Transmissible Spongiform Encephalopathies Surveillance Research Articles

Detection of Abnormal Prion Protein by Immunohistochemistry.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that usesprimary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Technical specifications on the collection, management and analysis of data on the surveillance of transmissible spongiform encephalopathies (TSEs) in ruminants and other animals in the EU

Technical specifications on the collection, management and analysis of data on the surveillance of transmissible spongiform encephalopathies (TSEs) in ruminants and other animals in the EU

Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy–Detecting Amyloid Seeding Assays

Transmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, invitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. Invitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed invitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE(+) and TSE(-) associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing invitro PrP conversion may be used to titrate TSEinfectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and invitro PrP conversion for elk CWD.

Neurological diseases of ruminant livestock in Australia. I: general neurological examination, necropsy procedures and neurological manifestations of systemic disease, trauma and neoplasia

Disease surveillance is an integral part of most veterinary practices in Australia. The aim of this series of invited reviews is to facilitate the differential and ultimately definitive diagnosis of some of the previously known, as well as the novel and emerging, neurological disorders of ruminant livestock, which is of particular importance in the surveillance for transmissible spongiform encephalopathies. General principles of a systematic neurological examination, necropsy procedures and the neurological manifestations of systemic disease, trauma and neoplasia are described here.

Surveillance and simulation of bovine spongiform encephalopathy and scrapie in small ruminants in Switzerland

BackgroundAfter bovine spongiform encephalopathy (BSE) emerged in European cattle livestock in 1986 a fundamental question was whether the agent established also in the small ruminants' population. In Switzerland transmissible spongiform encephalopathies (TSEs) in small ruminants have been monitored since 1990. While in the most recent TSE cases a BSE infection could be excluded, for historical cases techniques to discriminate scrapie from BSE had not been available at the time of diagnosis and thus their status remained unclear. We herein applied state-of-the-art techniques to retrospectively classify these animals and to re-analyze the affected flocks for secondary cases. These results were the basis for models, simulating the course of TSEs over a period of 70 years. The aim was to come to a statistically based overall assessment of the TSE situation in the domestic small ruminant population in Switzerland.ResultsIn sum 16 TSE cases were identified in small ruminants in Switzerland since 1981, of which eight were atypical and six were classical scrapie. In two animals retrospective analysis did not allow any further classification due to the lack of appropriate tissue samples. We found no evidence for an infection with the BSE agent in the cases under investigation. In none of the affected flocks, secondary cases were identified. A Bayesian prevalence calculation resulted in most likely estimates of one case of BSE, five cases of classical scrapie and 21 cases of atypical scrapie per 100'000 small ruminants. According to our models none of the TSEs is considered to cause a broader epidemic in Switzerland. In a closed population, they are rather expected to fade out in the next decades or, in case of a sporadic origin, may remain at a very low level.ConclusionsIn summary, these data indicate that despite a significant epidemic of BSE in cattle, there is no evidence that BSE established in the small ruminant population in Switzerland. Classical and atypical scrapie both occur at a very low level and are not expected to escalate into an epidemic. In this situation the extent of TSE surveillance in small ruminants requires reevaluation based on cost-benefit analysis.

The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries

BackgroundDuring the last decade, active surveillance for transmissible spongiform encephalopathies in small ruminants has been intensive in Europe. In many countries this has led to the detection of cases of atypical scrapie which, unlike classical scrapie, might not be contagious. EU legislation requires, that following detection of a scrapie case, control measures including further testing take place in affected flocks, including the culling of genotype susceptible to classical scrapie. This might result in the detection of additional cases. The aim of this study was to investigate the occurrence of additional cases in flocks affected by atypical scrapie using surveillance data collected in Europe in order to ascertain whether atypical scrapie, is contagious.ResultsQuestionnaires were used to collect, at national level, the results of active surveillance and testing associated with flock outbreaks in 12 European countries. The mean prevalence of atypical scrapie was 5.5 (5.0-6.0) cases per ten thousand in abattoir surveillance and 8.1 (7.3-9.0) cases per ten thousand in fallen stock. By using meta-analysis, on 11 out of the 12 countries, we found that the probability of detecting additional cases of atypical scrapie in positive flocks was similar to the probability observed in animals slaughtered for human consumption (odds ratio, OR = 1.07, CI95%: 0.70-1.63) or among fallen stock (OR = 0.78, CI95%: 0.51-1.2). In contrast, when comparing the two scrapie types, the probability of detecting additional cases in classical scrapie positive flocks was significantly higher than the probability of detecting additional cases in atypical scrapie positive flocks (OR = 32.4, CI95%: 20.7-50.7).ConclusionsThese results suggest that atypical scrapie is not contagious or has a very low transmissibility under natural conditions compared with classical scrapie. Furthermore this study stressed the importance of standardised data collection to make good use of the analyses undertaken by European countries in their efforts to control atypical and classical scrapie.

