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From: TSS ()
Subject: Nor98 scrapie identified in the United States
Date: July 5, 2009 at 1:19 pm PST

-------------------- BSE-L@LISTS.AEGEE.ORG --------------------


----- Original Message ----- From: Terry S. Singeltary Sr. To: TERRY SINGELTARY Sent: Tuesday, June 30, 2009 9:42 PM Subject: Nor98 scrapie identified in the United States

Nor98 scrapie identified in the United States

J Vet Diagn Invest 21:454-463 (2009)

Christie M. Loiacono,1 Bruce V. Thomsen, S. Mark Hall, Matti Kiupel, Diane Sutton, Katherine O'Rourke, Bradd Barr, Lucy Anthenill, Delwyn Keane

Abstract.

A distinct strain of scrapie identified in sheep of Norway in 1998 has since been identified in numerous countries throughout Europe. The disease is known as Nor98 or Nor98-like scrapie, among other names. Distinctions between classic scrapie and Nor98 scrapie are made based on histopathology and immunodiagnostic results. There are also differences in the epidemiology, typical signalment, and likelihood of clinical signs being observed. In addition, sheep that have genotypes associated with resistance to classic scrapie are not spared from Nor98 disease. The various differences between classic and Nor98 scrapie have been consistently reported in the vast majority of cases described across Europe. The current study describes in detail the pathologic changes and diagnostic results of the first 6 cases of Nor98 scrapie disease diagnosed in sheep of the United States.

Key words: Histopathology; Nor98; PrP immunolabeling; scrapie; sheep.

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The current study describes the diagnostic findings of the first 6 cases of Nor98 scrapie in sheep of the United States.

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Results

Case 1

The first case identified as consistent with Nor98 scrapie had nonclassic PrPSc distribution in brain tissue, no PrPSc in lymph tissue, and nonclassic migration of protein bands on a Western blot test. The animal was an aged, mottled-faced ewe that was traced back to a commercial flock in Wyoming. Individual animal records were not kept on the premises, so it was impossible to determine how long the ewe was on the farm, how many times she lambed on that farm, or her flock of origin. She was taken to a sale along with 88 other sheep and was tested at slaughter for scrapie as part of the RSSS program. No abnormal clinical signs were noted at the time of sale. The Prnp genotype of the case 1 ewe was AFRQ/ ALRQ (136 AA, 141 FL, 154 RR, 171 QQ). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve and in the dorsal aspect of the dorsal horns of the cervical spinal cord (Fig. 1A, B). There was no PrPSc immunolabeling in the cerebellum, in the dorsal motor nucleus of the vagus nerve, or in lymphoid tissue. ELISA results were positive when using cerebellum and brainstem by the TSE sheep and goat kitk and negative for the same tissues when using the scrapie antigen kitl (Table 1). On Western blot, multiple distinct protein bands were present, including an unglycosylated band at ,15 kDa (Fig. 2A, lane 1). Subsequently, the entire flock was depopulated, and all 317 adult animals tested negative for Nor98 scrapie and classic scrapie.

Case 2

The second case was a clinically normal 8-year-old Suffolk ewe that had been in a quarantined flock for 5 years at a USDA facility in Iowa. The USDA flock, over the 5-year period, contained several hundred classic scrapie-exposed sheep that had been acquired over time from numerous private flocks designated as scrapie-infected or source flocks. A significant portion of the sheep in this quarantined flock ultimately died or was euthanized because of classic scrapie. The ewe and the entire remaining flock of 95 sheep were euthanized and tested for scrapie at the completion of a study that evaluated the use of third eyelid tissue for scrapie testing. The Prnp genotype of the case 2 ewe was ALRR/ALRR (136 AA, 141 LL, 154 RR, 171 RR). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve (Fig. 1C) and in the dorsal aspect of the dorsal horns of the cervical spinal cord. There was also abundant PrPSc immunolabeling in the cerebellar molecular layer, mild-to-moderate immunolabeling in the cerebellar granular layer, and minimal scattered immunolabeling in the cerebellar white matter (Fig. 1D). PrPSc immunolabeling was absent from the dorsal motor nucleus of the vagus nerve and lymphoid tissue. ELISA results were positive when using cerebellum by both the TSE sheep and goat kitk and the scrapie antigen kitl (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2B, lanes 3 and 4). Digestion with PNGase after proteinase K treatment resulted in 3 unglycosylated bands (Fig. 2C, lane 4) comigrating with the lower 3 bands of the untreated sample (Fig. 2C, lane 3). A classic scrapie sample treated under identical conditions yielded a single band comigrating with the lowest band in the untreated sample (Fig. 2C, lanes 1 and 2). All sheep in this USDA-quarantined flock were tested for scrapie. No additional cases of Nor98 scrapie were detected, whereas 4 cases of classic scrapie were identified in the remaining 95 sheep.

