A case of atypical scrapie/Nor98 in a sheep from New Zealand

----- Original Message ----- From: Terry S. Singeltary Sr. To: TERRY SINGELTARY Sent: Sunday, November 22, 2009 9:21 PM Subject: A case of atypical scrapie/Nor98 in a sheep from New Zealand


A case of atypical scrapie/Nor98 in a sheep from New Zealand

Abnormal prion protein consistent with atypical scrapie/Nor98 has been detected in the brain of a New Zealand sheep that was part of a large consignment of brains sent to Europe for use as negative control material. Nature of atypical scrapie/Nor98 Accumulated scientific knowledge to date has demonstrated that atypical scrapie, also known as Nor98, is clinically, pathologically, biochemically and epidemiologically different from classical scrapie (1). The majority of atypical scrapie/Nor98 cases have been identified in clinically normal sheep sampled at slaughter (1-3). Cases have been reported from Norway, Sweden, Finland, the UK, Germany, France, Portugal, Belgium, the Netherlands, Ireland, Denmark(4), the United States (5) and Canada (6). The apparent prevalence of the cases in the European Union (EU) Member States is very low, for example for the UK it is less than 0.1%. Active surveillance programmes have led to the detection of atypical scrapie/Nor98 cases even in the absence of any case of classical scrapie in sheep in Portugal, Denmark, Sweden and Finland(4), and in the Falkland Islands(7), the first region in the Southern Hemisphere to report such a case. Atypical scrapie/Nor98 appears to be a non-contagious, sporadic degenerative condition of older sheep. A spontaneous aetiology, possibly with a genetic determinant, environmental influences and metabolic factors, has been suggested in a number of studies based on epidemiological evidence (2-4, 8). Contributing to this suggestion is the observation that atypical scrapie/Nor98 cases have been detected by active surveillance programmes, wherever such programmes have been initiated (4). Most cases which have been detected in a flock have been the only case and this lead to early speculation that the condition is not naturally infectious (1-3). Experimental oral transmission to sheep has been unsuccessful, to date. Experimental oral transmission into standard mice and into bank voles also failed (1). Only experimental intracerebral transmission to transgenic mice expressing the ovine (9, 10) or porcine (11) prion gene and experimental intracerebral challenge in sheep were successful (12). Unlike classical scrapie, abnormal prion protein has not been detected in peripheral tissues of sheep affected by atypical scrapie/Nor98 (1, 4). The OIE Terrestrial Animal Health Code chapter on scrapie specifically excludes atypical scrapie/Nor98 and explicitly states that the condition is unrelated to ‘classical’ scrapie, is probably not contagious and may be a spontaneously occurring condition (13). Draft for submission to Surveillance magazine 27/10/2009 2 It seems that wherever scrapie surveillance in sheep raising countries is intensified and suitable tests are used, atypical scrapie/Nor98 is found. Recently, three cases of atypical scrapie/Nor98 were reported in a UK research sheep flock derived from animals of New Zealand origin (14). Subsequent investigations in the UK did not find any evidence that the condition was either acquired in the UK or introduced from New Zealand. Thus the discovery of an atypical scrapie/Nor98 case in an otherwise healthy New Zealand sheep is not entirely unexpected (15); and reinforces the view that atypical scrapie/Nor 98 occurs spontaneously or naturally in very small numbers of older sheep in every population.


http://www.biosecurity.govt.nz/files/pests/atypical-scrapie/background-paper.pdf



2009 BSE AND SCRAPIE NZ


http://www.biosecurity.govt.nz/files/pests/atypical-scrapie/literature-review.pdf



Nevertheless, the incidence of atypical scrapie is much higher than that of sporadic CJD in humans. Long-term studies on the occurrence and genetics of atypical scrapie, combined with the development of more sensitive methods for detection of infectivity, are needed to elucidate these aspects. Atypical scrapie is not confined to sheep, as it has also been diagnosed in goats in a few European countries including Norway (Benestad et al. 2006). Currently the molecular characteristics of atypical scrapie in goats and sheep are indistinguishable (Le Dur et al. 2005). However, only a few cases have been found in goats to date, so any epidemiological link between the diseases in these two species is presently unknown. 4


http://www.vkm.no/dav/7f33267b4e.pdf



This position has been accepted by the OIE and the Animal Health Code Article 14.9.1 states “The chapter does not cover so-called ‘atypical’ scrapie which is clinically, pathologically, biochemically and epidemiologically unrelated to ‘classical’ scrapie, may not be contagious and may, in fact, be a spontaneous degenerative condition of older sheep.” This position has been supported by the scientific panel on biological hazards of the European Food Safety Authority 3.

