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Transmissible spongiform encephalopathy (TSE) surveillance in goats relies on tests initially approved for cattle, subsequently assessed for sheep, and approval extrapolated for use in "small ruminants." The current EU-approved immunodetection tests employ antibodies against various epitopes of the prion protein PrP Sc , which is encoded by the host PRNP gene. The caprine PRNP gene is polymorphic, mostly at codons different from the ovine PRNP . The EU goat population is much more heterogeneous than the sheep population, with more PRNP -related polymorphisms, and with marked breed-related differences. The ability of the current tests to detect disease-specific PrP Sc generated against these different genetic backgrounds is currently assumed, rather than proven. We examined whether common polymorphisms within the goat PRNP gene might have any adverse effect on the relative performance of EU-approved rapid tests. The sample panel comprised goats from the UK, Cyprus, France, and Italy, with either experimental or naturally acquired scrapie at both the preclinical and/or unknown and clinical stages of disease. Test sensitivity was significantly lower and more variable when compared using samples from animals that were preclinical or of unknown status. However, all of the rapid tests included in our study were able to correctly identify all samples from animals in the clinical stages of disease, apart from samples from animals polymorphic for serine or aspartic acid at codon 146, in which the performance of the Bio-Rad tests was profoundly affected. Our data show that some polymorphisms may adversely affect one test and not another, as well as underline the dangers of extrapolating from other species.

This report presents the results of experimental challenges of goats with scrapie by both the intracerebral (i.c.) and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by i.c. challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than those of animals of all other genotypes (chi-square value, 10.8; P = 0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN animals) succumbed to oral challenge. The results also indicate that any cases of infection in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the rapid enzyme-linked immunosorbent assay (ELISA) was more variable and dependent on the polymorphism. Together with data from previous studies of goats exposed to infection in the field, these data support the previously reported observations that polymorphisms at this codon have a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions. IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms that are associated with susceptibility or resistance to scrapie. However, in contrast to the polymorphisms in sheep, they are more numerous in goats and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. An initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after i.c. inoculation. The presence of a D or S residue prolonged incubation periods significantly, and prions were detected in peripheral tissues only in NN animals. In oral challenges, prions were detected only in NN animals, and the presence of a D or S residue at this position conferred resistance to the disease. This study provides an experimental transmission model for assessing the genetic susceptibility of goats to scrapie., (© Crown copyright 2017.)

Aims: Prion diseases exist in classical and atypical disease forms. Both forms are characterized by disease-associated accumulation of a host membrane sialoglycoprotein known as prion protein (PrP d ). In classical forms of prion diseases, PrP d can accumulate in the extracellular space as fibrillar amyloid, intracellularly within lysosomes, but mainly on membranes in association with unique and characteristic membrane pathology. These membrane changes are found in all species and strains of classical prion diseases and consist of spiral, branched and clathrin-coated membrane invaginations on dendrites. Atypical prion diseases have been described in ruminants and man and have distinct biological, biochemical and pathological properties when compared to classical disease. The purpose of this study was to determine whether the subcellular pattern of PrP d accumulation and membrane changes in atypical scrapie were the same as those found in classical prion diseases., Methods: Immunogold electron microscopy was used to examine brains of atypical scrapie-affected sheep and Tg338 mice., Results: Classical prion disease-associated membrane lesions were not found in atypical scrapie-affected sheep, however, white matter PrP d accumulation was localized mainly to the inner mesaxon and paranodal cytoplasm of oligodendroglia. Similar lesions were found in myelinated axons of atypical scrapie Tg338-infected mice. However, Tg338 mice also showed the unique grey matter membrane changes seen in classical forms of disease., Conclusions: These data show that atypical scrapie infection directs a change in trafficking of abnormal PrP to axons and oligodendroglia and that the resulting pathology is an interaction between the agent strain and host genotype., (© 2016 Crown copyright. Neuropathology and Applied Neurobiology © 2016 British Neuropathological Society. This article Altered trafficking of abnormal prion protein in atypical scrapie: prion protein accumulation in oligodendroglial inner mesaxons is written by M. Jeffrey et al. It is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

The results of the study reported here are part of an ongoing integrated research programme aimed at producing additional, robust, evidence on the genetic resistance to classical scrapie in goats, with particular reference to codon 146. The study targeted animals aged ⩾6 years, which were born and raised in infected herds and were being culled for management reasons. A total of 556 animals were tested, and all positive animals (n = 117) were of the susceptible NN genotype. A total of 246 goats heterozygous or homozygous for putatively resistant alleles (S146 and D146) were screened with no positive results. The outcome of this study supports the hypothesis that the D146 and S146 alleles could be used as the basis for a nationwide strategy for breeding for resistance in the Cypriot goat population.