Case 3

A 16-year-old, white-faced, cross-bred wether was born to a black-faced ewe. He lived his entire life as a pet on a farm in California, and was 1 of 6 wethers purchased as a group at 2 months of age. No other sheep were kept on the farm before this group, and none were introduced to the farm after the group was purchased. No clinical signs suggestive of scrapie disease were noted. The animal, with a history of having a large cervical mass, was submitted for necropsy. Samples were tested for scrapie as part of the national scrapie surveillance program through the National Animal Health Laboratory Network. The necropsy revealed a 15-cm cervical thymoma that had compressed the esophagus and resulted in aspiration pneumonia. The Prnp genotype of the case 4 wether was ALRQ/ALRQ (136 AA, 141 LL, 154 RR, 171 QQ). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve and in the dorsal aspect of the dorsal horns of the cervical spinal cord (Fig. 1E). PrPSc immunolabeling in the dorsal motor nucleus of the vagus nerve and in lymphoid tissue was absent. The cerebellum was unavailable for evaluation. ELISA and Western blot tests were not done, because fresh tissue was unavailable. Brain samples were available from one other aged flock mate, and all tests were negative for Nor98 and classic scrapie.

Case 4

The fourth case of Nor98 scrapie was identified in an approximately 8-year-old Dorset ewe that was born into a flock of approximately 20 ewes in Indiana. After a flock dispersal sale, the ewe went to slaughter and was tested for scrapie as part of the RSSS program. No clinical signs suggestive of scrapie disease were noted. The Prnp genotype of the case 4 ewe was ALRQ/ALHQ (136 AA, 141 LL, 154 RH, 171 QQ). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve and in the dorsal aspect of the dorsal horns of the cervical spinal cord. There was abundant PrPSc immunolabeling in the cerebellar molecular layer, moderate immunolabeling in the cerebellar granular layer, and minimal scattered immunolabeling in the cerebellar white matter (Fig. 1F), and no PrPSc immunolabeling in the dorsal motor nucleus of the vagus nerve or in lymphoid tissue. The ELISA results were positive for cerebellum by the TSE sheep and goat kitk and negative for the same tissue when using the scrapie antigen kitl (Table 1). Western blot results included multiple protein bands with an unglycosylated band at ,15 kDa (Fig. 2D, lane 6). Based on sale records, all of the animals sold at the dispersal sale went to slaughter, thus, no additional animals were available for scrapie testing.

Case 5

The fifth case was a clinically normal, approximately 3-year-old, white-faced, cross-bred ewe from an approximately 400 head commercial flock in Minnesota. The ewe was euthanized and tested for scrapie because the flock had previously been identified as a classic scrapie-infected flock. The Prnp genotype of the case 5 ewe was AFRQ/ALRQ (136 AA, 141 FL, 154 RR, 171 QQ). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve and in the dorsal aspect of the dorsal horns of the cervical spinal cord. Abundant PrPSc immunolabeling was present in the cerebellar molecular layer, mild immunolabeling in the cerebellar granular layer, and minimal scattered immunolabeling in the cerebellar white matter (Fig. 1G). PrPSc immunolabeling in the dorsal motor nucleus of the vagus nerve and in lymphoid tissue was absent. The ELISA results were positive when using cerebellum by both the TSE sheep and goat kitk and the scrapie antigen kitl (Table 1). When using brainstem, ELISA was positive by the TSE sheep and goat kitk and negative by the scrapie antigen kitl (Table 1). Western blot produced multiple protein bands, including an unglycosylated band at ,15 kDa (Fig. 2E, lane 4). The flock was depopulated, and no additional cases of Nor98 or classic scrapie were identified in the adult sheep.

Case 6

The sixth case of Nor98 scrapie was identified in a 4-year-old, white-faced ewe that was purchased and added to a commercial flock in Pennsylvania that consisted of approximately 700 head of sheep and goats. Individual animal records were not kept on the premises, so it was impossible to determine exactly how long the ewe was on the farm or her flock of origin. It was estimated that she remained in this flock for approximately 1 month, was sent to slaughter, and was tested for PrPSc as part of the RSSS program. No clinical signs suggestive of scrapie disease were noted. The Prnp genotype of the case 6 ewe was AFRQ/ALRQ (136 AA, 141 FL, 154 RR, 171 QQ). Evaluation of the brain by using HE revealed no lesions. IHC highlighted PrPSc bilaterally in the spinal nucleus of the trigeminal nerve (Fig. 1H) and in the dorsal aspect of the dorsal horns of the cervical spinal cord. PrPSc immunolabeling in the dorsal motor nucleus of the vagus nerve and in lymphoid tissue was absent. Cerebellum was unavailable for evaluation. ELISA and Western blot tests were not done because fresh tissue was unavailable. The commercial flock was depopulated, and adult animals exposed to this ewe were tested for scrapie. No additional cases of Nor98 or classic scrapie were identified. A summary of relevant findings from all cases is shown in Table 1.

