The 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.
From 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.
This 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.
In the wake of the epidemic of bovine spongiform encephalopathy the British government established a flock of sheep from which scrapie-free animals are supplied to laboratories for research. Three breeds of sheep carrying a variety of different genotypes associated with scrapie susceptibility/resistance were imported in 1998 and 2001 from New Zealand, a country regarded as free from scrapie. They are kept in a purpose-built Sheep Unit under strict disease security and are monitored clinically and post mortem for evidence of scrapie. It is emphasised that atypical scrapie, as distinct from classical scrapie, has been recognised only relatively recently and differs from classical scrapie in its clinical, neuropathological and biochemical features. Most cases are detected in apparently healthy sheep by post mortem examination.
The occurrence of atypical scrapie in three sheep in (or derived from) the Sheep Unit is reported. Significant features of the affected sheep included their relatively high ages (6 y 1 mo, 7 y 9 mo, 9 y 7 mo respectively), their breed (all Cheviots) and their similar PRNP genotypes (AFRQ/AFRQ, AFRQ/ALRQ, and AFRQ/AFRQ, respectively). Two of the three sheep showed no clinical signs prior to death but all were confirmed as having atypical scrapie by immunohistochemistry and Western immunoblotting. Results of epidemiological investigations are presented and possible aetiologies of the cases are discussed.
By process of exclusion, a likely explanation for the three cases of atypical scrapie is that they arose spontaneously and were not infected from an exterior source. If correct, this raises challenging issues for countries which are currently regarded as free from scrapie. It would mean that atypical scrapie is liable to occur in flocks worldwide, especially in older sheep of susceptible genotypes. To state confidently that both the classical and atypical forms of scrapie are absent from a population it is necessary for active surveillance to have taken place.
Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.
Following the bovine spongiform encephalopathy (BSE) crisis and the identification of its zoonotic properties, a sanitary policy has been implemented based on both eradication of transmissible spongiform encephalopathies (TSE) in food-producing animals and exclusion of known infectious materials from the food chain. Atypical/Nor98 scrapie is a prion disease of small ruminants identified worldwide. Currently it represents a significant part of the TSE cases detected in Europe. The restricted tissue distribution of Atypical/Nor98 scrapie agent in its natural host and the low detected prevalence of secondary cases in affected flocks meant that it is believed to be a poorly transmissible disease. This has led to the view that Atypical/Nor98 scrapie is a spontaneous disorder for which human and animal exposure risk remains low. In this study we demonstrate that in affected individuals, Atypical/Nor98 scrapie agent can disseminate in lymphoid tissues, nerves, and muscles, challenging the idea that it is a brain-restricted infectious agent. Evidence for the deficiencies in the current methods applied for monitoring Atypical/Nor98 scrapie is provided that would indicate an underestimation in the prevalence in the general population and in the affected flocks. These elements challenge the hypothesis on the biology of this recently identified TSE agent.
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.
In the view of concerns that bovine spongiform encephalopathy has entered the small ruminant population, comprehensive active surveillance programs for transmissible spongiform encephalopathies (TSEs) in sheep and goats were implemented worldwide. In these, previously unrecognized atypical scrapie cases were identified that to date represent the majority of detected small ruminant TSE cases in some countries. The pathogenesis and epidemiology of atypical scrapie, as well as its relevance to both animal health and food safety, is still poorly understood. In the present study, we performed a systematic neuropathological analysis of recently diagnosed atypical scrapie cases in Switzerland. Our results show that the neuropathological presentation in atypical scrapie–affected small ruminants varies remarkably, and the results indicate a biodiversity of TSEs in sheep and goats larger than expected, with some similarities to known human TSEs. These findings will form the basis for future research on TSE phenotypes and help to design experimental studies necessary to generate data for risk assessments and the implementation of appropriate disease-control strategies.
Following the bovine spongiform encephalopathy (BSE) crisis, the European Union has introduced policies for eradicating transmissible spongiform encephalopathies (TSEs), including scrapie, from large ruminants. However, recent European Union surveillance has identified a novel prion disease, ‘atypical’ scrapie, substantially different from classical scrapie. It is unknown whether atypical scrapie is naturally transmissible or zoonotic, like BSE. Furthermore, cases have occurred in scrapie-resistant genotypes that are targets for selection in legislated selective breeding programmes. Here, the first epidemiological study of British cases of atypical scrapie is described, focusing on the demographics and trading patterns of farms and using databases of recorded livestock movements. Triplet comparisons found that farms with atypical scrapie stock more sheep than those of the general, non-affected population. They also move larger numbers of animals than control farms, but similar numbers to farms reporting classical scrapie. Whilst there is weak evidence of association through sheep trading of farms reporting classical scrapie, atypical scrapie shows no such evidence, being well-distributed across regions of Great Britain and through the sheep-trading network. Thus, although cases are few in number so far, our study suggests that, should natural transmission of atypical scrapie be occurring at all, it is doing so slowly.
