus are absent or easily overlooked, and these sheep are more likely to present as recumbent cases. This is similar to the situation with atypical scrapie where clinical cases are hardly reported, most likely due to the absence of signs of pruritus.

To determine the metabolic status, blood samples were supplied to IGER Aberystwyth as part of a collaboration to investigate metabolic changes throughout the course of the disease.

Measurement of lactate, 3-hydroxybutyrate and citrate by Nuclear magnetic resonance spectroscopy

Samples were collected from nine Cheviot lambs orally challenged with 5 g of pooled scrapie brain homogenate (RBP1). Controls comprised nine lambs orally challenged with 3 g of scrapie-free brain homogenate (derived from Horkstow farm), one of which was re-dosed with RBP1 250 days after the first oral challenge to be re-used for another part of the study.

EDTA blood samples were collected at 83 days post challenge from all animals (control samples from controls, pre-clinical samples from RBP1-challenged sheep) and prior to cull from the RBP1-challenged sheep, which were culled due to the development of clinical disease (clinical samples; disease confirmed by postmortem tests). Subsequently, four of the sheep dosed with scrapie-free homogenate and the re-dosed sheep developed clinical signs of scrapie, which was confirmed on post-mortem examination, although none of the sheep displayed clinical signs at the time of the blood sampling (approximately 646 days prior to cull).

Measurement of L-lactic acid and free amino acids by colorimetric assay and isotope dilution gas chromatography/ mass spectroscopy

Blood samples were collected monthly until development of clinical signs from 16 Cheviot lambs orally challenged with 1 g of pooled scrapie brain homogenate (RBP1) and of different prion protein genotypes (five VRQ/VRQ, six VRQ/ARR and five ARQ/ARQ). Blood samples for comparison were taken from 12 one month-old lambs (six VRQ/VRQ, six ARQ/ARQ) and 10 six month-old lambs (six ARQ/ARQ, four ARQ/VRQ) from a classical scrapie-free flock (SE1931), which also provided the lambs for study SE1953.

Results and conclusionsThe analysis of the samples was not funded by this project. The results and conclusion have been published elsewhere [see below] and are here only briefly summarised for completeness.

Measurement of lactate, 3-hydroxybutyrate and citrateThere was a tendency of the clinical and pre-clinical blood samples to have higher citrate and 3-hydroxybutyrate concentrations than control samples. Lactate concentrations were lower in clinical cases compared to controls and pre-clinical sheep. None of the markers on their own were reliable to distinguish infected from control animals. The results suggested that the energy metabolism was altered in scrapie, and changes may occur already in the pre-clinical stage.

Measurement of L-lactate and free amino acidsThere was a significant decrease in the plasma concentration of most free-amino acids (asparagine, aspartate, leucine, lysine, methionine, phenylalanine, proline, tyrosine, valine) as the disease progressed, which this was attributed to a decreased feed intake and resulting cachexia. A significant increase in alanine and serine with disease progression was believed to be caused by alterations in the energy metabolism although no significant differences were found for L-lactate, contrary to the finding above.

As these initial findings did not indicate that metabolic monitoring would advance our understanding of the disease, it was subsequently discontinued in this project.

These metabolic data were published in:

Charlton AJ, Jones S, Heasman L, Davis AM, Dennis MJ: Scrapie infection alters the distribution of plasma metabolites in diseased Cheviot sheep indicating a change in energy metabolism. Res Vet Sci 2006, 80:275-280.

Allison G, Rees Stevens P, Heasman L, Davis A, Jackman R, Moorby J: Effect of scrapie incubation on the concentrations of plasma amino acids and L-lactate in infected lambs. Vet Res Commun 2008, 32:591-597.

Objective 4: Necropsy and examination of timed kills and clinical cases

A. Clinical cases:

The mean incubation periods for VRQ/VRQ Cheviot sheep orally challenged with 1g or 5g of RBP1 were respectively 199 days (SEM, 2) and 189 days (SEM, 2). The difference in these means are highly significant (P=0.0002). However, the incubation periods are much shorter than occurs in most natural disease cases and the standard errors are extremely tight suggesting that both 1g and 5g represent a significantly higher and overwhelming challenge than occurs naturally.

Fig 1. Graph showing the mean incubation periods and SEM for 1g and 5 g doses of RBP1 in different sheep genotypes

Figure 2 Graph showing the incubation periods of different sheep PRNP genotypes following challenge with RBP1

A 1g dose of the RBP1 pool was also very effective in establishing disease in three other PRNP genotypes (see figure 2). ARQ/ARQ sheep had a mean incubation period of 504 days (SEM 3.1); VRQ/ARQ sheep, 259 days (SEM 1.9) and VRQ/ARR sheep, 366 days (SEM 3.2). These data indicated that the ARQ allele is relatively more resistant to infection with the RBP1 pool than is the VRQ allele. It is likely that the longer incubation period in the VRQ/ARR sheep when compared with the VRQ/ARQ sheep is the result of lower levels of amplification of disease associated forms of PrP that can be generated by when production is limited to one susceptible allele.