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Recent studies have shown experimental transmission of atypical scrapie in sheep and laboratory animals (Simmons et al 2007) but there is as yet no evidence to confirm that transmission can occur naturally in the field. Another recent paper by Espinosa et al (Espinosa, Herva, 2009) has shown that atypical scrapie can be transmitted with low efficiency to genetically engineered mice over expressing the porcine prion protein. They concluded there was a marked species transmission barrier. Further the agent appeared to undergo a strain phenotype shift upon transmission to the transgenic mice. This is the first report of this occurring.


http://www.biosecurity.govt.nz/files/pests/atypical-scrapie/literature-review.pdf



• Most critical is that atypical scrapie shows higher prevalence in so-called resistant ARR homozygote and heterozygote genotypes, compared with classical scrapie. • Atypical scrapie has not been found naturally in VRQ/VRQ sheep, although such sheep can be infected artificially. VRQ sheep are, in contrast, highly susceptible to classical scrapie. In the UK, one case of atypical scrapie has been found in VRQ heterozygote (AF141RQ/VRQ) sheep. It is important to ascertain whether or not VRQ-carrying sheep are significantly resistant to infection with atypical scrapie or whether the data might result from a failure to detect PrPres in atypical scrapie due to a different pattern of PrP distribution in tissues. • Increased incidence of atypical scrapie in sheep with PrP alleles carrying the variant phenylalanine (F) at position 141 (leucine(L)/phenylalanine) has also been observed compared with classical scrapie. • It will be important to clarify the genotype effect, particularly in relation to ARR and L141F in transmission studies. • In classical scrapie, there is clear evidence for a PrP genotype effect on tissue distribution patterns of PrPres. This might also be true for atypical scrapie although the data are less complete. 4. Transmission of atypical scrapie It has recently18 been demonstrated that atypical scrapie is experimentally transmissible to mice and sheep, primarily through intracerebral injection. There are some data suggesting that it may also be transmissible orally to sheep of different genotypes. The subgroup noted that challenge experiments with atypical scrapie in sheep were underway in the UK, with one successful intracerebral challenge to date. The subgroup was informed that positive transmission of infectivity from atypical scrapie isolated from sheep with a range of genotypes had been observed in mice. This included ovinised transgenic mice overexpressing the VRQ allele. Nor98 atypical scrapie had also transmitted to ARR ovinised mice, with transmission experiments in AF141RQ ovinised mice planned. Biochemical features of the isolates were maintained after transmission, and were distinct from BSE and classical scrapie. High infectivity titres were observed in brain tissue from atypical scrapie, including from ARR/ARR sheep. Brain transmission experiments in mice carrying the human PrP gene were at an early stage. 18 Le Dur A., Béringue V., Andréoletti O., Reine F., Laï T.H., Baron T., Bratberg B., Vilotte J.- L., Sarradin P., Benestad S.L. and Laude H.(2005) A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes. PNAS 102, 16031-16036


http://www.seac.gov.uk/pdf/positionstatement-sheep-subgroup.pdf



A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes

Annick Le Dur*,†, Vincent Béringue*,†, Olivier Andréoletti‡, Fabienne Reine*, Thanh Lan Laï*, Thierry Baron§, Bjørn Bratberg¶, Jean-Luc Vilotte?, Pierre Sarradin**, Sylvie L. Benestad¶, and Hubert Laude*,†† + Author Affiliations

*Virologie Immunologie Moléculaires and ?Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; ‡Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogène, 31066 Toulouse, France; §Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway Edited by Stanley B. Prusiner, University of California, San Francisco, CA (received for review March 21, 2005)

Next Section Abstract Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and “cases” that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.

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Our study demonstrates that an authentic TSE infectious agent is responsible in sheep and goats of sporadic atypical infections that remained unnoticed until recently. This raises important issues with regard to control of scrapie infection in small ruminants. Of major concern, ARR/ARR sheep can no longer be regarded as free of natural TSE infection. This finding challenges, at least to some extent, the foundation of the selective breeding programs engaged in several European Union member states (47, 48) and may call for a reappraisal of possible consequences of this strategy in the long term. Finally, more information about this newly discovered type of TSE agent, its prevalence in countries free of scrapie or BSE disease, and its potential to across-species transmission would be needed for a comprehensive evaluation of its implications in terms of public health.


http://www.pnas.org/content/102/44/16031.long



http://www.pnas.org/content/102/44/16031.figures-only




Transmissibility of Atypical Scrapie in Ovine Transgenic Mice: Major Effects of Host Prion Protein Expression and Donor Prion Genotype

Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ~0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ~6× the normal PrPc level died after a survival periods of 400 days, those with ~1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.

Article Metrics Related Content Comments: 0 Formal Correction: This article has been formally corrected to address the following errors.

To add a note, highlight some text. Hide notes Make a general comment Jump to Jean-Noël Arsac1, Dominique Bétemps1, Eric Morignat1, Cécile Féraudet2, Anna Bencsik1, Denise Aubert3, Jacques Grassi2, Thierry Baron1*

1 Agence Française de Sécurité Sanitaire des Aliments, Lyon, France, 2 Commissariat à l'Energie Atomique, Service de Pharmacologie et d'Immunoanalyse, Gif sur Yvette, France, 3 Ecole Normale Supérieure de Lyon, CNRS/INRA, Lyon, France

Abstract Top Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ~0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ~6× the normal PrPc level died after a survival periods of 400 days, those with ~1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.

