The risk of infection or clinical classical scrapie at population level has been studied much less frequently given the difficulties of selecting and following up scra- pie-affected flocks over time. Some attempts have been made with differing levels of success, but most of them were subject to bias in the genotype data sources or in the selection of flocks for study, mainly managed by research institutes . In some cases, it was unavoidable to prevent farmers from altering their breeding policy once the genotype of the animals had been revealed . The relationship between genetic, phenotypic factors and scrapie has been studied in some individual flocks in Great Britain during the last ten years. Descriptions of scrapie epidemics in flock with known genotypes are available in the literature like the scrapie epidemic in a Texel sheep flock  and in a closed flock of Romanov sheep . Tongue et al.  reported genotype distri- butions of 15 scrapie-affected flocks in Great Britain. Baylis et al.  described the greater scrapie risk for the ARQ/VRQ, ARH/VRQ and VRQ/VRQ genotypes and the resistance conferred by the ARR allele when com- paring surveillance scrapie data and the genotypes from
that were held in close contact in the same flock and that this flock comprised a high proportion of classical-scrapie-affected goats. This condition may therefore be contagious and addi- tionally may be etiologically related to classical scrapie. The diversity of TSE phenotypes also depends on the occurrence of different prion strains in a given host population. For classical scrapie it was postulated that at least 20 strains can be discrim- inated based on their phenotypic features in mouse transmis- sion studies (9). Recently, transmission studies of scrapie iso- lates in transgenic mice indicated that some isolates contained multiple prion strains, which resulted in different disease phe- notypes, including distinct PrP res banding patterns (29, 34).
One of the major features of prion disease susceptibility and transmissibility is the PrP-related genetic variability of both host and donor, which, e.g., is evident in sheep (4). The amino acid sequence of PrP is considered to be conserved between mammalian species, yet within species it can be polymorphic, as seen in humans, sheep, and goats, though not typically in cattle (29, 53, 63, 68). Susceptibility for TSE infection is highly influenced by single amino acid polymorphisms. In humans, this has become evident in individuals from Papua New Guinea who developed genetic resistance for kuru by the evo- lution of a unique resistance PrP allelotype (codon 127, glycine to valine [V]) (38). In sheep, variable levels of resistance to TSEs have been identified and found to be dependent on both prion strain and PrP polymorphisms. For classical scrapie and bovine spongiform encephalopathy (BSE) in sheep, three im- portant amino acid polymorphisms that influence susceptibility and transmission have been described, i.e., alanine (A) to V at codon 136, arginine (R) to histidine (H) at codon 154, and glutamine (Q) to R at codon 171 (3, 28, 29, 57). In atypical/ Nor98 scrapie, a form of scrapie that has poor transmission properties, susceptibility mainly correlates to a substitution of R to H at codon 154 or leucine (L) to phenylalanine (F) at codon 141 (19, 43, 53). Taking the major TSE transmission- * Corresponding author. Mailing address: Central Veterinary Insti-
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Flocks were eligible to join the IAH study if they had had at least one case of classical scrapie confirmed in the previous two years. Upon recruitment, all subsequent cases were reported by the farmers to the relevant authorities and suspect animals were confiscated. Tissue samples were sent to the Veterinary Labora- tories Agency (VLA) for routine analysis for evidence of classical scrapie . Importantly, all the cases would have been showing clinical signs of disease and were confirmed by laboratory diagnosis using methods which would not have misidentified these animals as having atypical scrapie. Upon confiscation, demo- graphic data for cases including the animal’s breed, sex, date of birth, date of death, PrP genotype and whether the case occurred in a homebred or purchased animal were recorded in the Scrapie Notification Database (SND) held at the VLA. Analysis of cases includes those of both homebred and purchased origin. It is important to note that in several flocks, cases had been reported for a number of years before joining the IAH study; here we use all the data on scrapie cases in our flocks held in the SND (both before and after joining the IAH study) and therefore report on the entire, officially-confirmed outbreaks within the flocks. Of the 415 scrapie cases that were reported in these flocks, 327 (79%) were genotyped.