B) Investigations of the sequential accumulation of PrP d in the viscera and CNS throughout the incubation of the disease.

B 1) VRQ/VRQ genotype sheep.

Forty-six TSE free sheep were orally inoculated with RBP1 and sequentially killed to study the progression of disease. Twenty sheep were challenged with 1g and 21 sheep were challenged with 5g. All sheep were challenged at 14 days of age and were of the VRQ/VRQ PRNP genotype. The mean incubation periods for these two groups were respectively 192 days (SD 6.9) and 185 days (SD7.4). These incubation periods are not significantly different and are much shorter than occurs in most natural disease cases suggesting that both 1g and 5g represent a significantly higher and overwhelming challenge than occurs naturally.

PrPd accumulation was initially detected in the lympho-reticular system (LRS) at 21 days post challenge and was extensively present in LRS tissues by 55 dpi. PrPd accumulation was found in many LRS tissues prior to the accumulation of PrPd within the enteric nervous system (ENS). These data suggest that the initial infection of lymphoid tissues occurs following transcytosis of infectivity across the intestinal mucosa and that the subsequent spread of infectivity from the alimentary tract to other lymphoid tissues is by haematogenous spread.

Evidence of PrPd accumulation in the ENS was not found until 110 days post challenge and was only detected in a fraction of sympathetic and parasympathetic ganglia at clinical stage of disease. These data are not consistent with the current hypothesis that infectivity ascends to the brain via the peripheral nervous tissue. Rather it suggests that ENS and ganglionic sites accumulate PrPd following centrifugal spread of infectivity from the brain.

This data has been published in BMC Veterinary Research 2009, 5:9 by Ryder et al., Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host.

The timing and number or percentage of visceral tissues showing PrPd accumulation provides an indication of the onset, distribution and dissemination of systemic infection. When the frequency of PrPd accumulation in different viscera were compared in sheep of VRQ/VRQ genotypes challenged by different routes or different doses was compared, in each case an early and widespread dissemination to viscera was detected in VRQ/VRQ genotype sheep (Figs 3-5).

Figure 3 percentage of lymphoid tissues positive for PrPd in 1g dosed VRQ/VRQ sheep according to incubation period (days)

%

Incubation period (days post-challenge)

Figure 4 percentage of lymphoid tissues positive for PrPd in 5g dosed VRQ/VRQ sheep according to incubation period (days)

%

Incubation period (days post-challenge)

Figure 5 percentage of lymphoid tissues positive for PrPd in VRQ/VRQ sheep challenged by conjuctival instillation according to incubation period (days)

%

Incubation period (days post-challenge)

Three important caveats should be placed on these data: firstly that the overwhelming doses of infectivity used in these studies may have resulted in disease that is not entirely typical of natural disease and secondly that the pathogenesis and spread of infectivity established in this VRQ/VRQ model may differ from the timing of disease or its pathogenesis in other sheep genotypes (see below). Thirdly, the Ripley brain pool material may differ in its transmission properties compared to other sources of sheep prions.

B 2) Sheep with genotypes other than VRQ/VRQ sheep.

In addition to VRQ/VRQ genotype sheep, sheep of three other genotypes were also killed at selected time points prior to the onset of clinical disease following oral challenge with 1g dose of RBP1. The percentage of PrPd

positive lymphoid tissues in sheep of 4 different genotypes according to time elapsed since challenge is shown in figure 6.

As discussed above incubation periods are shortest in VRQ/VRQ sheep and longest in ARQ/ARR sheep. Figure 6 shows that not only are the incubation periods different for different PRNP genotypes but the rate and magnitude of PrPd accumulation in lymphoid tissues is also influenced by genotype. In particular, sheep of the VRQ/ARR genotype, although having shorter incubation periods than ARQ/ARQ have slower and more incomplete systemic PrPd accumulation, presumably reflecting less consistent and lower levels of systemic infectivity. These data show that the dynamics and peripheral amplification of infectivity are genotype dependent.

Figure 6 percentage of lymphoid tissues positive for PrPd in sheep challenged according to incubation period (days)

C) Identification of optimal sites for application of PrP diagnostics based on pathogenesis

Data from serial examination of tissues obtained from experimentally challenged VRQ/VRQ sheep suggest that lymphoid tissues of the alimentary tract are amongst the earliest tissues to become infected. Both rectal mucosa (RAMALT) and pharyngeal tonsil contain lymphoid tissues that are accessible for biopsy, and these tissues offer potential sampling sites for pre- and post-mortem diagnosis of scrapie infection.

