Research and Development s15

Project The diagnosis and neuropathological monitoring of MAFF title suspect BSE cases. project code SE0225

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title The diagnosis and neuropathological monitoring of suspect BSE cases.

MAFF project code SE0225

Contractor organisation Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, and location Surrey. KT15 3NB

Total MAFF project costs £

Project start date 01/07/1998 Project end date 31/03/2002

Executive summary (maximum 2 sides A4)

It is clear from laboratory animal models of scrapie that intraspecific passage of scrapie-like pathogens (as has occurred with the recycling of BSE-infected material via meat and bone meal) may result in alterations in the nature, distribution and severity of lesions. Continued systematic monitoring of BSE through the epidemic is essential, therefore, to detect possible changes in the biology of the causative pathogen, and to ensure the continuous validation of the statutory diagnostic criteria.

Over the last 10 years a number of ‘diagnostic’ tests for transmissible spongiform encephalopathies have been developed, based on the demonstration of protease-resistant disease-specific PrP accumulation. These tests, notably PrP immunocytochemistry, and Western blotting are now commonplace research tools, but their sensitivity and specificity relative to the established statutory tests (histology and SAF) have never been systematically studied in the context of the diagnosis of suspect field cases of BSE.

This proposal forms a logical continuation of previous similar studies (SE 0203, SE 0206, SE 0216 and SE 0220). Broadly, the lesion distribution and severity (the lesion profile) in 100 positive cases from the 1994 birth cohort was determined by a previously validated semi-quantitative method. A sample of clinical suspects in which BSE was not confirmed were also examined for evidence of an alternative neurohistological diagnosis or an atypical presentation of BSE. All cases , and the equivalent samples from the 1993 birth cohort (SE0220) were also sampled for SAF and Western blotting analysis, and the obex section stained immunohistochemically (IHC) for protease-resistant PrP.

CSG 15 (Rev. 12/99) 1 Project The diagnosis and neuropathological monitoring of MAFF SE0225 title suspect BSE cases. project code

In total, the brainstems from 491 cases (200 positive and 291 ‘negative’) were examined independently by all four methods. Complete agreement among all four tests was found in 417 cases (84.7%), of which 142 (34%) were positive, and 275 (66%) were negative.

All cases diagnosed as ‘histopathology positive’ were confirmed by both immunohistochemistry and Western Blotting.

Of the cases that were negative by histopathology (i.e. no vacuolar lesions in the brainstem at the level of the obex), 14 (4.8%) were found to be positive by at least one PrP detection method.

SAF detection proved to be less consistent, with 65 (30.8%) of cases positive by at least one other method found negative for SAF, and 5 (1.8%) of cases negative by all other methods being SAF positive. There was good agreement between the two PrP detection methods, with the exception of only 6 (2.8%) of the positive cases, in which one was a ‘stand alone’ IHC positive result, and the remaining 5 were positive only by Western Blot. A clear hierarchy of diagnostic sensitivity is present, therefore, with SAF at the bottom, histology next, and PrP detection methods performing better than both.There is no absolute hierarchy between IHC and WB, although the majority of the small number of singleton results are WB ones.

The alternative differential diagnoses found in the ‘negative’ population is summarised below:

No significant lesions 114 (69%) Substantia nigra vacuolation 38 (23%) Listeriosis 12 (7.3%) Other inflammatory 4 (2.4%) Miscellaneous 1 (0.6%) BSE (negative at the obex ) 2 (1.2%) (N.B. More than one diagnosis was identified in 6 animals)

This examination of the cases which were not histologically confirmed as BSE (by statutory criteria) revealed that the proportions of other neurological diseases presenting clinically as BSE were remarkably consistent in both this and previous studies, and there was no indication of any atypical presentation of vacuolar lesions.

Early in this study, there was clear indication that a small proportion of cases considered to be negative on the basis of obex histology alone were positive by immunohistochemical techniques for the visualisation of PrPSc.This included the small number of cases which have vacuolar lesions rostral to the obex but which cannot be identified on examination of a single section (Wells et al 1989; Simmons et al 1996), and is in keeping with the known temporal development of PrPSc accumulation in experimental models of BSE in cattle (Wells et al 1998; Hawkins unpublished data [SE1736]). The occurrence of the anticipated pattern of lesions in the areas rostral to the medulla indicate that these cases confirm to the profile originally described for this population. This finding led directly to the inclusion, in 2000, of IHC as an integral component of the statutory diagnostic approach to all cases which were not clear-cut positives on histopathological examination alone.