The public health threat represented by a potential circulation of bovine spongiform encephalopathy agent in sheep population has led European animal health authorities to launch large screening and genetic selection programmes. If demonstrated, such a circulation would have dramatic economic consequences for sheep breeding sector. In this context, it is important to evaluate the feasibility of qualification procedures that would allow sheep breeders demonstrating their flock is free from scrapie. Classical approaches, based on surveys designed to detect disease presence, do not account for scrapie specificities: the genetic variations of susceptibility and the absence of live diagnostic test routinely available. Adapting these approaches leads to a paradoxical situation in which a greater amount of testing is needed to substantiate disease freedom in genetically resistant flocks than in susceptible flocks, whereas probability of disease freedom is a priori higher in the former than in the latter. The goal of this study was to propose, evaluate and compare several qualification strategies for demonstrating a flock is free from scrapie.
A probabilistic framework was defined that accounts for scrapie specificities and allows solving the preceding paradox. Six qualification strategies were defined that combine genotyping data, diagnostic tests results and flock pedigree. These were compared in two types of simulated flocks: resistant and susceptible flocks. Two strategies allowed demonstrating disease freedom in several years, for the majority of simulated flocks: a strategy in which all the flock animals are genotyped, and a strategy in which only founders animals are genotyped, the flock pedigree being known. In both cases, diagnostic tests are performed on culled animals. The less costly strategy varied according to the genetic context (resistant or susceptible) and to the relative costs of a genotyping exam and of a diagnostic test.
This work demonstrates that combining data sources allows substantiating a flock is free from scrapie within a reasonable time frame. Qualification schemes could thus be a useful tool for voluntary or mandatory scrapie control programmes. However, there is no general strategy that would always minimize the costs and choice of the strategy should be adapted to local genetic conditions.
Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrPSc, the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrPSc assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrPSc-positive sheep were identified through the passive surveillance program, and an additional three PrPSc-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrPSc-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.
The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.
The origin of the transmissible agent involved in the food-borne epidemic of bovine spongiform encephalopathy (BSE) remains a mystery. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic, form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. In this study we analyzed the molecular features of the disease-associated protease-resistant prion protein (PrPres) found in the brain of transgenic mice overexpressing the ovine prion protein after experimental infection with prions from bovine classical and L-type BSEs or from ovine scrapie. Scrapie cases included rare “CH1641-like” isolates, which share some PrPres molecular features with classical BSE and L-type BSE. Scrapie isolates induced in transgenic mouse brains the production of a C-terminally cleaved form of PrPres, which was particularly abundant from “CH1641-like” cases. In contrast, this C-terminal prion protein product was undetectable in ovine transgenic mice infected with bovine prions from both classical and L-type BSE. These findings add a novel approach for the discrimination of prions that may help to understand their possible changes during cross-species transmissions.
Scrapie is a chronic neurodegenerative disease affecting small ruminants and belongs to the transmissible spongiform encephalopathies. Scrapie is considered a serious animal disease and it has been notifiable in Norway since 1965. The clinical signs of scrapie might be vague and the farmers, if familiar with the signs of scrapie, are often in the best position for detecting scrapie suspects. In 2002, an anonymous questionnaire survey was conducted in order to assess Norwegian sheep farmers' vigilance of scrapie.
Although the potential detection of a scrapie-positive animal would lead to the destruction of the sheep flock concerned, almost all the farmers (97 %) expressed their willingness to report scrapie suspects. This was most certainly dependent on the Government taking the economic responsibility for the control programme as nearly all the farmers responded that this was an important condition. Listeriosis is relatively common disease in Norwegian sheep and a differential diagnosis for scrapie. In a multinomial logistic regression the reporting behaviour for non-recovering listeriosis cases, used as a measurement of willingness to report scrapie, was examined. The reporting of non-recovering listeriosis cases increased as the knowledge of scrapie-associated signs increased, and the reporting behaviour was dependent on both economic and non-economic values.