Neuropathologic Features of Aleutian Disease in Farmed Mink in Ireland and Molecular Characterization of Aleutian Mink Disease Virus Detected in Brain Tissues

A neuropathologic survey was conducted on mink brains from the 5 licensed mink farms in Ireland. The survey was part of a transmissible spongiform encephalopathy surveillance study. Aleutian disease (AD) was present on 4 of the 5 farms (80%). Neuropathologic features of nonsuppurative meningoencephalitis were common in mink from the 4 affected farms but were absent in the mink from the fifth farm, which was free of AD. The meningoencephalitis was characterized by infiltrates of lymphocytes and plasma cells, which were present in meninges, perivascular spaces, and the brain parenchyma. Fibrinoid necrotizing arteritis was seen in 11 mink brains, all of which were obtained from a single farm. Aleutian mink disease virus (AMDV) sequences for the capsid protein VP2 were obtained from brain samples from all affected farms. Although containing previously unreported amino acid residues, similarities with European and North American isolates were observed in the hypervariable regions within VP2, suggesting Irish AMDV is related to those isolates. The predicted amino acid residues, suspected of conferring pathogenicity at certain positions of the VP2 sequence, were present in the viral nucleic acid sequences.

Interlaboratory trial on TSE rapid tests for the control of the Italian scrapie surveillance network

Scrapie, a neurodegenerative disease of sheep and goats and one of several transmissible spongiform encephalopathies (TSEs) has been subject to mandatory active surveillance in EU through rapid testing since 2002. Regulation EC/999/2001 on TSE surveillance requires that each Member State's National Reference Laboratory for TSE periodically verifies diagnostic standards and methods by comparative testing. In 2007 the Italian Reference Centre carried out the first ring trial for classical scrapie on a set of 21 negative and 9 positive homogenated brainstems, the latter consisting of three replicates of an ARQ/ARQ scrapie positive sample diluted 1:10, 1:20 and 1:50. The purpose of the study was to verify the interlaboratory agreement in term of Cohen's kappa ( k) of the rapid systems currently used by the 25 national rapid test laboratories (RTLs) [laboratories: Biorad TeSeE (17 laboratories), Enfer TSE version 2.0 (4 laboratories), Idexx Herd chek BSE-scrapie antigen kit (3 laboratories) and Prionics check LIA SR (1 laboratory)]. Our results show that the agreement among the laboratories using the same rapid test varied between 0.84 and 1, while the estimated overall agreement among the 25 laboratories was very good ( k- combined = 0.87, 95% confidence interval 0.85–0.89). Nevertheless, as regards differences in analytical sensitivity among the rapid tests in use, under-recognition of pre-clinical scrapie cases by lower performing systems must be expected.

Field Performance of Two Rapid Screening Tests in Active Surveillance of Transmissible Spongiform Encephalopathies in Small Ruminants