Background: Previous studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples)., Results: In an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie. All sheep challenged with goat scrapie brain became infected based on the immunohistochemical detection of disease-associated PrP (PrP(sc)) in lymphoid tissue, with an ARQ/ARQ sheep being the first to succumb. Subsequent feeding of milk to eight pairs of new-born ARQ/ARQ lambs, with each pair receiving milk from a different scrapie-affected goat, resulted in scrapie in the six pairs that received the largest volume of milk (38-87 litres per lamb), whereas two pairs fed 8-9 litres per lamb, and an environmental control group raised on sheep milk from healthy ewes, did not show evidence of infection when culled at up to 1882 days of age. Infection in those 12 milk recipients occurred regardless of the clinical status, PrP(sc) distribution, caprine arthritis-encephalitis virus infection status and PRNP polymorphisms at codon 142 (II or IM) of the donor goats, but survival time was influenced by PRNP polymorphisms at codon 141. Serial PMCA applied to a total of 32 milk samples (four each from the eight donor goats collected throughout lactation) detected PrP(sc) in one sample each from two goats., Conclusions: The scrapie agent was present in the milk from infected goats and was able to transmit to susceptible species even at early preclinical stage of infection, when PrP(sc) was undetectable in the brain of the donor goats. Serial PMCA as a PrP(sc) detection method to assess the risk of scrapie transmission via milk in goats proved inefficient compared to the bioassay.

Introduction: 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., Results: 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., Conclusions: 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.

Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

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.

Classical scrapie is one of the transmissible spongiform encephalopathies (TSEs), a group of fatal infectious diseases that affect the central nervous system (CNS). Classical scrapie can transmit laterally from ewe to lamb perinatally or between adult animals. Here we report detection of infectivity in tissues of an unborn fetus, providing evidence that in utero transmission of classical scrapie is also possible.

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

Background: Evidence for scrapie transmission from VRQ/VRQ ewes to lambs via milk was first reported in 2008 but in that study there were concerns that lateral transmission may have contributed to the high transmission rate observed since five control lambs housed with the milk recipients also became infected. This report provides further information obtained from two follow-up studies, one where milk recipients were housed separately after milk consumption to confirm the validity of the high scrapie transmission rate via milk and the second to assess any difference in infectivity from colostrum and subsequent milk. Protein misfolding cyclic amplification (PMCA) was also used to detect prion protein in milk samples as a comparison with the infectivity data and extended to milk samples from ewes without a VRQ allele., Results: Seven pairs of lambs fed colostrum and milk individually from seven scrapie-affected sheep (pre-clinical or clinical) presented with disease-associated prion protein, PrPd, in rectal lymphoid tissue at 4-5 months of age. Five further pairs of lambs fed either colostrum or subsequent milk from five pre-clinical scrapie-affected sheep equally presented with PrPd in lymphoid tissue by 9 months of age. Nine sheep were lost due to intercurrent diseases but all remaining milk or colostrum recipients, including those in the original study with the lateral transmission controls, developed clinical signs of scrapie from 19 months of age and scrapie was confirmed by brain examination. Unexposed control sheep totalling 19 across all three studies showed no evidence of infection.Scrapie PrP was amplified repeatedly by PMCA in all tested milk samples from scrapie-affected VRQ/VRQ sheep, and in one scrapie-affected ARQ/ARQ sheep. By contrast, milk samples from five VRQ/VRQ and 11 ARQ/ARQ scrapie-free sheep did not have detectable scrapie PrP on repeated tests., Conclusions: Feeding of milk from scrapie-affected sheep results in a high transmission rate in VRQ/VRQ sheep and both colostrum and milk transmit scrapie. Detection of scrapie prion protein in individual milk samples from scrapie-affected ewes confirms PMCA as a valuable in vitro test.

Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrP(Sc) immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrP(Sc) deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrP(Sc) deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE., (© 2011 The Authors. Brain Pathology © 2011 International Society of Neuropathology.)