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of PrP Sc across the brain associated with different prion types  and as a method of discriminating classical scrapie and experimental BSE . For strain typing, the PET-blot method was used previously in C57BL/6 mice to compare the distribution of BSE from different spe- cies with ovine and mouse adapted scrapie . In the current study PET-blots proved a useful tool to clearly demonstrate the differences in neuroanatomical location of PrP Sc associated with different classical scrapie strains and in this respect correlated well with IHC staining. In agreement with previous studies, IHC showed compar- ably better microscopic resolution at the cellular and subcellular levels, enabling different PrP Sc types to be more clearly defined, although certain strain specific markers, i.e. perpendicular streaks of PrP Sc through the molecular layer of the cerebellum, associated with ME7, were also identifiable by PET-blot as were denser struc- tures including aggregates and plaques.
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A scrapie research flock was established in 1998 by purchasing clinically healthy sheep from scrapie-affected flocks with scrapie- susceptible genotypes and maintained through breeding sheep of susceptible genotypes (10). At the time of the project, more than 50% of the sheep in the flock were homozygous VRQ (valine at codon 136, arginine at 154, and glutamine at 171 of the ovine prion protein gene), which is associated with high susceptibil- ity to classical scrapie (13), and developed clinical disease from 18 months of age. Sheep were kept on pastures for many years, being kept in sheds just before lambing and occasionally in severe weather. The pasture was managed on a rotation basis and was plowed and reseeded as required. Manure and composted bedding from the lambing sheds was incinerated and not spread on the pasture. For logistical reasons, sheep with clinical signs suggestive of scrapie were predominantly kept on one particular pasture to enable closer monitoring until cull at clinical end-stage.
resistance [7-10]. Accordingly, the EU has implemented breeding programs to increase scrapie resistance in sheep populations. In compliance with regulation (EC) 999/2001, as amended, several Member States are now increasing the frequency of the ARR haplotype. A simi- lar approach has not yet been applied in goats, but it would be desirable in this species too, given that scrapie poses a problem for the economy and animal welfare, and that goats, often bred in mixed flocks with sheep, can play a role in maintaining the circulation of scrapie strains and the consequent sheep exposure. Genetic ana- lysis of the goat PRNP gene revealed 46 polymorphisms in the open reading frame [3,11], including silent muta- tions and a PRNP variant containing three instead of the five usual octapeptide repeats . Various European studies have suggested that several polymorphisms can modulate scrapie susceptibility in goats as well. The pre- sence of methionine (M) at codon 142 is associated with increased incubation periods after experimental chal- lenge of BSE and scrapie strains  and in natural scrapie outbreaks [14,15]. A reduced susceptibility to natural scrapie has also been reported for goats carrying arginine (R) at codon 143, histidine (H) at codon 154 [16,17] or glutamine (Q) at codon 211 . However, H154 has clearly been suggested to be a risk factor for Nor98 goat scrapie . The most promising results have been obtained for codon 146, carrying serine (S) or aspartic acid (D), which is linked to high resistance in Cyprus [19,20], and for codon 222, carrying lysine (K), which in Italy was first reported as conferring resistance and has only been associated with healthy animals [16,21]. An association of K222 with a protective effect was also later found in France and Greece [14,22]. Taken together, these results provide encouraging evi- dence for the support of breeding programs for resis- tance in goats against classical scrapie in all EU Member States, as stated by the EFSA Panel on Biological Hazards in the “ Opinion on genetic TSE resistance in goats in all EU Member States ” , and perhaps also in other non-EU countries. In such a prospective, it is essential to reinforce existing data from field studies with those from experimental studies, particularly those carried out with the experimental transmission of differ- ent TSE isolates in goats harbouring the PRNP alleles of interest.
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Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrP Sc in 7 of 15 and 14 of 14 sheep respectively. However PrP Sc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.
Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make- up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie- affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non- resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.
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Association studies between Prnp genetics and suscept- ibility to scrapie in goats are so far limited and often restricted in their statistical significance. Nonetheless, some caprine Prnp alleles have been implicated as provid- ing increased resistance to disease development relative to wild-type alleles. Importantly, novel polymorphisms that increase susceptibility have not yet been discovered, although goats share with sheep the association of codon 154 histidine (H154) with increased susceptibility to atypi- cal scrapie . The PrP variant encoding lysine in codon 222 (K222) has been associated with highly significant pro- tection from scrapie in Italian and French studies [14,15], whereas the codon 146 serine (S146) and 146 aspartic acid (D146) variants have been similarly associated with scrapie resistance in Cypriot herds [16,17]. Modulation of classical scrapie by polymorphisms in codons 143, 154 and 211 has been suggested by studies from France, and Greece [14,18,19]. The association of 142 methionine (M142) with lengthening of the incubation periods after experimental scrapie or BSE challenge was shown by us some years ago  whereas recently the M142 allele was shown to mod- ulate genetic susceptibility in a single high incidence herd in the UK [21,22].