Discussion

Nor 98-like scrapie has significantly different diagnostic features when compared with classic scrapie.1,10 In the past, classic scrapie disease was confirmed by examination of the brain tissue for a triad of salient histopathologic changes, including vacuolation limited to gray matter referred to as spongiform change, loss of neurons, and accompanying gliosis.26 However, more recently, a presumptive histopathologic diagnosis was largely replaced by the more sensitive and confirmatory IHC and biochemical methods for detection of PrPSc in the brain or lymphoid tissues. In the case of Nor98 scrapie, there is generally little or no vacuolation in the brain, and, to date, no lymphoid accumulation of PrPSc has been detected.1 Classic scrapie has the earliest and most intense PrPSc immunolabeling at the motor nucleus of the vagus nerve as well as often abundant immunolabeling in numerous areas that contained lymphoid tissue, including retropharyngeal lymph nodes, tonsil, third eyelid, and rectal submucosa. Conversely, Nor98 scrapie-infected animals have no immunolabeling for PrPSc in the motor nucleus of the vagus nerve or in lymphoid tissues, but there typically is immunolabeling for PrPSc in the spinal nucleus of the trigeminal nerve and variable but often intense, immunolabeling for PrPSc in the cerebellum.1 Thus, the diagnosis of Nor98 disease can be based on IHC identification of PrPSc in specific regions of the brain, primarily the spinal nucleus of the trigeminal nerve and the cerebellum (Fig. 1) if there is a concurrent lack of immunolabeling for PrPSc in the dorsal motor nucleus of the vagus nerve or in any lymphoid tissues. In addition, there is a distinct diagnostic Western blot pattern for Nor98 scrapie that consists of multiple protein bands, including an unglycosylated band at ,15 kDa. This pattern remains unchanged for the lower 2-3 bands after enzymatic deglycosylation and is distinct from classic scrapie, in which the 3 protein bands that range from 20 to 30 kDa are reduced to a single unglycosylated 20 kDa band after treatment with PNGase.1,10,15

Nor98 scrapie was diagnosed in each of the 6 cases described in the present study based on IHC results (Table 1). In 4 of the cases that included fresh tissue, Western blot and ELISA results supported the diagnosis. All 6 cases had striking IHC features, including positive PrPSc immunolabeling in the spinal nucleus of the trigeminal nerve and positive PrPSc immunolabeling in the dorsal horns of the cervical spinal cord (Fig. 1). Positive PrPSc immunolabeling was less consistent in sections of cerebellum in the 4 cases that included this tissue for evaluation. Three of the 4 cases that included cerebellum were strongly positive with a similar distribution of PrPSc. The majority of PrPSc immunolabeling was present in the cerebellar molecular layer. Low-to-moderate PrPSc immunolabeling was present in the cerebellar granular layer, with only minimal scattered immunolabeling in the cerebellar white matter. The Purkinje cells were negative for immunolabeling in each of these cases. The fourth case, including cerebellum had no PrPSc immunolabeling present. None of the 6 cases had PrPSc immunolabeling in the dorsal motor nucleus of the vagus nerve, and none had PrPSc immunolabeling in lymphoid tissues. Western blot produced similar results for the 4 cases that had fresh tissue available for testing. In each case, there were multiple protein bands, including an unglycosylated band that measured ,15 kDa (Fig 2A, lane 1; B, lanes 3 and 4; D, lane 6; E, lane 4). A distinct feature in the analysis of Nor98 scrapie by Western blot testing is the presence of 3 different unglycosylated bands after PNGase treatment15 as observed in case 2 (Fig. 2C, lane 4), whereas, with classic scrapie, just 1 band is detected (Fig. 2C, lane 2). ELISA results were positive by the TSE sheep and goat kitk for all 4 cases that included fresh tissue but were positive by the scrapie antigen kitl for only 2 of these cases (Table 1).