To investigate the possibility of oral transmission of atypical scrapie in sheep and determine the distribution of infectivity in the animals' peripheral tissues, we challenged neonatal lambs orally with atypical scrapie; they were then killed at 12 or 24 months. Screening test results were negative for disease-specific prion protein in all but 2 recipients; they had positive results for examination of brain, but negative for peripheral tissues. Infectivity of brain, distal ileum, and spleen from all animals was assessed in mouse bioassays; positive results were obtained from tissues that had negative results on screening. These findings demonstrate that atypical scrapie can be transmitted orally and indicate that it has the potential for natural transmission and iatrogenic spread through animal feed. Detection of infectivity in tissues negative by current surveillance methods indicates that diagnostic sensitivity is suboptimal for atypical scrapie, and potentially infectious material may be able to pass into the human food chain.

The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep., (© 2011 The Author(s))

In a consignment of sheep brains from New Zealand, to be used in Europe as negative control material in scrapie rapid screening test evaluations, brain samples from 1 sheep (no. 1512) gave the following initially confusing results in various screening tests: the brainstem repeatedly produced negative results in 2 very similar screening kits (enzyme-linked immunosorbent assay [ELISA]-1, ELISA-2), a macerate made from brainstem and cerebellum returned a clearly positive result in ELISA-2, and the macerate and a brainstem sample gave negative results in a third screening test (ELISA-3). In subsequent testing, cerebellum tissue alone tested strongly positive in ELISA-1 and produced a banding pattern very similar to atypical scrapie/Nor98 in a confirmatory Western blot (WB). The macerate showed weak staining in the confirmatory WB but presented a staining pattern identical to atypical scrapie/Nor98 in the scrapie-associated fibril WB. The latter test confirmed conclusively the first case of atypical scrapie/Nor98 in a sheep from New Zealand. Other parts of the brain either tested negative or very weak positive in ELISA-2 and in WBs, or tested with negative results by histopathology and immunohistochemistry. It appears that sheep no. 1512 is a case of atypical scrapie/Nor98 in which the abnormal prion protein was detected mainly in the cerebellum. This case emphasizes the need to retain brainstem, and cerebral and cerebellar tissues, as frozen and fixed materials, for conclusive confirmatory testing. Furthermore, consideration should be given to which screening method to use.

It is currently believed that primary transmission of classical scrapie to wild-type mice is inefficient and characterized by low attack rates and variable incubation periods and lesion profiles. Consequently, strain characterization of classical scrapie in these mice relies on subpassage. The aim of this study was to perform a retrospective analysis of lesion profiles and immunohistochemistry patterns after transmission of a large number of classical scrapie sources to wild-type mice and to investigate trends that might be used to characterize the agent without subpassaging. Scrapie field cases (n = 31) collected from individual farms between 1996 and 1999 were inoculated into RIII, C57BL, and VM mice and profiled using standard methodology and analyzed by immunohistochemistry. Using cluster analysis to resultant lesion profiles produced groups of similar lesion profiles in RIII and C57BL mice. We observed correlations between lesion profile clusters and the ovine prion protein (PrP) genotype. Immunohistochemistry indicated donor-mediated trends in the PrP pattern. These results indicate that ovine PrP genotype is a factor that is linked to both the lesion profile and the pattern of PrP deposition on primary transmission of classical scrapie to wild-type mice.

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

Background: Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease.However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007., Results: This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype., Conclusions: Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.

Background: As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation., Results: The sensitivity and specificity of the short clinical protocol in detecting a scrapie case in the scrapie-affected herds was 3.9% and 99.6%, respectively, based on the presence of tremor, positive scratch test, extensive hair loss, ataxia and absent menace response. All BSE- and scrapie-affected goats displayed abnormalities in sensation (over-reactivity to external stimuli, startle responses, pruritus, absent menace response) and movement (ataxia, tremor, postural deficits) at an advanced clinical stage but the first detectable sign associated with scrapie or BSE could vary between animals. Signs of pruritus were not always present despite similar prion protein genotypes. Clinical signs of scrapie were also displayed by two scrapie cases that presented with detectable disease-associated prion protein only in lymphoid tissues., Conclusions: BSE and scrapie may present as pruritic and non-pruritic forms in goats. Signs assessed for the clinical diagnosis of scrapie or BSE in goats should include postural and gait abnormalities, pruritus and visual impairment. However, many scrapie cases will be missed if detection is solely based on the display of clinical signs. PrPd accumulation in the brain appeared to be related to the severity of clinical disease but not to the display of individual neurological signs.