SNIP...

Discussion Top Characterization of the infectious agents involved in TSEs has historically required transmission in inbred wild-type mice, such as C57Bl, VM or RIII [4], [22], [23]. However some TSEs, such as the CH1641 experimental scrapie source [24] or some human TSEs [25] fail to transmit to wild-type mice of any genotype. The recent availability of transgenic mice expressing the prion gene of the natural host of the disease has facilitated TSE transmission studies, as in the use of ovine transgenic mice for scrapie or BSE [5], [20], [21]. The strain-specific molecular diversity of the classical scrapie or BSE sources was faithfully reproduced in the TgOvPrP4 ovine transgenic mouse model that we have developed. However, a novel TSE, called atypical scrapie or Nor98, has recently been identified in sheep and goats throughout Europe [12]. In several countries this disease is now more frequent than classical scrapie and in some countries atypical scrapie has been recognized in the absence of classical scrapie [26]–[28]. The experimental transmissibility of this disease was established in a transgenic mouse model (tg338) that strongly over-expresses (8- to 10-fold) the VRQ allele of ovine prion protein, from a series of 10 French atypical scrapie cases and 2 Norwegian Nor98 isolates [17]. In contrast, experimental transmission was not achieved in wild-type mice [12], [17] or in bank voles [29]. Overall this clearly confirmed that atypical scrapie is a genuine TSE associated with infectious prions.

In this study we confirmed the transmissibility of atypical scrapie, after intracerebral challenge, in another ovine transgenic mouse model (TgOvPrP4), but failed to transmit the disease from 2 cases into wild-type mice. Twelve cases, including the 10 French isolates previously transmitted to the tg338 mouse line [17], were successfully transmitted to TgOvPrP4 mice that over-expressed the AL141RQ prion protein. Although this prnp genotype of TgOvPrP4 mice is only rarely identified in sheep with atypical scrapie [13]–[16], [26], [30]–[33], most of the inoculated mice accumulated the pathological prion protein in their brain. Biochemical analysis of PrPres showed a fully conserved pattern comparable to that previously described in the natural host [15], i.e. a similar complex pattern including 5 major bands derived from 3 distinct PrPres products. Both PrPres fragments cleaved at both N- and C-terminal ends, and the uncleaved (or marginally cleaved) PrPres, were consistently maintained after transmission into ovine transgenic mice (Figure 4). The consistency of the Western blot profiles, whatever the isolate, and in line with previous studies in tg338 ovine transgenic mice, does not suggest any diversity in atypical scrapie. This consistent PrPres glycoprofile is somewhat reminiscent of certain PrPres features in patients with Gerstmann-Straüssler-Scheinker syndrome [34]–[36].

This study enabled us to extend the descriptive profile of our TgOvPrP4 mouse model and examine TSE transmission factors in relation to the level of expression of the prion protein by the infected host. After long-term breeding of the mouse line we indeed found three TgOvPrP4 mice subpopulations expressing in their brain ~0.25, 1.5 or 6× the PrPc level measured in a sheep brain control. We then decided to evaluate the possible influence of these differences in PrPc levels in the central nervous system on TSE development, following the inoculation of 3 mouse-adapted strains (BSE, C506M3, 87V), 3 experimental small ruminants isolates (SSBP1, CH1641, BSEOVINE) and 22 small ruminant natural isolates collected by active surveillance of TSEs in sheep and goats (10 classical scrapie isolates, including 3 usual isolates as shown by PrPres molecular analysis and 7 “CH1641-like” isolates; 12 atypical scrapie isolates). This panel of samples thus represents the molecular diversity of TSEs in small ruminants, especially with the three basic PrPres phenotypes possibly demonstrated by Western blot in natural scrapie. Strikingly the influence of the PrPc expression level was much more obvious for the atypical scrapie isolates, and a considerable delay in disease onset was associated with a decrease in PrPc expression levels,. TgOvPrP4 mice with higher PrPc levels (~6×) died after shorter periods (around 400 days) than mice with lower PrPc levels (~1.5×) (around 700 days). Comparisons of different transgenic mouse lines already indicated that the expression levels of the prion protein were inversely related to the incubation period in classical scrapie [6]. Nevertheless, to the best of our knowledge, the variable influence of PrPc expression levels according to the form of prion disease defined by its essential molecular features, has never been described and is here reported between different animals in a same transgenic mouse line. However, although the development of atypical scrapie appears to be much more facilitated by increased PrPc expression, this also suggests that the risk of transmission in animals expressing physiological levels of the protein would be relatively low. This characteristic, in addition to the species barrier, would help to explain the failure to transmit atypical scrapie to wild-type mice (RIII, VM and C56Bl mice) [17] or bank voles [29], unlike classical scrapie. Besides, a major influence of the PrPc expression levels has been demonstrated in transgenic mice over-expressing (i) mutated human prion protein linked to Gerstmann–Sträussler-Scheinker syndrome [37], [38] or (ii) a prion protein with a nine-octapeptide insertion associated with a human familial prion disease [39], [40]. In both cases disease development was strongly conditioned by the levels of transgene expressions, not only following inoculations of brain tissues from affected patients, but also spontaneously.