The 6 U.S. cases were identified in a variety of testing scenarios that ranged from routine slaughter surveillance of healthy appearing animals to surveillance testing in an aged animal diagnosed with thoracic neoplasia and secondary pneumonia. None of the 6 cases had a clinical history that suggested the presence of a TSE. This is consistent with the findings in many European countries that have found Nor98 disease on a regular basis through slaughter surveillance of apparently healthy animals and fallen stock.1 Three of the sheep (cases 1, 4, and 6) were identified through the RSSS program. Of the remaining 3 animals, 2 sheep (cases 2 and 5) were found in flocks in which classic scrapie had been previously diagnosed, and the last case (case 3, a 16-year-old California wether) was presented for a routine diagnostic necropsy in which scrapie testing was included. Clinical signs are uncommon in the Nor98 strain of scrapie but most often include ataxia without pruritis, which is more commonly associated with classic scrapie.2 None of the 6 sheep described in the present study were reported to have had clinical signs, but it might be that subtle signs were not detected. This is a possibility with cases 1, 4, 5, and 6, because they were each part of large flocks with high rates of turnover. With regard to case 3, signs, including ataxia, may have been masked by other clinical signs associated with the unrelated disease processes of pneumonia and an obstructing tumor. Clinical signs in case 2 were unlikely to be missed, because the animal was closely scrutinized for scrapie signs as part of a scrapie study flock. Both Nor98 and classic scrapie can be diagnosed in a wide variety of age groups, but Nor98 appears to be more commonly found in older animals. In contrast to classic scrapie, which was diagnosed most commonly in 3-5-year-old sheep, Nor98 scrapie was found most frequently in animals more than 5 years of age.17 The U.S. cases described in the current report ranged in age from approximately 3 to 16 years, with a mean age of 7.8 years.

The 3 codons of the ovine Prnp gene at positions 136, 154, and 171, which are consistently reported to influence classic scrapie disease,14 also appear to have some influence on Nor98 scrapie. Sheep with genotypes known to be underrepresented in the classic scrapie-infected population are not spared from the Nor98 strain. Nor98 scrapie has been found in sheep that carry the A136H154Q171 haplotypes (A 5 alanine, H 5 histidine, and Q 5 glutamine) known to be less susceptible to classic scrapie and in sheep that carry the A136R154R171 haplotype (R 5 arginine) known to be associated with natural resistance to classic scrapie.8,16 Case 4 in the current study had the AHQ haplotype, and case 2 was homozygous for the ARR allele. The V136R154Q171 haplotype (V 5 valine) has been associated with higher susceptibility to classic scrapie but seems to offer at least partial resistance to Nor98 scrapie.14,16,17,24 None of the 6 cases reported in the present study had the VRQ haplotype. In addition, a fourth codon at position 141 has been identified that also appears to influence the presence of Nor98 scrapie disease.18 When phenylalanine (F) is present at position 141 with the ARQ haplotype (AF141RQ), the risk for developing Nor98 scrapie is highest, particularly when a sheep has 2 of this particular allele or has 1 AHQ allele along with the AFRQ allele.17,18,24 Cases 1, 5, and 6 presented in the current study each had an AFRQ haplotype with F residue at the 141 codon. Of the 6 cases presented, 4 had susceptible alleles. Cases 1, 5, and 6 had the AFRQ haplotype, and case 4 had the AHQ haplotype. Furthermore, case 2 had the ARR alleles reported to be susceptible to Nor98 scrapie but resistant to classic scrapie. In total, 5 of the 6 sheep identified in the current study had genotypes consistent with susceptibility to Nor98 scrapie. The sixth case, case 3, is homozygous for the ALRQ allele, which has been reported to be present in the Nor98 scrapie-infected population but is considered to have a low risk of susceptibility.17,18

The incidence of Nor98 scrapie is also reportedly different from that of classic scrapie. In a study that included 104 scrapie-infected flocks in Germany, the incidence of Nor98 scrapie was 0.2%, whereas the incidence of classic scrapie was 1.7%.17 The same study also found that, in more than 90% of the flocks infected with Nor98 scrapie, there was only a single infected animal within the flock. Similarly, each of the U.S. Nor98-positive animals was the only animal identified in their respective flock with this unique strain of scrapie. In another study that compared the prevalence of classic scrapie and Nor98 scrapie in 20 European countries, it was reported that, although the overall prevalence was low for both classic and Nor98 scrapie, there was greater variation in the prevalence of classic scrapie. Nor98 scrapie was relatively consistently present in all countries that participated in the study.12 Given this information and when considering how closely the 6 U.S. cases of Nor98 scrapie reported herein parallel European cases with regard to epidemiology and pathology, it is likely that a low but relatively consistent prevalence of Nor98 scrapie will be found wherever rigorous surveillance occurs.