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Background: Although the epidemiology of scrapie has been broadly understood for many years, attempts to introduce voluntary or compulsory controls to eradicate the disease have frequently failed. Lack of precision in defining the risk factors on farm has been one of the challenges to designing control strategies. This study attempted to define which parts of the annual flock management cycle represented the greatest risk of infection to naive lambs exposed to the farm environment at different times., Results: In VRQ/VRQ lambs exposed to infected sheep at pasture or during lambing, and exposed to the buildings in which lambing took place, the attack rate was high and survival times were short. Where exposure was to pasture alone the number of sheep affected in each experimental group was reduced, and survival times were longer and related to length of exposure., Conclusion: At the flock level, eradication and control strategies for scrapie must take into account the need to decontaminate buildings used for lambing, and to reduce (or prevent) the exposure of lambs to infected sheep, especially in the later stages of incubation, and at lambing. The potential for environmental contamination from pasture should also be considered. Genotype selection may still prove to be the only viable tool to prevent infection from contaminated pasture, reduce environmental contamination and limit direct transmission from sheep to sheep.

Background: 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., Results: 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., Conclusion: 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.

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.

Background: 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., Results: 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., Conclusion: 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.

Immunohistochemistry for PrPSc is used widely in scrapie diagnosis. In natural scrapie cases the use of immunohistochemistry (IHC) has revealed the existence of up to 12 different morphological types of immunostained deposits. The significance of this pattern variability in relation to genotype has not been studied extensively in natural disease. In this study we recorded in detail PrPSc patterns at the obex level of the medulla oblongata from 163 animals derived from 55 flocks which presented through passive surveillance in the UK and Italy. A strong association was seen between PrPSc patterns and PrP genotype, particularly in relation to codon 136. In a blind assessment of this association we were able to predict, with over 80% accuracy, the genotype of 151 scrapie cases which were presented through passive surveillance from 13 farms. The genotype of these cases was ARQ/ARQ or VRQ/VRQ. The association of PrPsc patterns with genotype was generally stronger in those farms where all the affected animals belonged to a single genotype compared with farms where both genotypes were identified, with the exception of one farm in which the genotype of all affected sheep was ARQ/ARQ and the PrPSc patterns were of the VRQ/VRQ type. Our observations support the hypothesis that the observed association between specific IHC patterns and genotypes may in fact be strain driven but in natural disease individual scrapie strains may demonstrate a genotypic tropism.

Background: Atypical scrapie is a recently recognised form of transmissible spongiform encephalopathy of sheep that differs from classical scrapie in its neuropathological and biochemical features. Most cases are detected in apparently healthy sheep and information on the clinical presentation is limited., Case Presentation: This report describes the clinical findings in two sheep notified as scrapie suspects and confirmed as atypical scrapie cases by immunohistochemistry and Western immunoblotting. Although both sheep displayed signs suggestive of a cerebellar dysfunction there was considerable variation in the individual clinical signs, which were similar to classical scrapie., Conclusion: Any sheep presenting with neurological gait deficits should be assessed more closely for other behavioural, neurological and physical signs associated with scrapie and their presence should lead to the suspicion of scrapie.

Vascular amyloidosis in the brain is a pathological feature of ovine scrapie. Its occurrence varies between sheep, but whether this variation reflects differences in the host or the infecting scrapie strain (or both) is not clear. To investigate whether amyloidosis, like vacuolation and PrPsc distribution, is associated with genotype, the brains from 131 sheep representing a range of genotypes commonly associated with scrapie were examined histologically and immunohistochemically. Vascular amyloidosis was absent in 66 sheep, 59 of which were of the ARQ/ARQ genotype and seven the ARQ/AHQ genotype. In contrast, it was found in four of 39 ARQ/VRQ sheep (10.2%) and in 10 of 26 VRQ/VRQ sheep (38.4%). The distribution of amyloid was highly variable, but the most severely affected areas were the lateral geniculate nuclei (five cases) and the ventral thalamic nuclei (four cases). No amyloidosis was found in the medulla or in the basal nuclei. From this preliminary study it was concluded that amyloidosis is relatively rare in sheep with scrapie. Moreover, its occurrence appeared to depend on the presence of at least one valine at codon 136.

