Variation in the prion protein sequence in Dutch goat breeds.

Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined for six goat breeds in the Netherlands. Overall frequencies in Dutch goats were determined from 768 brain tissue samples in 2005, 766 in 2008 and 300 in 2012, derived from random sampling for the national scrapie surveillance without knowledge of the breed. Breed specific frequencies were determined in the winter 2013/2014 by sampling 300 breeding animals from the main breeders of the different breeds. Detailed analysis of the scrapie-resistant K222 haplotype was carried out in 2014 for 220 Dutch Toggenburger goats and in 2015 for 942 goats from the Saanen derived White Goat breed. Nine haplotypes were identified in the Dutch breeds. Frequencies for non-wild type haplotypes were generally low. Exception was the K222 haplotype in the Dutch Toggenburger (29%) and the S146 haplotype in the Nubian and Boer breeds (respectively 7 and 31%). The frequency of the K222 haplotype in the Toggenburger was higher than for any other breed reported in literature, while for the White Goat breed it was with 3.1% similar to frequencies of other Saanen or Saanen derived breeds. Further evidence was found for the existence of two M142 haplotypes, M142 /S240 and M142 /P240 . Breeds vary in haplotype frequencies but frequencies of resistant genotypes are generally low and consequently selective breeding for scrapie resistance can only be slow but will benefit from animals identified in this study. The unexpectedly high frequency of the K222 haplotype in the Dutch Toggenburger underlines the need for conservation of rare breeds in order to conserve genetic diversity rare or absent in other breeds.

Prion Type-Dependent Deposition of PRNP Allelic Products in Heterozygous Sheep.

UNLABELLED: Susceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrP(res)) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrP(res) material from BSE-infected ARR/VRQ sheep. PrP(res) in BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrP(res) accumulation process in prion formation as well as the disease-associated phenotypic expressions in the host. IMPORTANCE: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

Evaluation of two commercial, rapid, ELISA kits testing for scrapie in retro-pharyngeal lymph nodes in sheep.

AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes. METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID). RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3-75.3)% for ELISA-BR and 91.6 (95% CI=88.2-94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID. CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity. CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

[Active surveillance for scrapie in the Netherlands: effect of a breeding programme on the prevalence of scrapie in sheep (2002-2010)?]. / Acht jaar actieve scrapiesurveillance in Nederland: het effect van het fokprogramma op de prevalentie van scrapie bij het schaap (2002-2010).

The susceptibility of sheep to scrapie is modulated by the prion protein (PrP) genotype of the animal. An ambitious voluntary scrapie control programme was started in the Netherlands in 1998, based on selection of rams with theARR/ARR genotype for breeding. This programme was followed by an obligatory programme in 2004; the programme has been voluntary since 2007. We monitored the prevalence of PrP genotype frequencies and the prevalence of scrapie in the Dutch sheep population between 2002 and June 2010. Results showed that selection for scrapie-resistant sheep resulted in an increase in the ARR allele frequency in the Dutch national flock from 37.5% in 2005 to 61.4% in 2009. Moreover, surveillance data showed that there was a significant decrease in the prevalence of scrapie a few years after the start of the obligatory breeding programme, from more than 0.2% in 2004 to 0.015% in 2009. This decrease is a consequence of the increased number of scrapie-resistant sheep in the Dutch sheep population. To date, the results and the models based on the data show that the selective breeding programme should be continued for several years in order to successfully eradicate scrapie. It will be important to monitor the PrP frequency and scrapie prevalence in the Dutch sheep population in the coming years.

Differentiation of ruminant transmissible spongiform encephalopathy isolate types, including bovine spongiform encephalopathy and CH1641 scrapie.

With increased awareness of the diversity of transmissible spongiform encephalopathy (TSE) strains in the ruminant population, comes an appreciation of the need for improved methods of differential diagnosis. Exposure to bovine spongiform encephalopathy (BSE) has been associated with the human TSE, variant Creutzfeldt-Jakob disease, emphasizing the necessity in distinguishing low-risk TSE types from BSE. TSE type discrimination in ruminants such as cattle, sheep, goats and deer, requires the application of several prion protein (PrP)-specific antibodies in parallel immunochemical tests on brain homogenates or tissue sections from infected animals. This study uses in a single incubation step, three PrP-specific antibodies and fluorescent Alexa dye-labelled anti-mouse Fabs on a Western blot. The usual amount of brain tissue needed is 0.5 mg. This multiplex application of antibodies directed towards three different PrP epitopes enabled differential diagnosis of all established main features of classical scrapie, BSE and Nor98-like scrapie in sheep and goats, as well as the currently known BSE types C, H and L in cattle. Moreover, due to an antibody-dependent dual PrP-banding pattern, for the first time CH1641 scrapie of sheep can be reliably discriminated from the other TSE isolate types in sheep.

Pathogenesis of bovine spongiform encephalopathy in sheep.

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrP(Sc)) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrP(Sc) was detected after 6 months in the tonsil and the ileal Peyer's patches. At 9 months postinfection, PrP(Sc) accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrP(Sc) accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrP(Sc) was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7-L1. At subsequent time points, PrP(Sc) was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrP(Sc) involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.

Immunohistochemical distinction between preclinical bovine spongiform encephalopathy and scrapie infection in sheep.

Sheep are susceptible experimentally to bovine spongiform encephalopathy (BSE), the clinical signs being indistinguishable from those of scrapie. Because of the possibility of natural ovine BSE infection, laboratory tests are needed to distinguish between scrapie and BSE infection. The objectives of this study were to determine whether (1) PrPSc accumulates in biopsy samples of the tonsil or third eyelid, or both, of BSE-infected sheep before the appearance of clinical disease, and (2) such samples from BSE- and scrapie-infected sheep differ in respect of PrPSc accumulations. Homozygous ARQ sheep (n = 10) were dosed orally at 4-5 months of age with a brain homogenate from BSE-infected cattle. Third eyelid and tonsillar biopsy samples were taken at < or = 6 monthly intervals post-infection and examined immunohistochemically for PrPSc. Third eyelid protuberances were difficult to identify, resulting in many unsuitable samples; however, third eyelid samples shown to contain lymphoid follicles were invariably negative for PrPSc. In contrast, tonsillar biopsy samples became positive for PrPSc from 11 to 20 months post-infection. Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBMs) between BSE- and scrapie-infected sheep were detected with anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion protein: thus, PrPSc appeared as single granules in TBMs of tonsillar sections from BSE-infected sheep, whereas clusters of PrPSc granules were observed within TBMs in the tonsils of scrapie-infected sheep. In contrast, antibodies against epitopes situated N- and C-terminally from the 93-106 region of the ovine prion protein revealed no differences between BSE- and scrapie-infected sheep in terms of PrPSc granules in TBMs.

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein.

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

Early and late pathogenesis of natural scrapie infection in sheep.

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.