Our demonstration that atypical scrapie was much more influenced by PrPc expression levels than classical scrapie or BSE was possible in this mouse model solely because our approach was based on serum PrPc measurements. These were indeed found to reflect the levels of PrPc expression in the brain. In this model protein expression was controlled by the neuron-specific enolase promoter and neither RT-PCR or Western blot analyses were able to detect expression of the prnp gene in non nervous tissues [5], [41]. One explanation of the close relationship between serum and cerebral PrPc might be related to a release of PrPc from the central nervous system into the blood, as was also shown for some other neuronal proteins [42]. It is noteworthy that the plasma concentration of PrPc in wild-type C57Bl mice (~70+/-15 ng/ml) is much higher than in TgOvPrP4 mice, even in [TgOvPrP4 (x6)] with the highest level of expression (48+/-10 ng/ml). This suggests that a fraction of the PrPc in the serum of wild-type mice could originate from the nervous system, under physiological conditions. Importantly, direct evidence that PrPc readily crosses the blood brain barrier in both brain-to-blood and blood-to-brain directions has recently been reported [43]. Regarding the TgOvPrP4 mouse line, the presence of [TgOvPrP4 (x0.25)] animals which express low levels of PrPc clearly represents a drawback for transmission studies of TSEs. This can however be circumvented by assessing the PrPc expression levels in each individual mouse prior to its use in animal experiments. Furthermore, although this subpopulation increased following long-term random breeding of the mouse line, blood testing is also used to monitor PrPc expression levels during breeding in order to eliminate those breeders that produce progeny with a high proportion of poorly expressing animals. It should be noted that no indication of variation of transgene expression was found in a similarly produced mouse line (TgOvPrP59) originating from different founders [44], [45], which makes it unlikely that our observations in the TgOvPrP4 mouse line result from factors intrinsic to the transgene construct and/or the genetic background of the mice. However, the loss of transgene expression in a transgenic line is not unprecedented and two types of explanation are generally provided to account for such a loss. These include (1) integration of the transgene in an heterochromatin-rich region which results in silencing of the transgene in some cells (position effect variegation) [46] or (2) the repetitive nature of the transgene arrays which induces the formation of heterochromatin at the sites of integration and leads to gene silencing [47]. In addition, our genetic construction does not contain any insulators, which suggests that heterochromatin propagation might be a cause of transgene extinction in some animals [48].

Concerning the factors involved in the transmission of atypical scrapie, we were also surprised to demonstrate the influence of the prnp genotypes of the sheep or goat donors on development of the disease in TgOvPrP4 inoculated mice. This had not been observed in tg338 mice, which overall showed more rapid incubation of the disease [17]. Lower PrPc expression levels in TgOvPrP4 mice were required to transmit atypical scrapie, when the isolates carried the AF141RQ or AHQ alleles associated with increased susceptibility to the disease in sheep [14]. The same analysis of possible differences associated with the susceptible or resistant genotype, as here reported in atypical scrapie, could not be done for classical scrapie or BSE considering the available isolates. Among these sources only one classical scrapie isolates (Classusual 2) carried the ARR allele known to be associated with resistance to clinical scrapie [49], [50]. Although the ARR allele is rarely observed in classical scrapie we here confirmed, by both PrPres analyses in sheep and bioassays in wild-type and ovine transgenic mice, that it is possible to detect classical scrapie in relatively resistant animals during the implementation of flock analysis of scrapie cases [51]. Overall, given the limited number of samples available we cannot exclude a possible influence of the prnp genotypes for classical scrapie or BSE. The results observed with atypical scrapie are difficult to explain and we cannot exclude differences in infectious titres between the two groups of samples examined, in relation to (i) possible differences in the distribution of infectivity within the CNS [52] and/or (ii) different stages in the incubation periods in sheep and goats. On the other hand the AF141RQ or AHQ prnp alleles were associated with a lower conformational stability of the ovine PrPc protein [53], which might be associated with an increased capacity of the PrPsc of such genotypes to transconform PrPc. It is noteworthy that only one successful experimental transmission of atypical scrapie has so far been reported in sheep, which was obtained in an AHQ homozygous sheep intracerebrally inoculated with an atypical scrapie isolate from an AHQ homozygous sheep [18]. However the apparently spontaneous occurrence of three cases of atypical scrapie in genetically susceptible sheep was also reported in a flock derived from sheep imported from New Zealand, a country regarded as scrapie-free [54].