On immunohistochemical examination several morphological types of disease-specific prion protein (PrP(d)) accumulation are recognised in the brain of sheep suffering from scrapie. The present study examined the relationship between the type of PrP(d) deposits seen by light microscopy and ultrastructural changes in the olivary nuclei and the dorsal motor nucleus of the vagus (DMNV) in naturally infected sheep with clinical scrapie. The nature and magnitude of sub-cellular morphological changes found in the olivary nuclei differed from the patterns of degeneration previously described in the DMNV. In the olivary nuclei, lamellar bodies in the neuronal perikaryon were found to correlate with marked intraneuronal PrP(d) accumulation. Bizarre, coated, spiral invaginations of the plasmalemma were only found in A(136) homozygous sheep in this nucleus, where few coated pits were usually observed. Neuropil vacuolation in the olivary nuclei was mild and correlated with sparse extracellular PrP(d) deposition. In the DMNV, the magnitude of extracellular immunolabelling in the neuropil was prominent. These extracellular PrP(d )aggregates coincided with intense neuropil vacuolation, increased numbers of coated pits, and with the presence of pre-amyloid changes and infrequent short fibrils in the extracellular space. Scrapie-infected neurons in the two neuroanatomic sites examined, therefore, appear to process and respond to the presence of PrP(d) differently. We hypothesise that vacuolation, coated pits and spiral invaginations of the plasmalemma may be responses to extracellular PrP(d) molecules, and that lamellar bodies are changes associated with the high levels of intraneuronal PrP(d).

An accurate estimate of the prevalence of scrapie infection in the Great Britain (GB) sheep flock is essential when assessing any potential risk to human health through exposure to sheep transmissible spongiform encephalopathies (TSEs). One method for assessing the prevalence is to sample sheep intended for human consumption using a diagnostic test capable of detecting infected animals prior to the onset of clinical signs. An abattoir survey conducted in Great Britain in 1997-1998 tested brain samples from 2809 apparently healthy sheep of which none was found to be positive for scrapie by histopathology or immunohistochemistry (IHC) although 10 were positive for scrapie-associated fibrils (SAF). Subsequently, the tonsils from a subset of the animals sampled were examined using IHC, one of which tested positive. To interpret these results we use a likelihood-based approach, which accounts for the variation in the prevalence of infection with age and test sensitivity and specificity with stage of infection. Combining the results for all of the diagnostic tests yields an estimate of the prevalence of scrapie infection in the GB sheep flock of 0.22% (95% confidence interval: 0.01-0.97%). Moreover, our analysis suggests that all of the diagnostic tests used are very specific (greater than 99%). Indeed, only SAF detection yields a specificity estimate of less than 100%, which helps to account for the high number of samples found to be positive for SAF.

Sub-cellular studies of transmissible spongiform encephalopathies (TSEs) have been carried out on several animal species and human beings. However, studies of optimal perfusion-fixed tissues have largely been confined to examination of rodents. Using a recently developed technique, heads of scrapie-affected sheep and controls were perfusion fixed with mixed aldehydes. The obexes were immunohistochemically labelled with PrP antibodies, and the dorsal motor nucleus of the vagal nerve was examined by electron microscopy. Irregular neuritic profiles with highly invaginated membranes, associated with coated pits were found in all scrapie-affected sheep, but not in controls. Interestingly, they were consistently more frequent in the homozygous A(136) sheep. This is the first report describing sub-cellular differences in pathology associated with different PrP genotypes. Rarely, amorphous material, or sparse fibrillar structures, were present in the extracellular space. The changes were often associated with irregular plasmalemma and frequent coated pits. Vacuolation typical of TSEs, dystrophic neurites and variable gliosis were present. Herniation of membranes and organelles from apparently healthy processes into adjacent vacuoles and dendrites was also observed. We suggest that the increase in coated pits and plasmalemma invagination is related to an attempted internalisation of aggregated disease-specific PrP, or protofilaments, from the extracellular space.