None of the non-NN animals challenged orally developed any signs of scrapie even after survival periods in excess of 6 years (an increase of the mean incubation period of approximately 250%), which is approaching the commercial life span of a Cypriot goat. A similar study (16) used the same approach of inoculating goats of different genotypes orally or intracerebrally with the same inoculum. That report did not offer any statistical analysis of the differences in incubation periods between genotypes, but it showed that it is possible for the same allele to provide resistance to oral challenge and suscepti- bility following intracerebral challenge, as is observed in the present study. These data suggest that the molecular mechanisms that are involved in digestive tract uptake, peripheral distribution, and the propagation and transport of prions to the central nervous system may differ from those that are involved in prion propagation in the CNS. Therefore, data from intracerebral studies alone are not representative of the allelic or species molecular barriers to TSE (14).
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The titre of classical scrapie in the intestines (duodenum and jejunum) was not found in the literature. The titre is likely to be heterogeneously distributed depending on concentrations of gut-associated lymphoid tissue. In the absence of data, the titre was assumed to be the same as the ileum, with all three tissues ranked at the same level by the WHO as ‘lower infectivity tissues’ (WHO, 2010). This assumption and the heavy weight of these tissues, not classed as SRM, result in the tissues contributing the most to the edible fraction of an infected carcase. The estimate is likely to be an over- estimate within the risk assessment, decreasing the absolute infectivity estimates, but would not impact the SRM scenario relative values. The WHO ranked list also lists a number of other ‘lower infectivity tissues’, which due to lack of data are not included in the risk assessment, but would form part of the edible fraction of the carcase. Inclusion of these tissues, if quantitative values could be gained, would slightly increase the absolute infectivity estimates, potentially balancing the overestimate for the intestines.
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It has been speculated that the origin of CWD in North American cervids may be associated with classical scra- pie because some scrapie infected sheep had been penned together with deer at a research center between 1968 and 1971 [4, 15, 16]. To support this hypothesis, Tamgüney  reported the successful transmission of one classical scrapie isolate into transgenic mice carrying the elk prion protein gene [Tg(ElkPrP) mice], but it is noteworthy that the agent signature (as defined by lesion profile) in these mice was different from that in mice inoculated with CWD, suggesting that the scrapie and CWD agent were distinct strains. Norway has had a scrapie surveillance program in place since 1997 with a total of 264 000 small ruminants analyzed. Few cases of classical scrapie have been diagnosed in Norway and the last case was identified in 2009. There are no reports of classical scrapie within the range of the Nordfjella reindeer sub-population, but as sheep traditionally are transported over long distances to graze in mountain pastures, it cannot be formally excluded that reindeer in this or a nearby subpopulation could have been exposed to sheep with classical scrapie at some point of time.
ovine BSE compared to classical scrapie that was not influenced by host genotype or scrapie type . Recently, there have been further developments in the differentiation of BSE from natural scrapie with particular emphasis on differentiating BSE from CH1641 which is not possible using any of the criteria described above . Pirisinu and co-workers  demonstrated that ovine BSE was more stable to guanidinium hydrochloride (GndHCl) than classical scrapie or CH1641 scrapie and that treatment with 3.5 mol/L GndHCl before digestion in combination with P4/’core antibody’ binding ratios allowed the discrimination of both CH1641 and classical scrapie from ovine BSE. Alternatively, the probing of a single western blot with differently labelled SAF84 and L42 antibodies revealed a so-called dual glycotype aspect at the size of the monoglycosylated band which distinguished CH1641 from BSE in sheep; as CH1641 had a relatively high SAF84 to L42 binding ratio compared to BSE . In addition, a microwell immunofluorometric assay has been developed to differentiate between classical scrapie, atypical scrapie, CH1641 scrapie, experimentally infected ovine BSE and naturally infected bovine BSE . The assay is based on the capture of PrP Sc -res with three distinct antibodies