Based on our findings, the possibility that variations in the expression of ovine PrPc might be factors involved in susceptibility to atypical scrapie in sheep and goats, is open to question. A second proposal could question whether a failure of the protein quality-control systems might also be involved in susceptibility to atypical scrapie. Recent studies have provided new evidence for a role of these systems in the conversion of PrPc to an abnormal form of prion protein, with either impairment of proteasomal degradation or endoplasmic reticulum stress, leading to the generation of a misfolded form of prion protein [55]–[57]. Transgenic mice were also reported to develop a spontaneous neurological disease in the absence of PrPsc when preferentially expressing a transmembrane form of prion protein (PrPCtm) [58], [59], which would imply that CtmPrP is a key component in the pathway of neurodegeneration as in Gerstmann–Sträussler-Scheinker syndrome.

However our data do emphasize the unresolved questions regarding the origin of atypical scrapie, which as yet remains unknown. A spontaneous origin of the disease is suspected, and importantly, no evidence of natural transmissibility in small ruminants has been observed [33], [60]. The results of our novel approach, based on monitoring prion protein expression in animals from the same transgenic mouse line, suggest that the risk of transmission of atypical scrapie is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.


http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007300



P03.141

Aspects of the Cerebellar Neuropathology in Nor98

Gavier-Widén, D1; Benestad, SL2; Ottander, L1; Westergren, E1 1National Veterinary Insitute, Sweden; 2National Veterinary Institute,

Norway Nor98 is a prion disease of old sheep and goats. This atypical form of scrapie was first described in Norway in 1998. Several features of Nor98 were shown to be different from classical scrapie including the distribution of disease associated prion protein (PrPd) accumulation in the brain. The cerebellum is generally the most affected brain area in Nor98. The study here presented aimed at adding information on the neuropathology in the cerebellum of Nor98 naturally affected sheep of various genotypes in Sweden and Norway. A panel of histochemical and immunohistochemical (IHC) stainings such as IHC for PrPd, synaptophysin, glial fibrillary acidic protein, amyloid, and cell markers for phagocytic cells were conducted. The type of histological lesions and tissue reactions were evaluated. The types of PrPd deposition were characterized. The cerebellar cortex was regularly affected, even though there was a variation in the severity of the lesions from case to case. Neuropil vacuolation was more marked in the molecular layer, but affected also the granular cell layer. There was a loss of granule cells. Punctate deposition of PrPd was characteristic. It was morphologically and in distribution identical with that of synaptophysin, suggesting that PrPd accumulates in the synaptic structures. PrPd was also observed in the granule cell layer and in the white matter. The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.

***The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.


http://www.prion2007.com/pdf/Prion%20Book%20of%20Abstracts.pdf



PR-26

NOR98 SHOWS MOLECULAR FEATURES REMINISCENT OF GSS

R. Nonno1, E. Esposito1, G. Vaccari1, E. Bandino2, M. Conte1, B. Chiappini1, S. Marcon1, M. Di Bari1, S.L. Benestad3, U. Agrimi1 1 Istituto Superiore di Sanità, Department of Food Safety and Veterinary Public Health, Rome, Italy (romolo.nonno@iss.it); 2 Istituto Zooprofilattico della Sardegna, Sassari, Italy; 3 National Veterinary Institute, Department of Pathology, Oslo, Norway

Molecular variants of PrPSc are being increasingly investigated in sheep scrapie and are generally referred to as "atypical" scrapie, as opposed to "classical scrapie". Among the atypical group, Nor98 seems to be the best identified. We studied the molecular properties of Italian and Norwegian Nor98 samples by WB analysis of brain homogenates, either untreated, digested with different concentrations of proteinase K, or subjected to enzymatic deglycosylation. The identity of PrP fragments was inferred by means of antibodies spanning the full PrP sequence. We found that undigested brain homogenates contain a Nor98-specific PrP fragment migrating at 11 kDa (PrP11), truncated at both the C-terminus and the N-terminus, and not N-glycosylated. After mild PK digestion, Nor98 displayed full-length PrP (FL-PrP) and N-glycosylated C-terminal fragments (CTF), along with increased levels of PrP11. Proteinase K digestion curves (0,006-6,4 mg/ml) showed that FL-PrP and CTF are mainly digested above 0,01 mg/ml, while PrP11 is not entirely digested even at the highest concentrations, similarly to PrP27-30 associated with classical scrapie. Above 0,2 mg/ml PK, most Nor98 samples showed only PrP11 and a fragment of 17 kDa with the same properties of PrP11, that was tentatively identified as a dimer of PrP11. Detergent solubility studies showed that PrP11 is insoluble in 2% sodium laurylsorcosine and is mainly produced from detergentsoluble, full-length PrPSc. Furthermore, among Italian scrapie isolates, we found that a sample with molecular and pathological properties consistent with Nor98 showed plaque-like deposits of PrPSc in the thalamus when the brain was analysed by PrPSc immunohistochemistry. Taken together, our results show that the distinctive pathological feature of Nor98 is a PrP fragment spanning amino acids ~ 90-155. This fragment is produced by successive N-terminal and C-terminal cleavages from a full-length and largely detergent-soluble PrPSc, is produced in vivo and is extremely resistant to PK digestion.