In general, good agreement was seen between the PrP detection methods. However, a lack of an absolute diagnostic performance hierarchy highlights one of the biggest problems facing any diagnostic test for TSE – the difference between analytical sensitivity and diagnostic sensitivity. If samples cannot be accurately collected to reflect the current state of understanding of the pathogenesis of the disease, then the analytical sensitivity of any test is irrelevant, and the result meaningless.

Comparison of the positive profiling data with that obtained from a similar sample of BSE-affected cattle from early in the epidemic (1987/89) showed that the distribution and severity of vacuolation in BSE has so far remained unchanged, i.e. the BSE epidemic appears to have been sustained by a single stable strain of the agent. It must, however, be borne in mind that all animals identified as suspect conform to a well-defined clinical presentation. It is possible that an alternative strain of agent would produce a clinical disease so different from that currently recognised as BSE, that it would not present as a suspect.

Scientific report (maximum 20 sides A4) CSG 15 (1/00) 2 Project The diagnosis and neuropathological monitoring of MAFF SE0225 title suspect BSE cases. project code

INTRODUCTION

It is well known that the transmissible spongiform encephalopathies (TSE) are caused by an agent(s) of uncertain aetiology, of which there are numerous ‘strains’. When sheep scrapie, the oldest of the naturally-occurring TSE is experimentally transmitted through multiple generations of mice, a number of different disease phenotypes can be identified. These are determined by two variables; the strain of the agent (Bruce and Fraser 1991) and the recipient mouse genotype (Bruce and others 1991). The distinctive patterns of neurohistological lesions, particularly vacuolar changes, seen within the brains of these mice can be subjectively assessed and 'scored' semiquantitatively depending on the severity of vacuolation in each of a number of specific neuroanatomical locations. The resulting graph of 'score' versus 'location' is called the 'lesion profile', and is specific (in mice) for each strain/host combination (Fraser and Dickinson 1968). This is an important component of the technique known as 'strain typing', which has been extensively used in scrapie research for a number of years, to characterise different isolates of the infectious agent. More recently, lesion profiling has been adapted to study naturally-occurring spongiform encephalopathies in other species, for example CWD (Williams and Young 1993), FSE (Wells and others 1994), BSE (Simmons et al 1996) and scrapie (Ligios et al, in press; Begara-McGorum et al, in press).

When BSE was first identified, a detailed assessment was made of the severity and distribution of lesions in a total of 88 neuroanatomical sites from 100 cattle over the period 1987-89 (Wells and others 1992; Wells and Wilesmith 1995). This revealed that, "unlike scrapie, BSE had a stereotypic lesion profile from which it was concluded that host and agent factors, including probably the strain of agent,... are constant in this disease". Since this initial study, profiling of a selected sub-group of these areas has been performed for monitoring purposes on a number of groups of naturally-occuring BSE throughout the epidemic (Simmons et al 1996 and this report), and in some experimentally challenged animals for comparison (Hawkins et al 1996). Temporally corresponding samples of clinical suspects in which BSE was not confirmed by histopathological examination of the brainstem at the level of the obex have also been examined for evidence of any alternative neurohistological diagnoses or any atypical presentations of BSE.

Although early in the epidemic the diagnosis of BSE relied entirely on histopathological criteria, the demonstration of scrapie associated fibrils (SAF) (Scott et al 1990) and, more recently the PrP- immunodetection methods such as immunohistochemistry (Haritani et al 1994) and Western blotting (Schaller et al 1999) have become increasingly prevalent, and combinations of these methods form the basis of the current statutory confirmation of BSE in Great Britain. A number of ‘diagnostic’ tests for transmissible spongiform encephalopathies, based on the demonstration of protease-resistant disease-specific PrP accumulation, are now commonplace research and surveillance tools. However, their sensitivity and specificity relative to the originally established statutory tests (histology and SAF) need to be systematically studied in the context of robustness when applied to suspect field cases of BSE, to ensure the continuous validation of the statutory confirmatory diagnostic criteria.

This report details the sensitivity and specificity of these four diagnostic methods in 491 naturally- occurring clinical suspects of BSE born in 1993 and 1994. It also presents the histopathological profile and differential diagnostic breakdown of the more recent birth cohort (1994), and compares it with earlier studies. This work also provides a background against which the profiles of other cases of BSE (e.g. cases arising outwith the UK) can be compared, to assess if the same strain of agent is responsible.