Acknowledgements

The authors would like to thank Julie Lease, Sharon Lund, Dongyue Zhuang, Patricia Meinhardt, Troy Boyle, Jennifer Lamoreux, Donna Lester, and Jim Fosse for technical support. The authors also wish to thank the state and university veterinary diagnostic laboratories and their directors, especially Dr. James Collins, who test under contract to NVSL for TSE diseases and screen U.S. sheep for the presence of scrapie disease. Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. Sources and manufacturers

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full text ;

PDF Nor98 scrapie identified in the United States Loiacono et al. J Vet Diagn Invest.2009; 21: 454-463

http://jvdi.org/cgi/reprint/21/4/454?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=prion&searchid=1&FIRSTINDEX=0&volume=21&issue=4&resourcetype=HWCIT

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

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)

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.

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

Monday, December 1, 2008 When Atypical Scrapie cross species barriers

Authors

Andreoletti O., Herva M. H., Cassard H., Espinosa J. C., Lacroux C., Simon S., Padilla D., Benestad S. L., Lantier F., Schelcher F., Grassi J., Torres, J. M., UMR INRA ENVT 1225, Ecole Nationale Veterinaire de Toulouse.France; ICISA-INlA, Madrid, Spain; CEA, IBiTec-5, DSV, CEA/Saclay, Gif sur Yvette cedex, France; National Veterinary Institute, Postboks 750 Sentrum, 0106 Oslo, Norway, INRA IASP, Centre INRA de Tours, 3738O Nouzilly, France.

Content

Atypical scrapie is a TSE occurring in small ruminants and harbouring peculiar clinical, epidemiological and biochemical properties. Currently this form of disease is identified in a large number of countries. In this study we report the transmission of an atypical scrapie isolate through different species barriers as modeled by transgenic mice (Tg) expressing different species PRP sequence.

The donor isolate was collected in 1995 in a French commercial sheep flock. inoculation into AHQ/AHQ sheep induced a disease which had all neuro-pathological and biochemical characteristics of atypical scrapie. Transmitted into Transgenic mice expressing either ovine or PrPc, the isolate retained all the described characteristics of atypical scrapie.

Surprisingly the TSE agent characteristics were dramatically different v/hen passaged into Tg bovine mice. The recovered TSE agent had biological and biochemical characteristics similar to those of atypical BSE L in the same mouse model. Moreover, whereas no other TSE agent than BSE were shown to transmit into Tg porcine mice, atypical scrapie was able to develop into this model, albeit with low attack rate on first passage.

Furthermore, after adaptation in the porcine mouse model this prion showed similar biological and biochemical characteristics than BSE adapted to this porcine mouse model. Altogether these data indicate.

(i) the unsuspected potential abilities of atypical scrapie to cross species barriers

(ii) the possible capacity of this agent to acquire new characteristics when crossing species barrier

These findings raise some interrogation on the concept of TSE strain and on the origin of the diversity of the TSE agents and could have consequences on field TSE control measures.

http://www.neuroprion.org/resources/pdf_docs/conferences/prion2008/abstract-book-prion2008.pdf

NOR-98 ATYPICAL SCRAPIE 5 cases documented in USA in 5 different states USA 2007

http://nor-98.blogspot.com/2008/04/seac-spongiform-encephalopathy-advisory.html

Tuesday, June 3, 2008 SCRAPIE USA UPDATE JUNE 2008 NOR-98 REPORTED PA

http://nor-98.blogspot.com/2008/06/scrapie-usa-update-june-2008-nor-98.html

http://nor-98.blogspot.com/

Monday, September 1, 2008

RE-FOIA OF DECLARATION OF EXTRAORDINARY EMERGENCY BECAUSE OF AN ATYPICAL T.S.E. (PRION DISEASE) OF FOREIGN ORIGIN IN THE UNITED STATES [No. 00-072-1] September 1, 2008

http://foiamadsheepmadrivervalley.blogspot.com/2008/09/re-foia-of-declaration-of-extraordinary.html

SEAC Position Statement

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Food standards agency atypical scrapie contingency plan Issue 1. In June 2006 the Food Standards Agency (FSA) Board considered current precautionary risk management measures for small ruminants. The Board agreed that current precautionary measures were sufficient. However, they wished to develop a contingency plan in case SEAC's understanding of the risk of atypical scrapie for human health changed. To inform this contingency plan the FSA requested SEAC advice on potential outcomes of research on atypical scrapie, or surveillance results, that may lead to a change in SEAC's estimate of the risk to human health.