Recently, screening tests for monitoring the prevalence of transmissible spongiform encephalopathies specifically in sheep and goats became available. Although most countries require comprehensive test validation prior to approval, little is known about their performance under normal operating conditions. Switzerland was one of the first countries to implement 2 of these tests, an enzyme-linked immunosorbent assay (ELISA) and a Western blot, in a 1-year active surveillance program. Slaughtered animals (n = 32,777) were analyzed in either of the 2 tests with immunohistochemistry for confirmation of initial reactive results, and fallen stock samples (n = 3,193) were subjected to both screening tests and immunohistochemistry in parallel. Initial reactive and false-positive rates were recorded over time. Both tests revealed an excellent diagnostic specificity (>99.5%). However, initial reactive rates were elevated at the beginning of the program but dropped to levels below 1% with routine and enhanced staff training. Only those in the ELISA increased again in the second half of the program and correlated with the degree of tissue autolysis in the fallen stock samples. It is noteworthy that the Western blot missed 1 of the 3 atypical scrapie cases in the fallen stock, indicating potential differences in the diagnostic sensitivities between the 2 screening tests. However, an estimation of the diagnostic sensitivity for both tests on field samples remained difficult due to the low disease prevalence. Taken together, these results highlight the importance of staff training, sample quality, and interlaboratory comparison trials when such screening tests are implemented in the field.

A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries

BackgroundThe development of active surveillance programmes for transmissible spongiform encephalopathies of small ruminants across Europe has led to the recent identification of a previously undetected form of ovine prion disease, 'atypical' scrapie. Knowledge of the epidemiology of this disease is still limited, as is whether it represents a risk for animal and/or public health.The detection of atypical scrapie has been related to the use of only some of the EU agreed rapid tests. Information about the rapid tests used is not, as yet, available from public reports on the surveillance of transmissible spongiform encephalopathies in small ruminants. We collected detailed results of active surveillance from European countries to estimate and to compare the prevalence of atypical scrapie and classical scrapie in sheep for each country stratified by each surveillance stream; healthy slaughtered and found dead adult sheep.ResultsFrom the 20 participating countries, it appeared that atypical scrapie was detected in Europe wherever the conditions necessary for its diagnosis were present. In most countries, atypical scrapie and classical scrapie occurred at low prevalence level. The classical scrapie prevalence estimates were more variable than those for atypical scrapie, which appeared remarkably homogeneous across countries, surveillance streams and calendar years of surveillance. Differences were observed in the age and genotype of atypical scrapie and classical scrapie cases that are consistent with previous published findings.ConclusionThis work suggests that atypical scrapie is not rare compared to classical scrapie. The homogeneity of its prevalence, whatever the country, stream of surveillance or year of detection, contrasts with the epidemiological pattern of classical scrapie. This suggests that the aetiology of atypical scrapie differs from that of classical scrapie.

Experimental transmission of atypical scrapie to sheep

BackgroundActive surveillance for transmissible spongiform encephalopathies in small ruminants has been an EU regulatory requirement since 2002. A number of European countries have subsequently reported cases of atypical scrapie, similar to previously published cases from Norway, which have pathological and molecular features distinct from classical scrapie. Most cases have occurred singly in flocks, associated with genotypes considered to be more resistant to classical disease. Experimental transmissibility of such isolates has been reported in certain ovinised transgenic mice, but has not previously been reported in the natural host. Information on the transmissibility of this agent is vital to ensuring that disease control measures are effective and proportionate.ResultsThis report presents the successful experimental transmission, in 378 days, of atypical scrapie to a recipient sheep of homologous genotype with preservation of the pathological and molecular characteristics of the donor. This isolate also transmitted to ovinised transgenic mice (Tg338) with a murine phenotype indistinguishable from that of Nor 98.ConclusionThis result strengthens the opinion that these cases result from a distinct strain of scrapie agent, which is potentially transmissible in the natural host under field conditions.

Lack of PrP sc immunostaining in intracranial ectopic lymphoid follicles in a sheep with concomitant non-suppurative encephalitis and Nor98-like atypical scrapie: A case report

During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an initial reactor was detected using a rapid test on a brain sample. Immunohistochemistry confirmed an atypical TSE presentation that closely resembled the previously described Nor98 cases. Sequencing of the prnp gene confirmed the ARQ/AHQ genotype with the L141F mutation at codon 141 associated with this phenotype. The head, including the brain and cranial lymphoid tissues, was sampled and examined thoroughly. Non-purulent encephalitis, with ectopic lymphoid follicle formation within the brain, was diagnosed concomitant to the TSE. When scrapie-associated prion protein (PrP sc) deposition was studied by immunohistochemistry there was a noticeable lack of lymphotropism. The distribution of PrP sc in the brain differed considerably from that of classical scrapie cases. Astrogliosis and microgliosis were demonstrated by histochemical procedures.

Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrPsc). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K–resistant core fragment of PrPsc distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrPsc distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.

An evaluation of scrapie surveillance in the United States

Animal health surveillance systems should reflect national disease control priorities and promote the best use of public resources by maximizing effectiveness and efficiency. A surveillance system should be routinely evaluated to assess the degree to which the system accomplishes these goals, fulfills its stated objectives, and meets accepted surveillance standards. In the United States, there are a number of disparate endemic disease surveillance and eradication programs. The National Animal Health Surveillance System is a federal initiative designed to combine animal health surveillance and monitoring activities into a comprehensive and coordinated system. A protocol has been developed to facilitate the evaluation of animal health surveillance systems and investigate opportunities for coordination between the different surveillance and eradication programs. The evaluation protocol was based largely on protocols developed for public health but adapted for the specific needs and goals of animal health surveillance. The evaluation process was designed to identify program strengths and areas for improvement and facilitate the system's adaptability to changing situations. The evaluation protocol was applied to the scrapie surveillance system in the United States; scrapie surveillance was found to be an important part of surveillance for transmissible spongiform encephalopathies. Results from the evaluation of sensitivity, sampling methods and representativeness are presented.

Atypical Scrapie in a Swiss Goat and Implications for Transmissible Spongiform Encephalopathy Surveillance

Different types of transmissible spongiform encephalopathies (TSEs) affect sheep and goats. In addition to the classical form of scrapie, both species are susceptible to experimental infections with the bovine spongiform encephalopathy (BSE) agent, and in recent years atypical scrapie cases have been reported in sheep from different European countries. Atypical scrapie in sheep is characterized by distinct histopathologic lesions and molecular characteristics of the abnormal scrapie prion protein (PrP(sc)). Characteristics of atypical scrapie have not yet been described in detail in goats. A goat presenting features of atypical scrapie was identified in Switzerland. Although there was no difference between the molecular characteristics of PrP(sc) in this animal and those of atypical scrapie in sheep, differences in the distribution of histopathologic lesions and PrP(sc) deposition were observed. In particular the cerebellar cortex, a major site of PrP(sc) deposition in atypical scrapie in sheep, was found to be virtually unaffected in this goat. In contrast, severe lesions and PrP(sc) deposition were detected in more rostral brain structures, such as thalamus and midbrain. Two TSE screening tests and PrP(sc) immunohistochemistry were either negative or barely positive when applied to cerebellum and obex tissues, the target samples for TSE surveillance in sheep and goats. These findings suggest that such cases may have been missed in the past and could be overlooked in the future if sampling and testing procedures are not adapted. The epidemiological and veterinary public health implications of these atypical cases, however, are not yet known.

Molecular profiling and comparison of field transmissible spongiform encephalopathy cases diagnosed in Catalunya

Molecular profiling of the proteinase K resistant prion protein (PrP res) is a technique that has been applied to the characterisation of transmissible spongiform encephalopathy (TSE) strains. An interesting example of the application of this technique is the ability to differentiate, at the experimental level, between bovine spongiform encephalopathy (BSE) and scrapie infection in sheep, and to distinguish between classical and atypical BSE and scrapie cases. Twenty-six BSE cases and two scrapie cases from an active TSE surveillance program and diagnosed at the PRIOCAT, TSE Reference Laboratory (Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona, Catalunya, Spain) were examined by Western blotting. Molecular profiling was achieved by comparing the glycosylation profile, deglycosylated PrP molecular weight and 6H4/P4 monoclonal antibody binding ratio. The results obtained during the characterisation of these field cases indicated an absence of atypical BSE cases in Catalunya.

Recognition of the Nor98 Variant of Scrapie in the Swedish Sheep Population

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.

Discriminating Scrapie and Bovine Spongiform Encephalopathy Isolates by Infrared Spectroscopy of Pathological Prion Protein

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.

Opinion of the Scientific Panel on biological hazards (BIOHAZ) on the interpretation of results of EU surveillance of transmissible spongiform encephalopathies (TSEs) in ovine and caprine animals, culling strategies for TSEs in small ruminants and the TSE

Opinion of the Scientific Panel on biological hazards (BIOHAZ) on the interpretation of results of EU surveillance of transmissible spongiform encephalopathies (TSEs) in ovine and caprine animals, culling strategies for TSEs in small ruminants and the TSE

European Union's rapid TSE testing in adult cattle and sheep: implementation and results in 2001 and 2002.