The results indicate that in 2002 almost all sheep farmers showed willingness to report any scrapie suspects. Nevertheless there is an underreporting of scrapie suspects and the farmers' awareness and hence their vigilance of scrapie could be improved. Furthermore, the results suggest that to ensure the farmers' compliance to control programmes for serious infectious diseases, the farmers' concerns of non-economic as well as economic values should be considered.
Active 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.
This 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.
This 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.
Data from the Compulsory Scrapie Flocks Scheme (CSFS), part of the compulsory eradication measures for the control of scrapie in the EU, have been used to estimate the within-holding prevalence of classical scrapie in Great Britain (GB). Specifically data from one of the testing routes within the CSFS have been used; the initial cull (IC), whereby two options can be applied: the whole flock cull option by which the entire flock is depopulated, and the genotyping and cull of certain genotypes.
Between April 2005 and September 2007, 25,316 suitable samples, submitted from 411 flocks in 213 scrapie-affected holdings in Great Britain, were tested for scrapie. The predicted within-holding prevalence for the initial cull was 0.65% (95% CI: 0.55–0.75). For the whole cull option was 0.47% (95% CI: 0.32–0.68) and for the genotype and cull or mixed option (both options applied in different flocks of the same holding), the predicted within-holding prevalence was 0.7% (95% CI: 0.6–0.83). There were no significant differences in the within-flock prevalence between countries (England, Scotland and Wales) or between CSFS holdings by the surveillance stream that detected the index case. The number of CSFS flocks on a holding did not affect the overall within-holding prevalence of classical scrapie.
These estimates are important in the discussion of the epidemiological implications of the current EU testing programme of scrapie-affected flocks and to inform epidemiological and mathematical models. Furthermore, these estimates may provide baseline data to assist the design of future surveillance activities and control policies with the aim to increase their efficiency.
Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres.
Optimal PrPres detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrPresconcentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype – VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171 – allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002–2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrPres, but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrPres in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrPres bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrPres, BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrPres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study.
The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products.
Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known - classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie.
The risk of scrapie infection increases with increased duration and proximity of contact between sheep at lambing. Scrapie infectivity has not been detected in milk but cellular prion protein, the precursor of disease-associated prion protein PrPd, has been found in milk from ruminants. To determine whether milk is able to transmit scrapie, 18 lambs with a prion protein genotype associated with high susceptibility to scrapie (VRQ/VRQ) were fed milk from twelve scrapie-affected ewes of the same genotype, and 15 VRQ/VRQ sheep reared on scrapie-free dams served as controls.
Three lambs fed milk from scrapie-affected ewes were culled due to intercurrent diseases at 43, 44 and 105 days of age respectively, and PrPd was detected in the distal ileum of the first two lambs, whilst PrPd was not found in lymphoreticular tissues in the third lamb. A control lamb, housed in a separate pen and culled at 38 days of age, was also negative for PrPd in a range of tissues. Samples of recto-anal mucosa associated lymphoid tissue collected from the remaining 15 live lambs at seven months of age (between five to seven months after mixing) were positive for PrPd in the scrapie milk recipients, whereas PrPd was not detected in the remaining 14 controls at that time. A subsequent sample collected from control lambs revealed PrPd accumulation in two of five lambs eight months after mixing with scrapie milk recipients suggestive of an early stage of infection via lateral transmission. By contrast, the control sheep housed in the same building but not mixed with the scrapie milk recipients were still negative for PrPd.
The presence of PrPd in distal ileum and rectal mucosa indicates transmission of scrapie from ewe to lamb via milk (or colostrum) although it is not yet clear if such cases would go on to develop clinical disease. The high level of infection in scrapie-milk recipients revealed by rectal mucosal testing at approximately seven months of age may be enhanced or supplemented by intra-recipient infection as these lambs were mixed together after feeding with milk from scrapie-affected ewes and we also observed lateral transmission from these animals to lambs weaned from scrapie-free ewes.
After 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.
In 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.
In 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.
Since 2002, an active surveillance program for transmissible spongiform encephalopathy in small ruminants in European Union countries allowed identification of a considerable number of atypical cases with similarities to the previously identified atypical scrapie cases termed Nor98.