Background 2. In February 2006 the SEAC sheep subgroup published a position statement on atypical scrapie1 which concluded that atypical scrapie should be considered a distinct transmissible spongiform encephalopathy (TSE) of small ruminants and not simply a variant of classical scrapie. While there was no evidence that atypical scrapie could infect humans, a theoretical risk could not be excluded. SEAC concluded, however, that there were insufficient data available to adequately assess the potential risks to human health. The subgroup recommended that an adequate assessment of the potential risk to human health of atypical scrapie might come from studies on the prevalence, transmission in animal models, tissue distribution and human health surveillance.

3. SEAC considered the potential significance of the outcomes of these areas of research for the human health risk from atypical scrapie, based on scenarios put forward by the FSA2.

Prevalence 4. The SEAC sheep subgroup statement concludes that there could be around 82,000 sheep in the UK infected with atypical scrapie. Future surveillance and epidemiological studies along with the analysis of sheep brain tissue samples dating back to 1964 may confirm the historical presence of the disease, changes in prevalence over time, and whether or not atypical scrapie may occur in countries previously thought to be free from classical and atypical scrapie. SEAC considered that the identification of historical cases, new cases in scrapie-free countries, and changes in prevalence of atypical scrapie would be significant from a public health perspective. If such data indicate that atypical scrapie has been present for many years, and is not increasing in prevalence then, by analogy with classical scrapie the human health risk would be considered low. However, if atypical scrapie were found to be spreading rapidly, this would imply it is a new disease and any human health risk would be more uncertain. It is therefore important to continue to assess the historic prevalence of atypical scrapie, and for archived sheep samples be analysed for the presence of the disease.

5. A human health risk would only be confirmed by concomitant changes in the prevalence of new types of Creutzfeldt-Jakob Disease (CJD). Because of the long incubation periods of prion diseases, such data may not become apparent for many years, although atypical scrapie has been identified in a UK sheep from 1989, implying that humans may have been exposed to atypical scrapie via the dietary route for a number of years. In the absence of data suggesting a link to a new type of CJD SEAC would be unlikely to change its current assessment of the human health risk.

Transmission studies 6. Results from transmission studies using non-human primates, particularly via the oral route, would strongly inform the understanding of human health risk. The immune and lymphoreticular systems of non-human primates are closely related to those of humans and the peripheral pathogenesis of TSEs in non-human primates mimics that in humans. However, non-human primates only provide data relating to one genotype, MM, which comprises about 37% of the UK population3. Assessment of the level of risk would require comparison with transmissions of other TSEs in the same models, in particular BSE, some of which are already available.

7. Humanised mice can provide data on all three human prion protein genotypes. It is important to be aware of the possibility that such mice may not show any clinical sign of infection after primary transmission of atypical scrapie, yet a secondary transmission from these animals to others may result in clinical disease due to loss of the interspecies transmission barrier. Although humanised mice are a good model for human disease, it will be critical to compare the behaviour of atypical scrapie with other TSEs, especially classical scrapie and BSE, in several mouse models after secondary transmission in order to obtain the most reliable risk assessments.

8. The barrier to transmission of atypical scrapie between animal and human can be tested in vitro by cell free conversion assays. However, care is needed in interpreting the significance of such experiments as ex vivo data do not always correlate well with in vivo studies. Nevertheless, they could provide data on whether conversion of the normal prion protein in humans to the abnormal form by the atypical scrapie prion is or is not possible.

Tissue Distribution 9. Little is known about the tissue distribution of abnormal prion protein (PrPsc) and infectivity in sheep with atypical scrapie. If atypical scrapie is found to be a health risk to humans, data from studies to assess the tissue distribution of PrPsc and infectivity are essential to allow an assessment of the risk under specific control measures.

Human Health 10. Establishing a definitive link between an animal and a human TSE is extremely difficult. Animal model data will only be indicative, and not definitive, although if carried out appropriately could be strongly indicative of a human health risk. The emergence of a new type of CJD which shows the same transmission characteristics as atypical scrapie in non-human primates and humanised mice would provide a strong indication that transmission had occurred through the consumption of infected material. Thus, ongoing human surveillance is critical.

11. It is difficult to conclude that atypical scrapie is not a human health risk from negative experimental or surveillance results. However, negative results from current and retrospective surveillance and transmission studies, over a significant period of time to allow for possibly long incubation periods, would imply a negligible human health risk.

Conclusion 12. It is not possible to assess the human health risk from atypical scrapie, or changes in risk, in the absence of hard scientific data. No single data set is likely to be definitive and it would be essential to consider all the information available, rather than data from single studies in isolation. Studies comparing the properties of atypical scrapie and other TSE agents using the same animal model, especially humanised mice or non-human primates, would be most informative in the short term. Surveillance data to assess any association between forms of CJD and atypical scrapie prevalence would be most persuasive but are unlikely to become available in the short term. SEAC would have to review experimental methods and results, should they emerge, before any conclusion of a change to the risk to human health from atypical scrapie could be made.

