The Scrapie Prevalence in a Goat Herd Is Underestimated by Using a Rapid Diagnostic Test.

Current European surveillance regulations for scrapie, a naturally occurring transmissible spongiform encephalopathy (TSE) or prion disease in sheep and goats, require testing of fallen stock or healthy slaughter animals, and outline measures in the case of confirmation of disease. An outbreak of classical scrapie in a herd with 2500 goats led to the culling of the whole herd, providing the opportunity to examine a subset of goats, take samples, and examine them for the presence of disease-associated prion protein (PrPSc) to provide further information on scrapie test sensitivity, pathology, and association with prion protein genotype. Goats were examined clinically prior to cull, and the brains examined post mortem by Bio-Rad ELISA, a rapid screening test used for active surveillance in sheep and goats, and two confirmatory tests, Western blot and immunohistochemistry. Furthermore, up to 10 lymphoid tissues were examined by immunohistochemistry. Of 151 goats examined, three (2.0%) tested positive for scrapie by ELISA on brain, confirmed by confirmatory tests, and a further five (3.3%) were negative by ELISA but positive by at least one of the confirmatory tests. Only two of these, both positive by ELISA, displayed evident signs of scrapie. In addition, 10 (6.6%) goats, which also included two clinical suspects, were negative on brain examination but had detectable PrPSc in lymphoid tissue. PrPSc was detected most frequently in the medial retropharyngeal lymph node (LN; 94.4% of all 18 cases) and palatine tonsil (88.9%). Abnormal behavior and circling or loss of balance when blindfolded were the best clinical discriminators for scrapie status. None of the goats that carried a single allele in the prion protein gene associated with increased resistance to scrapie (Q211, K222, S146) were scrapie-positive, and the percentage of goats with these alleles was greater than expected from previous surveys. Significantly more goats that were scrapie-positive were isoleucine homozygous at codon 142 (II142). The results indicate that the sensitivity of the applied screening test is poor in goats compared to the confirmatory tests as gold standard, particularly for asymptomatic animals. Sensitivity of surveillance could be improved by testing retropharyngeal LN or palatine tonsil in addition to brain.

Molecular characterisation of atypical BSE prions by mass spectrometry and changes following transmission to sheep and transgenic mouse models.

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Pyroglutamyl-N-terminal prion protein fragments in sheep brain following the development of transmissible spongiform encephalopathies.

Protein misfolding, protein aggregation and disruption to cellular proteostasis are key processes in the propagation of disease and, in some progressive neurodegenerative diseases of the central nervous system, the misfolded protein can act as a self-replicating template or prion converting its normal isoform into a misfolded copy of itself. We have investigated the sheep transmissible spongiform encephalopathy, scrapie, and developed a multiple selected reaction monitoring (mSRM) mass spectrometry assay to quantify brain peptides representing the "ragged" N-terminus and the core of ovine prion protein (PrP(Sc)) by using Q-Tof mass spectrometry. This allowed us to identify pyroglutamylated N-terminal fragments of PrP(Sc) at residues 86, 95 and 101, and establish that these fragments were likely to be the result of in vivo processes. We found that the ratios of pyroglutamylated PrP(Sc) fragments were different in sheep of different breeds and geographical origin, and our expanded ovine PrP(Sc) assay was able to determine the ratio and allotypes of PrP accumulating in diseased brain of PrP heterozygous sheep; it also revealed significant differences between N-terminal amino acid profiles (N-TAAPs) in other types of ovine prion disease, CH1641 scrapie and ovine BSE. Variable rates of PrP misfolding, aggregation and degradation are the likely basis for phenotypic (or strain) differences in prion-affected animals and our mass spectrometry-based approach allows the simultaneous investigation of factors such as post-translational modification (pyroglutamyl formation), conformation (by N-TAAP analysis) and amino-acid polymorphisms (allotype ratio) which affect the kinetics of these proteostatic processes.

A single step multiplex immunofluorometric assay for differential diagnosis of BSE and scrapie.

Although there is no evidence that the European sheep population has been infected with bovine spongiform encephalopathy (BSE), distinguishing this from scrapie is paramount, given the association between BSE exposure and the human transmissible spongiform encephalopathy (TSE), variant Creutzfeldt-Jakob disease. The capability to differentially diagnose TSEs in sheep is thus essential in order to safeguard the food chain and human health. Biochemical methods for differentiating BSE and scrapie are largely reliant on assessment by Western blot (WB) analysis of the abnormal disease associated prion protein PrP(D) following partial proteolytic digestion. WB banding patterns obtained using a panel of antibodies enable different strain specific conformations of PrP(D) to be distinguished. This approach provides a robust confirmatory test but one which is not appropriate for high throughput screening. A simple, one step, bead array flow cytometry based multiplex immunofluorometric assay has been developed which is suitable for simultaneous screening and confirmation. Using a combination of antibodies directed towards three PrP epitopes enabled differential diagnosis of scrapie and BSE. Proof of principle studies indicated a high predictive value (100%) when applied to brain samples from control animals, BSE infected cattle and sheep naturally infected with scrapie or experimentally infected with BSE.

Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results.

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.