Major determinants of the pathological phenotype of natural scrapie are considered to be the agent strain and host prion protein (PrP) genotype, but the relationship between these is far from clear. Little is known about the strains that produce natural scrapie. A method of brain vacuolation profiling was developed which enables this aspect of disease phenotype to be characterized in detail. This method distinguished at least two distinct pathological phenotypes in sheep of a single genotype (ARQ/ARQ) from different flocks in the UK. Great similarity was also demonstrated between one of these phenotypes and the phenotype of sheep from a flock in Sardinia. The profile of four sheep of the same ARQ/ARQ genotype experimentally infected with bovine spongiform encephalopathy (BSE) was determined for comparison. It would appear from these preliminary observations that the application of lesion profiling techniques to ovine transmissible spongiform encephalopathy (TSE) may contribute to the definition of a particular scrapie phenotype within a flock. It may, therefore, have potential for improving our understanding of current TSE phenotypes in sheep, with regard to the possibility of identifying those of bovine origin.

Seventeen clinically suspect scrapie sheep, and twelve suspected BSE-affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at -70 degrees C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25 degrees C or 37 degrees C. All samples were tested by Western immunoblotting for detection of PrP(Sc) using the Prionics - Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect BSE cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37 degrees C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrP(Sc) protein. The abnormal protein could not be detected in any of the control animals by either technique. PrP(Sc) could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37 degrees C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or BSE by either of these diagnostic methods.

In 1997/1998, an abattoir survey was conducted to determine the likely exposure of the human population to transmissible spongiform encephalopathy (TSE) infection in sheep submitted for slaughter in Great Britain. The survey examined brain material from 2809 sheep processed through British abattoirs. Sampling was targeted by age: 45% of animals tested were > or =15 months old. All samples of adequate quality (98%) were tested for signs of scrapie infection using histopathology and scrapie-associated fibril (SAF) detection and 500 were tested using immunohistochemistry (IHC). No conclusive positive animals were found using either histology or IHC. Ten animals were positive by SAF. Standard statistical analyses suggest (with 95% confidence) that the prevalence of detectable (by histopathology) infection in the slaughter population was < or =0.11%. However, the incubation period of scrapie is long (usually around 2-3 years) and none of the tests used in the survey is capable of detecting scrapie infection in the early stages of infection. We present an age-structured stochastic model incorporating parameters for the incubation period of scrapie, prevalence of infection by age and test sensitivity. Using the model, we demonstrate that the negative results obtained for all samples using IHC and histopathology are consistent with a true prevalence of infection in the slaughter population of up to 11%. This suggests that up to 300 of the animals tested might have been infected but the infection was not sufficiently advanced in these animals to be detectable by IHC or histopathology. The survey was designed to detect a prevalence of 1% with a precision of +/-0.5% and a confidence level of 95% in each age group assuming that diagnostic tests were 100% specific and sensitive from a known stage in the incubation period. The results of the model demonstrate that to estimate a true prevalence of scrapie infection of 1% with an accuracy of +/-0.5% would have required a far larger sample size. An accurate estimate of the required sample size is complicated by uncertainty about test sensitivity and the underlying infection dynamics of scrapie. A pre-requisite for any future abattoir survey is validation of the diagnostic tests used in relation to both stage of incubation and genotype. Sampling in the <15-month age group was of no value in this survey because the diagnostic tests used were thought to be ineffective in most of the animals in this age group.

A randomised sample of 2,809 apparently healthy sheep, 55 per cent of them less than 15 months of age, which were slaughtered for human consumption at abattoirs in Great Britain in 1997/98, was taken to establish the prevalence of scrapie infection. The medulla oblongata of each sheep was examined histopathologically at the level of the obex, and fresh brain tissue was examined for scrapie-associated fibrils (SAF) to establish whether there was evidence of scrapie. In addition, histological sections of the medulla from 500 of the sheep were immunostained with an antiserum to PrP, and the same technique was also applied to any animal found positive or inconclusive by the histological or SAF examinations. Any sheep which was positive by any of these diagnostic methods was also examined by Western immunoblotting, for the detection of the disease-specific protein PrP(Sc). A total of 2,798 sheep (99.6 per cent) were negative by all the methods applied. Ten animals were SAF-positive but negative by all the other methods, and in one animal there was immunohistochemical staining which could not be interpreted unequivocally as disease-specific. A mathematical model was used to estimate the prevalence of scrapie infection in the national slaughtered sheep population which would be consistent with these results. By this model, the absence of unequivocally substantiated cases of scrapie in the sample was consistent with a prevalence of infection in the slaughter population of up to 11 per cent.