1 http://www.seac.gov.uk/pdf/positionstatement-sheep-subgroup.pdf (97 KB) 2 http://www.seac.gov.uk/papers/97-3.pdf (110 KB) 3 Palmer and Collinge (1993) Mutations and polymorphisms in the prion protein gene. Hum. Mutat. 2, 168-173.

Page updated: 6 September, 2007

http://www.seac.gov.uk/statements/fsa-scrapieplan.htm

Atypical scrapie

What does the term atypical scrapie mean?

Mankind has been aware of scrapie for over 250 years. In the past, infected sheep were usually detected when they became clinically affected, and were subjected to examination of brain tissue after death.

In the past 15-20 years, through the study of the genetics of sheep clinically affected with scrapie, it appeared that some were susceptible to scrapie, but others were apparently not as they were never represented in the clinical population (see TAFS Position paper on BSE in sheep for more extensive discussion). This absence of sheep of particular genotypes from the population of clinically affected sheep led to a belief that such sheep were at least resistant to clinical disease, and possibly resistant to infection by natural routes.

The expansion of research programmes due to concerns about BSE has demonstrated that such sheep are not totally resistant as they can be infected by inoculation directly into the brain (23).

Investigations into sheep that were ataxic (walking with difficulty, in an uncoordinated manner) in Norway identified a form of scrapie that seemed to differ from that seen in other flocks and countries. It occurred in genotypes of sheep that were rarely affected elsewhere. This form of scrapie was termed Nor98 (5). It is the first of the "atypical scrapie" isolates to be recognized.

When rapid tests were introduced into surveillance programmes in Europe in 2002, many of the sheep being tested were clinically healthy. Test results therefore had to be interpreted in isolation from data concerning clinical signs. Scientists in several countries faced a challenge to confirm positive results obtained by one test in particular, the BioRad Platelia® or TeSeETM test. Traditional test methods seemed ineffective in confirming the primary results. Equally challenging was the fact that the unconfirmed positive results were in genotypes of sheep that were previously assumed to be resistant. There were doubts about whether or not the fact that the tests had not been evaluated with sheep scrapie was a factor. The results could have been false

TAFS 3

positives due to reactions that would not have been recognized or anticipated by their prior evaluation solely on cattle BSE.

Verification of the results occurred quickly, but uncertainty as to their meaning led to the adoption of various terms - unclassified, atypical, discordant - to describe the cases, and eventually they collectively became known as "atypical".

In a report to the European Commission, the Community Reference Laboratory Strain Typing Expert Group advised that it was likely that sheep were susceptible to infection with a spectrum of prion strains, only some of which had previously been detected and associated with what was known as classical scrapie. While it was possible that the atypical scrapie isolates were simply part of that spectrum, and had previously not been recognized because appropriate tests had not been available, there was also a possibility, albeit less likely, that they were a new phenomenon.

This history is extensively discussed in two EFSA Opinions. One, in October 2005(16), describes attempts to classify isolates in small ruminants to ensure that there is consistency of approach between countries in terms of names applied to isolates. The second, in July 2006(17), describes much of the background especially in relation to the interrelationship with genotype and the likely impact on programmes to breed for resistance.

What are the differences between classical and atypical scrapie?

This is still an incomplete area of science, and therefore difficult to fully explain.

Both types of scrapie clearly involve the presence of abnormal prion protein, and are recognized as prion diseases.

Classical scrapie, identified in clinically affected sheep, occurs in certain genotypes of sheep. Historically there was only one recorded incident of a positive case in what was thought to be a fully resistant genotype (ARR/ARR) (24). There were doubts about the diagnosis in that case. Most classical scrapie cases have traditionally been in sheep carrying the VRQ and ARQ alleles (homozygous or heterozygous), occasionally in combination with ARR and AHQ. While it is still a point of debate as to whether a sheep can be classified as infected with classical scrapie if it is not exhibiting clinical signs, there is no doubt that the prion protein found, and the range of genotypes affected, are identical in healthy, found-dead or clinically affected sheep.

Atypical scrapie has been found predominantly in sheep carrying the AHQ, ARQ and ARR alleles, either homozygous or heterozygous. Full detail is given in the EFSA Opinions(1, 3, 4, 5, 9, 10, 13, 17, 18, 19, 20, 28, 29, 30, 31, 32, 33, 35).