In initial experiments RAMALT tissues were obtained at necropsy from 41 clinically affected sheep and from 280 in-contact sheep that were culled. None of the cull sheep were positive while 100% of the clinically affected sheep were shown to have PrPd in follicles (when combined with data from other sources, the analysis of RAMALT tissues was shown to be 97.1% sensitive and 100 % specific for confirmation of clinical scrapie in post-mortem collected RAMALT tissues).

Further investigations were carried out on biopsies taken from 24 pre-clinically affected sheep that were sampled once between 4 and 78 months. Tissues from Se1953 were also used to investiagte the feasibility of rapid testing RAMALT tissue for PrPres using ELISA technology. Further details of the efficiency of RAMALT and pharyngeal tonsil biopsy have been published as part of our Report on Defra-funded project, Se1788.

These data- which were in part also primarily funded in Se1788- have been published in the: Veterinary Record 2006, 158, 325-331. González et al., post-mortem diagnosis of pre-clinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa.

Veterinary Record 2008 162, 397-403. González et al., Diagnosis of pre-clinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies.

Journal of Veterinary Diagnostic Investigation (2008) 20, 203-208. González et al., Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa.

D) Provision of an archive of tissue of known infection status at different stages throughout the incubation period for comparison of the sensitivity of PrPd detection by IHC with detection of infectivity by bioassay.

A further requirement of the Se1953 project was to provide an archive of scrapie infected tissues for research. Extensive tissue and bodily fluid samples have been collected and both fixed and frozen samples have been archived. From this archive two requests for material have been received from 3rd parties to supply 39 tissues. A further 13 requests have been made by VLA personnel to supply materials for internal projects and other Defra funded studies. A total of 428 samples were supplied to these requests.

Objective 5: compilation of data and completion of final reports.

Annual and final reports have been completed. Data have been compiled for publication but there are extensive data sets that remain incompletely analysed. It is hoped that there will be opportunity to analyse these data in the future.

Objective 6: Challenge of sheep by conjunctival inoculation

Thirty-five two week old VRQ/VRQ lambs were dosed with approximately 200μl of RBP1 into the conjunctiva of the left eye once daily for 5 days. Thus a total challenge of about 1g of RBP1 inoculum was delivered to each lamb.

Seven sheep were allowed to progress to clinical disease. These animals showed clinical signs of scrapie from 212-288 days and were killed with a mean incubation period of 236 days.

Twenty eight lambs were killed in groups of 3 or 4 between 9 and 203 days post challenge (see table). Evidence of peripheral amplification of infectivity was first detected at 86 days post challenge which had spread to affect most LRS tissues by 113 days post challenge. The LRS tissues that accumulated PrPd at 86 days post challenge were the pharyngeal tonsil, the parotid lymph node, the retropharyngeal lymph node, the mesenteric lymph node and Payer’s patch of the ileum or jejunum: these are all lymphoid tissues that sample antigens in the upper alimentary tract.

Table showing the numbers of sheep and times of challenge and the results of immunohistochemistry in brain and viscera

Reason for kill

No sheep

MIP [days] CNS

Mean % LRS* pos

Timed Kill 3 9 Not sampled 0.00Timed Kill 3 17 Not sampled 0.00Timed Kill 3 30 Not sampled 0.00Timed Kill 3 43 Not sampled 0.00Timed Kill 3 86 Not Examined 28.21Timed Kill 3 113 Not Examined 76.92Timed Kill 4 132 Not Examined 84.62Timed Kill 3 177 Positive 94.87Timed Kill 3 203 Positive 95.83

Clinical scrapie 7 236 Positive 96.52

*Mean % LRS pos, refers to the percentage of lymphoid tissues that were positive for PrPd relative to the number of lymphoid tissues examined. Between 11 and 13 LRS tissues were examined for each sheep.

This experiment confirms the previously theoretical possibility that scrapie could spread by conjunctival instillation following for example splashing or production of aerosols from the bodily fluids of infected sheep, particularly during parturition.

The primary location of PrPd was seen within LRS tissues of the alimentary tract. This provokes intriguing idea that infectivity may have been absorbed via the alimentary canal after inoculum reached the pharynx via tear ducts or lymphatic channels; however, as yet, not all available tissues have been analysed; this is required before we can unequivocally determine whether infectivity reached the brain from the conjunctiva following ascending infection via the optic nerve, following haematogenous spread or following prior amplification in lymphoid tissues connected with lymphatic drainage of the gut or eye.

Objective 7 Investigation of the age at challenge on incubation period:

In addition to groups of lambs challenged at 14 days of age, a further group of sheep that had previously been challenged with normal brain homogenate were re-challenged with 1g of RBP1. All eight of these sheep succumbed to scrapie with mean incubation periods of 345 days. Individual incubation periods were highly variable and ranged from198 -639 days.