MATERIALS AND METHODS

Over the period 1997-1999, intact cattle brains, which had been fixed in 10% formal saline for at least two weeks, and unfixed brainstem samples, were sent to the Neuropathology Section at VLA Weybridge. These were all from cows that were clinical suspects of BSE, born in 1994. The CSG 15 (1/00) 3 Project The diagnosis and neuropathological monitoring of MAFF SE0225 title suspect BSE cases. project code

brainstem was sampled at the level of the obex and routinely processed for Statutory diagnostic examination, and a diagnosis of BSE 'negative' (i.e. histologically unconfirmed) or BSE 'positive' was reached using the (then) current statutory diagnostic criteria (Bradley and Matthews 1992).

As in previous studies of this kind, 100 positive cases and approximately 150 negative cases were retrospectively subselected from this group for further study.

As described in detail elsewhere (Simmons et al 1996), the 100 positive cases were lesion profiled to establish the severity of vacuolation in 17 specific neuroanatomical locations (Fig 1). In cases diagnosed as BSE 'negative' a total of eleven blocks, representing seven different levels of the brain were taken (Simmons,1994) and fixed for a further week in fresh fixative. The blocks were processed using a 3-day schedule, embedded in paraffin wax and 5µm sections cut and stained with modified haematoxylin eosin (HE) (Bradley and Matthews 1992). Each of the BSE 'negative' brains was examined for histological changes and, where possible, an alternative diagnosis was recorded.

The obex blocks from all positive and negative cases from this group and the 1993 birth cohort previously described in SE 0220) were immunostained for PrP using the polyclonal antibody 486 by a modification of the method described by Haritani and others (1994). The sections were deparaffinished in xylene and rehydrated to water, endogenous peroxidase activity was blocked with hydrogen peroxide, and the sections were pre-treated in 96 per cent formic acid (Merck) for 20 minutes followed by hydrated autoclaving at 121C for 30 minutes. The primary antiserum was applied at a dilution of 1:2500 and detected by avidin-biotin-horseradish peroxidase (Vector Elite), the colour being developed with diaminobenzidine.

The SAF extraction technique was a modification of the method of Hilmert and Diringer (1984), as described in detail by Scott and others (1992). The resulting negatively stained samples were examined in either a Philips 410 or CM10 transmission electron microscope at magnifications greater than 25,000. A positive result was recorded for any case in which there was one or more undisputed fibrils of any type within the definition of Merz and others (1981). Each sample was examined for 20 mintues before being declared negative.

For Western blotting, all the samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were immunostained with the monoclonal antibody 6H4 (Prionics) at a dilution of 1:5000, and visualised by means of an enhanced chemiluminescence system (Western Light; Tropix).

RESULTS

In total, the brainstems from 491 cases (200 positive and 291 ‘negative’) were examined by all four diagnostic methods. Complete agreement among all four tests was found in 417 cases (84.7%), of which 142 (34%) were positive, and 275 (66%) were negative. All cases diagnosed as ‘histopathology positive’ were confirmed by both immunohistochemistry and Western Blotting. Of the cases that were negative by histopathology (i.e. no vacuolar lesions in the obex), 14 (4.8%) were found to be positive by at least one PrP detection method (see Table 1). SAF detection proved to be less consistent, with 65 (30.8%) of cases positive by at least one other method found negative for SAF, and 5 (1.8%) of cases negative by all other methods being SAF positive. There was good agreement between the two PrP detection methods, with the exception of only 6 (2.8%) of the positive cases, in which one was a ‘stand alone’ IHC positive result, and the remaining 5 were positive only by Western Blot. A clear hierarchy of diagnostic sensitivity is present, therefore, with SAF at the bottom, histology next, and PrP detection methods performing better than both (Table 3). There is no absolute hierarchy between IHC and WB, although the majority of the small number of singleton results are WB ones (see Table 1).

The mean lesion profiles of positive cases born in 1994 are remarkably similar to those of previously profiled groups (Fig 2).

CSG 15 (1/00) 4 Project The diagnosis and neuropathological monitoring of MAFF SE0225 title suspect BSE cases. project code

The alternative differential diagnoses found in the ‘negative’ population is summarised in Table 2 which also includes data from other studies for comparison.