*** Intriguingly, these conclusions suggest that some pathological features of Nor98 are reminiscent of Gerstmann-Sträussler-Scheinker disease.

119


http://www.neuroprion.com/pdf_docs/conferences/prion2006/abstract_book.pdf



Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)


http://www.pnas.org/cgi/content/abstract/0502296102v1





>>> So far, there is no evidence for spontaneous PrPSc formation in any animal or human TSE.



Prions: Protein Aggregation and Infectious Diseases

ADRIANO AGUZZI AND ANNA MARIA CALELLA

Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland

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3. Sporadic Creutzfeldt-Jakob disease Approximately 85% of all human prion diseases are sporadic forms of CJD. For sCJD, there is no association with a mutant PRNP allele, nor is there any epidemiological evidence for exposure to a TSE agent through contact with people or animals infected with TSEs. sCJD cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the size and glycoform ratio of proteaseresistant prion protein identified on western blot (type 1 or type 2) (174). Heterozygosity (Met/Val) at PrP codon 129 appears to be associated with a lower risk (378) and/or prolonged incubation time (119, 387). The lack of routine laboratory testing for preclinical diagnosis makes the search for agent sources and other risk factors extremely difficult. At present, the means of acquisition of a TSE agent in these patients remains a mystery. So far, there is no evidence for spontaneous PrPSc formation in any animal or human TSE. In humans, the peak age incidence of sporadic CJD is 55–60 years. However, if spontaneous misfolding were the primary event, one might expect a continuously increasing incidence with age because more time would allow more opportunity for rare misfolding events.

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Physiol Rev • VOL 89 • OCTOBER 2009 • www.prv.org


http://physrev.physiology.org/cgi/content/abstract/89/4/1105





Monday, December 1, 2008

When Atypical Scrapie cross species barriers


http://nor-98.blogspot.com/2008/12/when-atypical-scrapie-cross-species.html




EVIDENCE OF SCRAPIE IN SHEEP AS A RESULT OF FOOD BORNE EXPOSURE

This is provided by the statistically significant increase in the incidence of sheep scrape from 1985, as determined from analyses of the submissions made to VI Centres, and from individual case and flock incident studies. ........



http://web.archive.org/web/20010305222246/www.bseinquiry.gov.uk/files/yb/1994/02/07002001.pdf





1: J Infect Dis 1980 Aug;142(2):205-8

Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.

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The successful transmission of kuru, Creutzfeldt-Jakob disease, and scrapie by natural feeding to squirrel monkeys that we have reported provides further grounds for concern that scrapie-infected meat may occasionally give rise in humans to Creutzfeldt-Jakob disease.


PMID: 6997404


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract




12/10/76 AGRICULTURAL RESEARCH COUNCIL REPORT OF THE ADVISORY COMMITTE ON SCRAPIE Office Note CHAIRMAN: PROFESSOR PETER WILDY

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A The Present Position with respect to Scrapie A] The Problem

Scrapie is a natural disease of sheep and goats. It is a slow and inexorably progressive degenerative disorder of the nervous system and it ia fatal. It is enzootic in the United Kingdom but not in all countries.

The field problem has been reviewed by a MAFF working group (ARC 35/77). It is difficult to assess the incidence in Britain for a variety of reasons but the disease causes serious financial loss; it is estimated that it cost Swaledale breeders alone $l.7 M during the five years 1971-1975. A further inestimable loss arises from the closure of certain export markets, in particular those of the United States, to British sheep.

It is clear that scrapie in sheep is important commercially and for that reason alone effective measures to control it should be devised as quickly as possible.

Recently the question has again been brought up as to whether scrapie is transmissible to man. This has followed reports that the disease has been transmitted to primates. One particularly lurid speculation (Gajdusek 1977) conjectures that the agents of scrapie, kuru, Creutzfeldt-Jakob disease and transmissible encephalopathy of mink are varieties of a single "virus". The U.S. Department of Agriculture concluded that it could "no longer justify or permit scrapie-blood line and scrapie-exposed sheep and goats to be processed for human or animal food at slaughter or rendering plants" (ARC 84/77)" The problem is emphasised by the finding that some strains of scrapie produce lesions identical to the once which characterise the human dementias"

Whether true or not. the hypothesis that these agents might be transmissible to man raises two considerations. First, the safety of laboratory personnel requires prompt attention. Second, action such as the "scorched meat" policy of USDA makes the solution of the acrapie problem urgent if the sheep industry is not to suffer grievously.

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76/10.12/4.6




http://web.archive.org/web/20010305223125/www.bseinquiry.gov.uk/files/yb/1976/10/12004001.pdf







Nature. 1972 Mar 10;236(5341):73-4.

Transmission of scrapie to the cynomolgus monkey (Macaca fascicularis). Gibbs CJ Jr, Gajdusek DC.