The interpretation of this study is confounded by the presence of disease in both environmental controls and normal brain homogenate control groups (see objective 8). While it is possible that the age of challenge may have impact on the incubation period and attack rate, the wide range of incubation periods and the frequency of disease in other controls groups suggests that some or all of these sheep may already have been incubating scrapie as a consequence of horizontal exposure to infectivity at the time they were re-challenged with RBP1. Conclusions regarding the effects of age at challenge cannot be made with any conviction from this experiment.

Objective 8 Challenge of resistant genotypes and control groups.

A) Scrapie resistant genotypes:

Classical scrapie is very rarely recorded in several PRNP genotypes of sheep and in particular ARR/ARR and ARQ/ARR sheep appear highly resistant to disease. Two week old lambs were derived by embryo transfer and challenged with RBP1 at 2 weeks of age.

a) 33 ARR/ARR lambs were orally dosed with 1g daily for 5 days. b) 30 ARR/ARQ lambs were dosed orally with 1g daily for 5 daysc) 7 ARR/ARQ lambs were un-dosed and served as environmental controlsd) 3 ARR/ARR lambs were un-dosed and served as environmental controls

None of the ARR/ARR sheep has developed clinical disease and none have shown convincing evidence of abnormal PrP accumulation either by immunohistochemistry or by Western blotting. The longest survival time is 2761 days (> 7 years).

Eight of 19 ARQ/ARR sheep examined between 2 and 7 years post challenge have shown evidence of PrPd accumulation but without evidence of any clinical disease. Abnormal PrP accumulation was found in brain both by immunocytochemistry and by Western blotting. Immunocytochemical labelling showed widespread PrPd accumulation in spinal cord and was also detected in ganglia of the peripheral nervous system and cranial nerves. PrPd has not been convincingly detected in lymphoid tissues. A further three clinical cases of scrapie have been identified in ARQ/ARR sheep at more than six years post challenge.

B) Other procedure control groups

Groups of sheep were challenged with 3g of negative sheep brain or were unchallenged as environmental controls. 100% of control sheep exposed to VRQ/VRQ sheep challenged with RBP1 scrapie developed disease with incubation periods significantly longer than the 5g or 1g scrapie challenged groups.

Table 2 showing outcome for control sheep groups

Group Age of exposureAttack rate mean days to death

environmental control 42 days 4/4 649 days

negative brain challenge 14 days 8/8 1085 days

The negative brain homogenate was created from sheep obtained from Horkstow flock. Neither clinical disease nor histological evidence of infection has been identified in cull sheep from this flock. The inoculum was made from ARR/ARR sheep and ARQ/ARR sheep which rarely develop scrapie under natural husbandry conditions. An

audit was carried out and confirmed the presumed origin of the origin of the tissues used to create the homogenate and that it lacked PrPres.

The environmental controls included in these experiments were used to evaluate whether infection could be transferred from infected sheep to others either directly or via environmental contaminations. Thus environmental controls were not separated from dosed sheep during a 28 days post-dosing period but remained with dosed sheep for varying lengths of time.

Access to the sheep compounds containing the procedural control and the environmental control sheep was via a barrier change area and each building had facilities to ensure changing of external clothing and footwear. Clothing was colour coded to avoid confusion between dirty and clean areas. For each pen staff (and visitors) were required to change into designated pen boots and boiler suits. Similarly each pen had designated cleaning equipment and other tools such as hoof knives.

In summary, although the biosecurity procedure used conforms to best practice, horizontal transmission from dosed to in contact animals occurred. There are several possible sources of animal- animal transfer of infectivity: via saliva, aerosols, faeces and urine. All of these bodily fluids as well as partial regurgitation of oral dose material may also have contributed to environmental contamination in the immediate post-dosing challenge period.

As described above the incubation periods of the VRQ/VRQ sheep were substantially shorter than occurs in natural disease indicating that the doses of infection given to these sheep were substantially greater than occurs naturally.

References to published material

9. 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.

The following papers have been published:

Allison G, et al., Changes in plasma metabolites and muscle glycogen are correlated to bovine spongiform encephalopathy in infected dairy cattle. Res Vet Sci 2007, 83:40-46.

Allison G, et al., : Effect of scrapie incubation on the concentrations of plasma amino acids and L-lactate in infected lambs. Vet Res Commun 2008, 32:591-597.

González L et al., Post-mortem diagnosis of pre-clinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa. Vet Rec 2006, 158, 325-331. González L et al., Diagnosis of pre-clinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies. Vet Rec 2008, 162:397-403.

González L et al., Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa. J Vet Diagn Invest 2008:20, 203-208.

Charlton AJ et al., Scrapie infection alters the distribution of plasma metabolites in diseased Cheviot sheep indicating a change in energy metabolism. Res Vet Sci 2006, 80:275-280.

Ryder SJ et al., Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host. BMC Vet Res 2009, 5:9.