DISCUSSION

Early in this study, there was clear indication that a small proportion of cases considered to be negative on the basis of obex histology alone were positive by immunohistochemical techniques for the visualisation of PrPSc.This included the small number of cases which have vacuolar lesions rostral to the obex but which cannot be identified on examination of a single section (Wells et al 1989; Simmons et al 1996), and is in keeping with the known temporal development of PrPSc accumulation in experimental models of BSE in cattle (Wells et al 1998), Hawkins unpublished data [SE1736]). The occurrence of the anticipated pattern of lesions in the areas rostral to the medulla indicate that these cases confirm to the profile originally described for this population. This finding led directly to the inclusion, in 2000, of IHC as an integral component of the statutory diagnostic approach to all cases that were not clear-cut positives on histopathological examination alone. In general, good agreement was seen between the PrP detection methods. This lack of an absolute diagnostic performance hierarchy highlights one of the biggest problems facing any diagnostic test for TSE – the difference between analytical sensitivity and diagnostic sensitivity. A whole range of new, rapid, diagnostic tests are currently being developed and evaluated for use in active surveillance around the world (Schimmel & Moynagh 1999 & 2002), all of them based on the use of PrP as a marker for disease. However, it is clear from all the different animal models of TSE that PrP accumulation is not detectable until at least half way through the incubation period in most models. When it finally occurs it starts to accumulate in very focal, neuroanatomically precise areas and initially the quantities are very small. Any detection method would therefore need to be able to detect small amounts of PrP (i.e. analytical sensitivity), but this would only be possible if the precise areas which contain this small amount of PrP are accurately sampled (diagnostic sensitivity). If samples cannot be accurately collected to reflect the current state of understanding of the pathogenesis of the disease, then the analytical sensitivity of any test is irrelevant, and the result meaningless. This problem becomes more critical when active surveillance is considered, with young healthy animals being tested prior to their entry into the food chain, or fallen stock which may have been dead for several hours (or even a day or more) before sampling is possible. The level of accuracy/repeatability achievable by trained pathologists in a research environment is rarely reproducible in multiple commercial outlets such as abattoirs and incineration plants, with many different staff who may be presented with carcasses which might not have been optimally handled with respect to sampling requirements. Fallen stock present a particular problem, because not only may autolysis compromise the analytical sensitivity of a test, but complete (or even partial) liquefaction of the brain makes precise anatomical targeting impossible. SAF appears to be the least reliable test for confirmation of positives, and the limitations of this method have been discussed in detail elsewhere (Simmons et al 2000). It remains, however, one of the most robust tests when dealing with autolysed material (Scott et al 1992). Detailed examination of the cases which were not histologically confirmed as BSE (by statutory criteria) revealed that the proportions of other neurological diseases presenting clinically as BSE were remarkably consistent in both this and previous studies, and there was no indication of any atypical presentation of vacuolar lesions. Comparison of the positive profiling data with that obtained from a similar sample of BSE-affected cattle from early in the epidemic (1987/89) showed that the distribution and severity of vacuolation in BSE has so far remained unchanged, i.e. the BSE epidemic appears to have been sustained by a single stable strain of the agent. It must, however, be borne in mind that all animals identified as suspect conform to a well-defined clinical presentation. It is possible that an alternative strain of agent would produce a clinical disease so different from that currently recognised as BSE, that it would not present as a suspect. Further information on the range of clinical and pathological presentations identifiable in cattle challenged with TSE will be forthcoming from other ongoing DEFRA-funded projects (Ryder, unpublished data [SE1941]), and the incidence of previously undetected natural disease will be continually assessed in the context of cases detected through active, rather than passive, surveillance.

The findings of this study concur with those of previous studies, supporting the theory that the BSE

epidemic continues to be sustained by a single stable strain of the BSE agent. No atypical presentations of BSE were identified. These longitudinal studies have helped to establish whether the epidemic continues to be sustained by a single strain of the agent, and whether the 'recycling' of BSE through the feedstuffs in the early stages of the epidemic in any way affected the disease phenotype in cattle.

REFERENCES

Begara-McGorum, I., González, L., Simmons, M., Hunter, N., Houston, F. and Jeffrey, M. (2002). Vacuolar Lesion Profile in Sheep Scrapie: Factors Influencing its Variation and Relationship to Disease-specific PrP Accumulation. Journal Comparative Pathology (in press)

Bradley, R. and Matthews, D. (1992). Sub-acute, transmissible spongiform encephalopathies: current concepts and future needs. Rev. Sci. Tech. 11, 605-634

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

Bruce, M.E. and Fraser, H. (1991). Scrapie strain variation and its implications. Current Topics in Microbiology and Immunology 172, 125-137

Fraser, H. and Dickinson, A.G. (1968). The sequential development of the brain lesions of scrapie in three strains of mice. J. Comp. Pathol. 78, 301-311.