Nature 236, 73 - 74 (10 March 1972); doi:10.1038/236073a0

Transmission of Scrapie to the Cynomolgus Monkey (Macaca fascicularis)

C. J. GIBBS jun. & D. C. GAJDUSEK

National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland

SCRAPIE has been transmitted to the cynomolgus, or crab-eating, monkey (Macaca fascicularis) with an incubation period of more than 5 yr from the time of intracerebral inoculation of scrapie-infected mouse brain. The animal developed a chronic central nervous system degeneration, with ataxia, tremor and myoclonus with associated severe scrapie-like pathology of intensive astroglial hypertrophy and proliferation, neuronal vacuolation and status spongiosus of grey matter. The strain of scrapie virus used was the eighth passage in Swiss mice (NIH) of a Compton strain of scrapie obtained as ninth intracerebral passage of the agent in goat brain, from Dr R. L. Chandler (ARC, Compton, Berkshire).



http://www.nature.com/nature/journal/v236/n5341/abs/236073a0.html





Epidemiology of Scrapie in the United States 1977


http://www.bseinquiry.gov.uk/files/mb/m08b/tab64.pdf


http://web.archive.org/web/20030513212324/http://www.bseinquiry.gov.uk/files/mb/m08b/tab64.pdf



Tuesday, April 28, 2009

Nor98-like Scrapie in the United States of America


http://nor-98.blogspot.com/2009/04/nor98-like-scrapie-in-united-states-of.html



Scrapie USA


http://scrapie-usa.blogspot.com/



SCRAPIE

The United States is unable to support the proposed new draft Code Chapter on Scrapie. The draft chapter, as written, departs significantly from the existing chapter, is confusing and is difficult to understand. This version of the scrapie chapter uses much of the same wording as the BSE chapter and is written as if the predominance of evidence revealed that scrapie was a food-borne disease similar to BSE in cattle which is inappropriate. Moreover, several of the new changes are not supported by current scientific evidence. As a result, detailed comments on individual articles would not meaningful at this time.

The United States is not supportive of the proposed draft chapter for the following reasons:

1. Inclusion of “atypical” scrapie: The scientific evidence indicates that “atypical” scrapie, also referred to as Nor-98, Nor-98-like, or non-classical scrapie, is not the same disease as classical scrapie. Further, “atypical” scrapie does not meet the criteria for listing diseases of trade concern by the OIE, as described in Chapter 2.1.1 of the Code. The United States recommends that the scope of this chapter be limited to classical scrapie in sheep and goats. Further, the United States recommends that OIE clearly adopt the position that “atypical” scrapie represents a distinct disease entity from classical scrapie and that it not be a listed disease.

• There is no evidence that “atypical” scrapie is a contagious disease. If it is contagious, available evidence suggests that it has a much lower transmission efficiency. (Hopp, et al, 2006; Green, et al, 2007; Benestad, et al 2008; McIntyre, et al, 2008)

• The disease appears to be ubiquitous in that it has been found wherever sufficient surveillance has been conducted. (Buschmann et al, 2004; De Bosschere et al, 2004; Orge, et al, 2004; Everest et al, 2006; Arsac, 2007; Benestad, et al 2008; Fediaevsky, et al, 2008)

• The disease does not appear to be economically significant in that the prevalence of clinical disease is low and it typically occurs in older animals. (Luhken, et al., 2007; Benestad, et al 2008).

• The disease is as likely as not to be the result of a spontaneous conversion of normal prion protein. (Benestad, et al 2008, De Bosschere et al 2007)

• Removal of exposed sheep is unlikely to reduce the prevalence of “atypical” scrapie infection and removing only those exposed sheep that are phenylalanine (F) at codon 141 is scientifically unsound since the disease is known to affect sheep of most other genotypes. Further, sheep with AHQ alleles have a similar risk of infection with “atypical” strains as sheep with F at codon 141. (Luhken, et al., 2007).

• If “atypical” scrapie is included as a listed disease, the surveillance and diagnostic requirements which are needed to identify these cases should be described in detail in both this Chapter and the Manual of Diagnostic Tests and Vaccines for Terrestrial

2

Animals. Data from Europe illustrates that using the proper test(s) is essential for the identification of atypical scrapie (Fediaevsky et al., 2008).

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6. Overemphasis on importation and use of bovine meat and bone meal as a route of scrapie transmission: Given that the draft Chapter is not intended to address risk mitigation for BSE in small ruminants, we believe there is an over-emphasis on this potential route of transmission in the current draft.

The United States recommends that the requirements in this chapter be limited to the inclusion of products from sheep and goats (instead of from all ruminants) in feed or feed ingredients intended for consumption by animals

• The use of products from sheep and goats as feed or feed ingredients for ruminant or non-ruminant animals represent one possible route of transmission (Philippe, et al, 2005) and a source of environmental contamination with the classical scrapie agent. However, this is not the primary route of transmission for the scrapie agent.

• The need for the exclusion of cattle-derived protein or other animal protein to mitigate BSE risk should be based on a country’s BSE risk status and should be addressed in Chapter 2.3.13 of the Code.