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All tg110 mice challenged with the VRQ/VRQ source were TSE negative or, in the case of the ARQ/ARQ source, showed low at- tack rates with the first positive animal identified 581 dpi (Fig. 3A and 4A). In contrast, mixtures of BSE with either VRQ/VRQ or ARQ/ARQ classical scrapie produced clinical-stage TSE with in- cubation periods of 236 to 326 dpi (Fig. 3A and 4A). These incu- bation periods are comparable with those generated by the origi- nal BSE source (221 to 267 dpi) albeit slightly longer, probably as a result of the slightly reduced titer of BSE in the mixtures. These data suggest that in this mouse line only the BSE component was isolated from the mixtures. The lesion profiles from the tg110 mice (Fig. 3C and 4C) further support the conclusion that the isolated agent had only BSE properties although it was not possi- ble to construct lesion profiles from either scrapie source due to the lack of sufficient clinically positive mice diagnosed with TSE. (ii) Classical scrapie 1-4-7 ARQ/ARQ and ovine BSE. The two tests failed to identify the presence of BSE in the dilution series when its concentration in the mixture dropped below FIG 2 Discriminatory ELISA. The mixed samples were tested in duplicate in blinded conditions. Ovine BSE was mixed with atypical scrapie (A), classical scrapie (VRQ/VRQ) (B), classical scrapie (1-4-7 ARQ/ARQ) (C), or classical scrapie (ARQ/ARQ) (D). The normalized ratio for classical scrapie samples, which are highly PK resistant, is less than 0.3, intermediate scrapie samples present a normalized ratio comprised between 0.3 and 0.7, and experimental ovine BSE samples have a normalized ratio between 0.7 and 1.3. Atypical scrapie samples have a ratio greater than 1.3. According to these values, the blinded samples were categorized as scrapie (gray), intermediate scrapie (hatched), BSE (black), or atypical scrapie (white).
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of the obex, thereby suggesting that the sheep scrapie prion may gain its first CNS access exclusively by the neural network inner- vating the inoculated PT and RPLN in which evidence of PrP Sc deposition had been previously found at 3 months. In this respect, since no detectable PrP Sc occurred in the CCG, PGG, PVG, DVG, TG, and CTG ganglia, we could not identify the precise nervous pathway followed by the scrapie agent to reach the substantia re- ticularis. Nevertheless, we speculate that the vagal afferent fibers could serve as the prion entry route. In fact, sensory vagal termi- nations scattered in the substantia reticularis between the NPNV and the nucleus ambiguus in the ventrolateral medulla were ob- served after injection of CTB-HRP tracer into the DVG (also termed nodose ganglia) of lambs (26). Furthermore, vagal efferent neurons are located not only in the NPNV but also in the reticular formation of the ovine obex (27). Our classical scrapie-infected sheep exhibited a PrP Sc deposition phenotype associated with
and/or infectivity: 5 of 14 mice (13 to 65% within a confidence interval of 95%) tested positive by either Western blot analysis, infectivity bioassay, or both (16). However, clinically apparent scrapie was never detected in B-cell-deficient mice (n ⫽ 27), even 665 days after intraperitoncal (i.p.) inoculation with pri- ons (16), suggesting that a neurotoxic or catalytic factor se- creted by B cells may contribute to pathogenesis and lead to the manifestation of symptoms. If this were true, i.c. inocula- tion of MT or RAG-1 ⫺/⫺ mice with inoculum derived from
We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glu- tamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron- specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongi- form encephalopathies.
scrapie-afflicted farms (scrapie recently diagnosed) . Although only one or a few animals usually have clinical symptoms in a stricken flock when culled, most often 20- 40% of the asymptomatic sheep show pathological changes characteristic of scrapie in the central nervous system [; Chief Veterinary Officer, personal communi- cation]. It was therefore concluded that the possible pro- tective effect of high concentration of Mn in the forage against the occurrence of clinical scrapie might rather be confined to the gastrointestinal tract, which is considered the main port of entry for the prion protein in the sheep, than to any other internal organs. These authors empha- sized, however, that variables like seasonal changes and pregnancy might significantly affect the concentration of Mn and Cu in the blood of sheep .
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Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases, which include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD) in humans. Scrapie has ex- isted for more than two centuries, while BSE was first recog- nized in 1985, followed by a BSE epidemic in the United Kingdom (31). Epidemiological studies suggested that BSE was primarily caused by feeding meat and bone meal (MBM) contaminated with scrapie agent to cattle (32). Once BSE appeared, the causative agent spread through the cattle pop- ulation by the use of BSE agent-contaminated MBM. The appearance of feline spongiform encephalopathy (FSE) in do- mestic and captive cats (34, 35) and, more recently, variant CJD (vCJD) in humans in 1996 (33), has raised a global con- cern for the spread of the BSE agent to other species via the food chain.