Haritani, M., Spencer, Y.I. and Wells, G.A.H. (1994). Hydrated autoclave pretreatment enhancement of prion protein immunoreactivity in formalin-fixed bovine spongiform encephalopathy-affected brain. Acta Neuropathologica (Berlin) 87, 86-90.

Hawkins, S.A.C., Wells, G.A.H., Simmons, M.M., Blamire, I.W.H., Meek, S.C. & Harris, P. (1996). The topographic distribution pattern of vacuolation in the central nervous system of cattle infected orally with bovine spongiform encephalopathy. Cattle Practice 4, 365-368.

Hilmert, H. & Diringer, H. (1984) A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Bioscience Reports 4, 165-170.

Jeffrey, M., Simmons, M.M. and Wells, G.A.H (1994). Observations on the differential diagnosis of bovine spongiform encephalopathy in Britain. In: Transmissible Spongiform Encephalopathies. Proceedings of a Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities, 14-15 September 1993, Brussels, pp347-358

Ligios, C., Jeffrey, M., Ryder, S.J., Bellworthy, S.J. & Simmons, M.M.. Distinction of scrapie phenotypes in sheep by lesion profiling. Journal Comparative Pathology (in press).

McGill, I.S. and Wells, G.A.H. (1993). Neuropathological findings in cattle with clinically suspect but histologically unconfirmed bovine spongiform encephalopathy (BSE). Journal of Comparative Pathology 108, 241-260.

Merz, P.A., Somerville, R.A., Wisniewski, H.M. & Iqbal, K. (1981). Abnormal fibrils from scrapie- infected brain. Acta Neuropathologica 54, 63-74.

Miller, L.D., David, A.H. & Jenny, A.L. (1992) Surveillance for lesions of bovine spongiform encephalopathy in US cattle.. J Vet Diag Invest 4 338-339

Moynagh, J. & Schimmel, H. (1999). Tests for BSE evaluated. Nature 400, 105.

Pollin, M.M., McGill, I.S. & Wells, G.A.H. (1992). The differential neurohistological diagnoses of clinically suspect but unconfirmed BSE. Neuropathology and Applied Neurobiology 18, 638.

Schaller, O., Fatzer, R., Stack, M.J., Clark, J., Cooley, W., Biffiger, K., Egli, S., Doherr, M., Vandevelde, M., Heim, D., Oesch, B. & Moser, M. (1999) Validation of a Western immunoblotting procedure for bovine PrPSc detection and it’s use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathologica 98, 437-443

Schimmel, H. & Moynagh, J. (2002). The evaluation of five rapid tests for the diagnosis of transmissible spongiform encephalopathy in bovines (2nd study). www.irmm.jrc.be

Scott, A.C., Wells, G.A.H., Chaplin, M.J. & Dawson, M. (1992) Bovine spongiform encephalopathy: detection of fibrils in the central nervous system is not affected by autolysis. Research in Veterinary Science 52, 332-336

Scott, A.C., Wells, G.A.H., Stack, M.J., White, H. & Dawson, M. (1990) Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain. Veterinary Microbiology 23, 295-304

Simmons, M.M. (1994). Lafora disease in the cow? Journal of Comparative Pathology 110, 389-401.

Simmons, M.M., Harris, P., Jeffrey, M., Meek, S., Blamire, I.W.H. and Wells, G.A.H. (1996). BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases. Veterinary Record 138, 175-177.

Simmons, M.M., Ryder, S.J., Chaplin M.C., Spencer Y.I., Webb C.R., Hoinville L.J., Ryan, J., Stack, M.J., Wells G.A.H. and Wilesmith, J.W. (2000) Scrapie surveillance in Great Britain: results of an abattoir survey, 1997/98. The Veterinary Record. 146, 391-395.

Wells, G.A.H. and Wilesmith, J.W. (1995). The neuropathology and epidemiology of bovine spongiform encephalopathy. Brain Pathology 5, 91-103

Wells, G.A.H., Hancock, R.D., Cooley, W.A., Richards, M.S., Higgins, R.J. and David, G.P. (1989) Bovine spongiform encephalopathy: diagnostic significance of vacuolar change in selected nuclei of the medulla oblongata. Veterinary Record 125, 521-524

Wells, G.A.H., Hawkins, S.A.C., Cunningham, A.A., Blamire, I.W.H., Wilesmith, J.W., Sayers, A.R. and Harris, P. (1994). Comparative pathology of the new transmissible spongiform encephalopathies. In: Transmissible Spongiform Encephalopathies. Proceedings of a Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities, 14-15 September 1993, Brussels, pp327-346.