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14. Failure to provide scientific justification for the list of permitted commodities in Item 1 of Article 2.4.8.1. .

We recommend that the list be re-evaluated and those items that have not been substantiated as presenting no risk be excluded or those with some risk but where the intended use mitigates the risk the use be specified.

• There is no known human health risk associated with scrapie. As such, if meat and meat products for human consumption are included in this list, sheep and/or goat milk intended for human consumption should also be added to the list of permitted commodities in Item 1 of Article 2.4.8.1.

• In the vast majority of sheep infected with classical scrapie, actual infectivity or PrPres has been identified in most tissues including the lymphoreticular system (tonsils, spleen, lymph nodes), the gastrointestinal tract, brain, and spinal cord (Hadlow et. al. 1979; Hadlow et al., 1980; van Kuelen et al., 1996; van Kuelen et al., 1999, Andreoletti et al., 2000; Heggebø et al., 2002; Caplazi et al., 2004). Infectivity and/or PrPres has also been identified in the placenta (see Hourrigan et al., 1979; Onodera et al., 1993; Pattison et al., 1972; Pattison et al., 1974; Race et al., 1998), blood (Hunter et al., 2002; Houston et al. 2008); peripheral nerves (Groschup et al., 1996), muscle (Pattison and Millson, 1962; Andreoletti et al., 2004; Casalone et al., 2005), salivary gland (Hadlow et al., 1980; Vascellari et al., 2007), kidney (Siso et al., 2006), and skin ( Thomzig et al., 2007). In addition, recent work has shown milk and/or colostrum from scrapie infected ewes transmitted the disease to 17 of 18 lambs (Konold et al., 2008).

• The data on the risk of low protein tallow made from scrapie infected tissues particularly for use in milk replacer is limited and some epidemiologic studies suggest an association of milk replacer use with scrapie risk. Taylor et al., 1997 examined the inactivation capacity of different rendering system in regards to scrapie. The presence of infectivity was determined by bioassay into mice. From the onset of this study, it was assumed that tallow was not the vehicle for the transmission of TSE. Hence only 2 tallow samples were examined.


http://www.aphis.usda.gov/import_export/animals/oie/downloads/tahc_mar-sep08/tahc-scrapie-77-mar08_cmt.pdf



USDA's Deputy Secretary Talks with Sheep Producers

May 1, 2009 - "Life in rural America is intimately integrated with production agriculture," said Kathleen Merrigan, deputy secretary for the U.S. Department of Agriculture (USDA), when she addressed the more than 50 sheep producers from around the country who were in Washington, D.C., this week. "I am very interested in looking for new ways for farmers to market their products and for them to grab a little more of the food dollar. The connection between the food that consumers eat and the farms that the food is grown on must be closer, and I look forward to working with producers to affect that."

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According to Jere Dick, DVM, associate deputy administrator and chief of field operations for the Animal and Plant Health Inspection Service (APHIS), "We anticipate discussions and hopefully revisions of the scrapie chapter at the World Organization for Animal Health meeting this month. The revisions will likely exclude Nor-98 like scrapie from classical scrapie regulations. If this is done, APHIS will be able to exempt flocks with Nor-98 like scrapie cases, should they occur, from extensive flock depopulation actions."

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http://sheepusa.org/?page=site/newsandevents&nav_id=c806da768c6fcc2652eee0967bc96b5a#772efce63bd783bb5da167cf744f349a




Monday, November 23, 2009

BSE GBR RISK ASSESSMENTS UPDATE NOVEMBER 23, 2009 COMMISSION OF THE EUROPEAN COMMUNITIES AND O.I.E.


http://docket-aphis-2006-0041.blogspot.com/2009/11/bse-gbr-risk-assessments-update.html




Tuesday, November 17, 2009

SEAC NEW RESULTS ON IDIOPATHIC BRAINSTEM NEURONAL CHROMATOLYSIS (IBNC) FROM THE VETERINARY LABORATORIES AGENCY (VLA) SEAC 103/1


http://bse-atypical.blogspot.com/2009/11/seac-new-results-on-idiopathic.html



Tuesday, November 17, 2009

SEAC EFFECT OF AGE ON THE PATHOGENESIS OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES SEAC 103/2


http://downercattle.blogspot.com/2009/11/seac-effect-of-age-on-pathogenesis-of.html



Thursday, November 05, 2009

Incidence and spectrum of sporadic Creutzfeldt-Jakob disease variants with mixed phenotype and co-occurrence of PrPSc types: an updated classification


http://creutzfeldt-jakob-disease.blogspot.com/2009/11/incidence-and-spectrum-of-sporadic.html



J Neurol Neurosurg Psychiatry 1994;57:757-758 doi:10.1136/jnnp.57.6.757 Research Article

Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery.

C J Gibbs, Jr, D M Asher, A Kobrine, H L Amyx, M P Sulima, D C Gajdusek + Author Affiliations

Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892. Abstract Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.


http://jnnp.bmj.com/content/57/6/757.abstract




TSS