Here we report molecular and neuropathological features of eight atypical/Nor98 scrapie cases detected between 2002 and 2009. Significant features of the affected sheep included: their relatively high ages (mean age 7.9 years, range between 4.3 and 12.8), their breed (all Latxa) and their PRNP genotypes (AFRQ/ALRQ, ALRR/ALRQ, AFRQ/AFRQ, AFRQ/AHQ, ALRQ/ALRH, ALRQ/ALRQ). All the sheep were confirmed as atypical scrapie by immunohistochemistry and immunoblotting. Two cases presented more PrP immunolabelling in cerebral cortex than in cerebellum.
This work indicates that atypical scrapie constitutes the most common small ruminant transmissible spongiform encephalopathy form in Latxa sheep in the Spanish Basque Country. Moreover, a new genotype (ALRQ/ALRH) was found associated to atypical scrapie.
This paper explores the spatial distribution of sampling within the active surveillance of sheep scrapie in Great Britain. We investigated the geographic distribution of the birth holdings of sheep sampled for scrapie during 2002 – 2005, including samples taken in abattoir surveys (c. 83,100) and from sheep that died in the field ("fallen stock", c. 14,600). We mapped the birth holdings by county and calculated the sampling rate, defined as the proportion of the holdings in each county sampled by the surveys. The Moran index was used to estimate the global spatial autocorrelation across Great Britain. The contributions of each county to the global Moran index were analysed by a local indicator of spatial autocorrelation (LISA).
The sampling rate differed among counties in both surveys, which affected the distribution of detected cases of scrapie. Within each survey, the county sampling rates in different years were positively correlated during 2002–2005, with the abattoir survey being more strongly autocorrelated through time than the fallen stock survey. In the abattoir survey, spatial indices indicated that sampling rates in neighbouring counties tended to be similar, with few significant contrasts. Sampling rates were strongly correlated with sheep density, being highest in Wales, Southwest England and Northern England. This relationship with sheep density accounted for over 80% of the variation in sampling rate among counties. In the fallen stock survey, sampling rates in neighbouring counties tended to be different, with more statistically significant contrasts. The fallen stock survey also included a larger proportion of holdings providing many samples.
Sampling will continue to be uneven unless action is taken to make it more uniform, if more uniform sampling becomes a target. Alternatively, analyses of scrapie occurrence in these datasets can take account of the distribution of sampling. Combining the surveys only partially reduces uneven sampling. Adjusting the distribution of sampling between abattoirs to reduce the bias in favour of regions with high sheep densities could probably achieve more even sampling. However, any adjustment of sampling should take account of the current understanding of the distribution of scrapie cases, which will be improved by further analysis of this dataset.
It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrPSc using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrPSc within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).
Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE.
PrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification.
PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.
Prions; Transmissible spongiform encephalopathy; Scrapie; Sheep; PMCA
Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs) which naturally affect small and large ruminants respectively. However, small ruminants, which are susceptible to BSE under experimental conditions, have been exposed to the same or similar contaminated food additives as cattle. To date two natural cases of BSE in small ruminants have been reported. As a result surveillance projects, combined with appropriate control measures, have been established throughout the European Union (EU) to minimize the overall incidence of small ruminant TSEs. Although BSE can be differentiated from classical scrapie (subsequently referred to as scrapie) if appropriate discriminatory tests are applied, the value of these tests in BSE/scrapie co-infection scenarios has not been evaluated fully. Mouse bioassay is regarded as the gold standard regarding differentiation of distinct TSE strains and has been used as to resolve TSE cases were laboratory tests produced equivocal results. However, the ability of this method to discriminate TSE strains when they co-exist has not been examined systematically. To address this issue we prepared in vitro mixtures of ovine BSE and scrapie and used them to challenge RIII, C57BL/6 and VM mice.
Disease phenotype analysis in all three mouse lines indicated that most phenotypic parameters (attack rates, incubation periods, lesion profiles and Western blots) were compatible with scrapie phenotypes as were immunohistochemistry (IHC) data from RIII and C57BL/6 mice. However, in VM mice that were challenged with BSE/scrapie mixtures a single BSE-associated IHC feature was identified, indicating the existence of BSE in animals where the scrapie phenotype was dominant.
We conclude that wild type mouse bioassay is of limited value in detecting BSE in the presence of scrapie particularly if the latter is in relative excess.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0194-2) contains supplementary material, which is available to authorized users.