Wells, G.A.H., Hawkins, S.A.C., Green, R.B., Austin, A.R., Dexter, I., Spencer, Y.I., Chaplin, M.J., Stack, M.J. and Dawson, M. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Veterinary Record 142, 103-106.

Wells, G.A.H., Hawkins, S.A.C., Hadlow, W.J. and Spencer, Y.I. (1992). The discovery of bovine spongiform encephalopathy and observations on the vacuolar changes. In: Prion Disease of Humans and Animals. Eds. S.B. Prusiner, J. Collinge, J. Powell and B. Anderton. Ellis-Horwood, Chichester, pp 256-274

Wilesmith, J.W., Wells, G.A.H, Ryan, J.B.M., Gavier-Widen, D. and Simmons, M.M. (1997) A cohort study to examine maternally associated risk factors for bovine spongiform encephalopathy. Veterinary Record 141, 239-243.

Williams, E.S. & Young, S. (1993). Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni). Veterinary Pathology 30, 36-45.

TABLE AND FIGURE LEGENDS

Table 1. Results of diagnostic test comparisons (n=491). The unshaded cells represent animals which fulfil the current statutory criteria for confirmed BSE. The shaded rows at the bottom represent statutorily negative animals.

Table 2 Differential neurohistological diagnoses in cases which were statutorily negative on the basis of vacuolar histopathology Study numbers A-1 to A-4 represent previous successive longitudinal studies, A1-3 being random samples of the epidemic as a whole (SE 0203 & 0206), and A-4 being animals born in 1992 [Simmons et al, 1996 SE 0216]. A-5 is a continuation of the longitudinal studies, based on animals born in 1993 (SE 0220). B - Scottish submissions [Jeffrey et al, 1994]. C- submissions in England and Wales 87-89 [McGill & Wells, 1993] . D - First 100 negative suspects born after July 1988 [Pollin et al 1992]. E -suspect BSE and rabies submissions in the USA [Miller et al, 1992]. NS - not stated.

Table 3. Relative sensitivity and specificity of each test against a diagnosis of statutorily positive.

Figure 1. Schematic representation of the standard sampling sites of bovine brain. a) frontal; b) parietal; c) occipital, thalamus; d) rostral midbrain; e) caudal midbrain f) rostral medulla, cerebellum; g) medulla (obex).

Figure 2. Periodic sampling and lesion profiling of BSE field cases throughout the epidemic (n=100 for each sample) shows that the profile has remained consistent.

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Table 1

Histology IHC WB SAF Number + + + + 142 + + + - 55 - + + + 2 - + + - 6 - + - - 1 - - + + 2 - - + - 3 - - - + 5 - - - - 275

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Table 2

Frequency of diagnosis (%) Study number Diagnosis A-1 A-2 1991- 1992- 1993- 1994- B C D E born born b born or n No significant lesions 61.6 61.7 61 61.5 68.2 70.7 39 57.5 54 47 Focal spongiosis (sub.nigra) 26.7 21 20 25.5 25.2 12.7 18 23 29 NS Inflammatory disorders 8.1 11.1 12 14 6.5 10.8 15 17 11 30 Tumours 1.2 0.6 0.7 1.5 - - 2 2.5 - 2.5 Cerebrocortical necrosis 2 1.2 - - - - 0.5 2 2 NS Congenital dysplasias - - 0.7 - - 2.5 0. - 3 NS Idiopathic brainstem - 0.6 - - - - 7 - - NS neuronal chromatolysis BSE (Statutory negative) 1 1.2 - - 1.8 1.9 NS NS - - Other 1.2 2.4 5.4 1.5 1 2.5 10.5 5.5 7 NS Period of sample collection 92/93 93/94 94/95 95/96 97/99 96/99 88/93 87/89 1991 1990- Number of cases in sample 174 164 122 149 107 157 761 200 100 117 BSE Negative rate (%) 11.5 16.2 55 47.6 18 24.9 18.2 7-14 82 100

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Table 3

Sensitivity Specificity (%) (%) Histology 93.4 100 Immunohistochemistry 97.6 100 Western blotting 99.5 100 SAF 69.2 98.2

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Figure 1

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lesion profile summary

original year 1 e 2 r

o year 2 c s 1991 born n a

e 1992 born

m 1.5 1993 born 1994 born

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 area code

Figure 2

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