After the discovery of variant Creutzfeldt-Jakob disease (vCJD), scientific advances quickly led to post-mortem tests to identify late-stage bovine spongiform encephalopathy (BSE) disease. These were first used in Switzerland in 1999 for active BSE surveillance of a) fallen and emergency-slaughter bovines (risk stock) and b) 5% sample of routinely slaughtered cattle over 30 months of age. In 1999 and 2000, Switzerland's estimated 103 BSE positives per 1000000 adult cattle put it in the same BSE risk classification as UK and Portugal. In July 2000, the European Union's Scientific Steering Committee published its methodology (and first vetted results) for geographical BSE risk (GBR) assessment in cattle. Member states with no BSE cases found themselves, on rational assessment, classified as GBR III (BSE likely but not confirmed, or confirmed at a lower level). Because of Europe's thus highly assessed BSE risks, active BSE surveillance of adult cattle in all member states began in January 2001 using one of three validated post-mortem tests. Implementation was variable across member states in January to March 2001 but, where operational, active surveillance was typically achieved for around 13300 routinely slaughtered and 1000 risk stock per month per 1000000 adult cattle; BSE positive rates were 60 and 600 per 1000000 routinely slaughtered and risk cattle, respectively. By the second half of 2001, active BSE surveillance was operating reasonably in most member states, although anomalies persisted. Performance and results for July to December 2001 and for January to June 2002 are considered in detail. The BSE positive rate decreased substantially in UK, Portugal and Ireland between semesters, whereas Spain's rates increased for both routinely slaughtered and risk bovines. Based on 1450000 routinely slaughtered and 135000 risk stock as standard, France could have expected 153 BSE positives in July to December 2001 (109 in January to June 2002); Italy 154 (67); and Germany only 39 (48). When sample-based surveillance data were scaled up and combined with clinical BSE cases, Great Britain's BSE positives were estimated at around 400 per 1000000 adult cattle in 2002 compared with over 1000 per 1000000 adult cattle in 2000. Age distributions for cattle subject to active BSE surveillance have been underexploited. The major transmissible spongiform encephalopathy (TSE) which affects sheep and goats is scrapie. Passive surveillance of scrapie is associated with substantial under-reporting. Susceptibility to scrapie depends strongly on sheep genotype; but resistance to scrapie does not necessarily confer resistance of sheep to BSE. Because of uncertainty about the true prevalence of scrapie-infected adult sheep and concern that BSE in sheep may be missed, the European Union pre-empted its planned evaluation of rapid post-mortem TSE tests in sheep by requiring the rapid TSE testing of small ruminants from April 2002 with one of the three cattle-validated tests. Basic requirements for active TSE surveillance in sheep were: random sample of 6000 fallen sheep and of 60000 routinely slaughtered adult native sheep to be tested per member state by end March 2003. Lower surveillance targets were set for countries with under 1000000 adult sheep. Adequately to map scrapie-susceptible genotypes and identify resistant genotypes, a random sample of 500 routinely slaughtered native adult sheep was to be genotyped, together with each TSE rapid test positive adult sheep and two sets of three suitably sampled controls. By the end of August 2002, when 41% of the initial surveillance time had elapsed, only 20% of the European joint target for routinely slaughtered adult sheep had been completed, but that for fallen sheep was exceeded. Except in Ireland, the upper 95% confidence bound on TSE prevalence exceeded 500 per 1000000 routinely slaughtered adult sheep in reporting-compliant countries with more than 1000000 adult sheep. The UK, Greece, Italy and France were likely to approach the goal of 100 TSE rapid test positives on completion of their assigned first-year surveillance target for sheep. Results from the recommended genotyping of TSE positive adult sheep and controls for use in inferring differential TSE-positive susceptibility by genotype are awaited. Only by genotyping 5000-50000 TSE-positive adult sheep, a massive undertaking even on the European scale, will it become clear whether scrapie resistance is relative rather than absolute. This paper details Europe's quantitative evolution in TSE surveillance.