BSE; Scrapie; Co-infection; Bioassay; Mouse
Most previous analyses of scrapie outbreaks have focused on flocks run by research institutes, which may not reflect the field situation. Within this study, we attempt to rectify this deficit by describing the epidemiological characteristics of 30 sheep flocks naturally-infected with classical scrapie, and by exploring possible underlying causes of variation in the characteristics between flocks, including flock-level prion protein (PrP) genotype profile. In total, the study involved PrP genotype data for nearly 8600 animals and over 400 scrapie cases.
We found that most scrapie cases were restricted to just two PrP genotypes (ARQ/VRQ and VRQ/VRQ), though two flocks had markedly different affected genotypes, despite having similar underlying genotype profiles to other flocks of the same breed; we identified differences amongst flocks in the age of cases of certain PrP genotypes; we found that the age-at-onset of clinical signs depended on peak incidence and flock type; we found evidence that purchasing infected animals is an important means of introducing scrapie to a flock; we found some evidence that flock-level PrP genotype profile and flock size account for variation in outbreak characteristics; identified seasonality in cases associated with lambing time in certain flocks; and we identified one case that was homozygous for phenylalanine at codon 141, a polymorphism associated with a very high risk of atypical scrapie, and 28 cases that were heterozygous at this codon.
This paper presents the largest study to date on commercially-run sheep flocks naturally-infected with classical scrapie, involving 30 study flocks, more than 400 scrapie cases and over 8500 PrP genotypes. We show that some of the observed variation in epidemiological characteristics between farms is related to differences in their PrP genotype profile; although much remains unexplained and may instead be attributed to the stochastic nature of scrapie dynamics.
So-called atypical scrapie was first identified in Great Britain (GB) in 2002 following the introduction of wide-scale scrapie surveillance. In particular, abattoir and fallen stock surveys have been carried out in GB since 2002, with a total of 147 atypical positives identified by the end of 2006. The results of these surveys provide data with which to assess temporal trends in the prevalence of atypical scrapie in sheep in Great Britain between 2002 and 2006.
Using the results of abattoir and fallen stock surveys, the prevalence of atypical scrapie (percentage of samples positive) was estimated. The prevalence in the abattoir and fallen stock surveys, for all years combined, was 0.09% (95% confidence interval (CI): 0.08%–0.11%) and 0.07% (95% CI: 0.05%–0.11%), respectively. There were no significant temporal trends in either survey. Comparing the surveys' results, there were no significant differences in annual prevalence or the prevalence within PrP genotypes. For the abattoir survey, the PrP genotype with the highest prevalence was AHQ/AHQ, which was significantly higher than all other genotypes, except ARR/AHQ, AHQ/ARH and ARH/ARQ.
The estimated prevalence of atypical scrapie was similar in both the abattoir and fallen stock surveys. Our results indicate there was no significant temporal trend in prevalence, adding to evidence that this atypical form of scrapie may be a sporadic condition or, if it is infectious, that the force of infection is very low.
The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein (PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.
Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.
Since 2002, active surveillance programmes have detected numerous atypical scrapie (AS) and classical scrapie cases (CS) in French sheep with almost all the PrP genotypes. The aim of this study was 1) to quantify the genetic risk of AS in French sheep and to compare it with the risk of CS, 2) to quantify the risk of AS associated with the increase of the ARR allele frequency as a result of the current genetic breeding programme against CS.
We obtained genotypes at codons 136, 141, 154 and 171 of the PRNP gene for representative samples of 248 AS and 245 CS cases. We used a random sample of 3,317 scrapie negative animals genotyped at codons 136, 154 and 171 and we made inferences on the position 141 by multiple imputations, using external data. To estimate the risk associated with PrP genotypes, we fitted multivariate logistic regression models and we estimated the prevalence of AS for the different genotypes. Then, we used the risk of AS estimated for the ALRR-ALRR genotype to analyse the risk of detecting an AS case in a flock homogenous for this genotype.
Genotypes most at risk for AS were those including an AFRQ or ALHQ allele while genotypes including a VLRQ allele were less commonly associated with AS. Compared to ALRQ-ALRQ, the ALRR-ALRR genotype was significantly at risk for AS and was very significantly protective for CS. The prevalence of AS among ALRR-ALRR animals was 0.6‰ and was not different from the prevalence in the general population.
In conclusion, further selection of ALRR-ALRR animals will not result in an overall increase of AS prevalence in the French sheep population although this genotype is clearly susceptible to AS. However the probability of detecting AS cases in flocks participating in genetic breeding programme against CS should be considered.