The abnormal form of prion protein in atypical cases has been shown to be more susceptible to enzymatic digestion than in classical scrapie. Because most of the rapid diagnostic tests use enzymes to prepare the sample for testing, some at higher concentrations than others, they were unable to detect atypical scrapie while others could. The protein had been totally or partially digested during the processing for some tests, to the point where antibodies used in the tests were no longer able to recognize and bind to target sites on the prion protein. This phenomenon has subsequently been demonstrated more precisely(21).

Other methods based on ELISA or Western Blot techniques are now available which clearly can detect atypical scrapie, but as demonstrated by the evaluation of rapid tests for small ruminants conducted by EFSA in 2005, they are certainly not equally capable(14, 15).

TAFS 4

How was atypical scrapie found?

Nor98 was identified through the investigation of unusual clinical signs in sheep in Norway.

In the EU it was entirely as a result of the introduction of rapid testing for active surveillance, and initially particularly through the use of one test manufactured by a company called BioRad. Other tests have subsequently demonstrated that they can also detect atypical scrape following the evaluation of tests specifically for use on small ruminants.

Where has atypical scrapie been found?

This has been detailed in the EFSA Opinions described above, but it is clear that apart from Norway, where Nor98 was first identified, atypical scrapie has now been identified in several European and other countries - including the United Kingdom, France, Germany, Belgium, Finland, Iceland, Italy, Ireland, The Netherlands, Portugal, Sweden, Switzerland and the Falkland Islands(1, 3, 4, 5, 9, 10, 13, 17, 18, 19, 20, 28, 29, 30, 31, 32, 33, 35).

As rapid tests used for active surveillance are not all equally capable of detecting atypical scrapie, this will inevitably influence the apparent geographical distribution. In other words, countries that only use tests that are either unable or less able to detect atypical scrapie are unlikely to have reported cases. Only when the tests evaluated specifically on small ruminant tissues replace those previously evaluated on cattle tissue, will they be able to detect atypical cases with any certainty. This has already occurred in the European Union.

Is there more than one strain of atypical scrapie?

At the moment it is not possible to confirm this until further characterization has been completed, especially by bioassay in laboratory rodents.

While there is a degree of conformity between western immunoblotting results in most countries, there is also evidence for variability between animals when whole brains are examined by immunohistochemistry. Whether this is determined by the strain or the genotype of the sheep or goat, is not yet known.

Immunohistochemically the distribution of staining in the brain is quite different to classical scrapie, with only faint, focal, staining of the brain stem at the level of the obex, but more substantial staining of cerebellum of most animals, although in some it is the frontal cortex that is targeted. In some animals focal plaques are also seen in both grey and white matter.

Many of the cases are similar to Nor98 in terms of diagnostic criteria that have been characterized so far. The western blot below demonstrates how the banding pattern for classical and atypical scrapie differ.

Can atypical scrapie cause clinical signs?

Yes. In addition to Nor98 cases reported in Norway, a small number of cases have been detected in the UK. In Norway the most common sign was progressive incoordination (ataxia), together with some weight loss. There was no evidence of scratching. Scratching is not universally seen in classical scrapie however, and may depend on the strain of classical scrapie with which the sheep is infected(5).

In the British sheep the predominant sign was again ataxia, with inconsistent recording of changes in temperament, loss of condition, or circling, and in one case scratching(26, 27). Video clips of clinically affected sheep can be viewed via reference 24. Similar clinical cases have also been recorded in Ireland(32).

Is it transmissible?

Experimentally, it has been shown that it can be transmitted to genetically modified mice(28), and by intracerebral inoculation to sheep (unpublished work in progress). ?? These transmissions do not prove that it will transmit naturally from sheep to sheep, but studies involving oral infection of sheep are under way.

Although most atypical cases occur singly in flocks, there are some instances where two affected sheep have been identified in flocks. This may indicate that natural transmission may occur, or that the sheep were infected from a common alternative source(22, 29). Possible indications of an association with the feeding of vitamins and mineral feed supplements were detected in Norway, but remain to be proven(22). Does it represent a risk to human health?

This is currently unknown, but if atypical scrapie is not a new phenomenon, and has simply been discovered recently, then the lack of epidemiological association between

TAFS 6

prion diseases in humans and sheep, or consumption of sheep products, suggest that atypical scrapie does not represent a risk to humans. This is not however demonstration of absolute safety.

see full text ;

http://www.tseandfoodsafety.org/position_papers/TAFS_POSITION_PAPER_ON_ATYPICAL_SCRAPIE_AND_%20ATYPICAL_BSE_070516.pdf


experimental transmission of 171RR Nor98 brain to 5 RR recipients (Jan 2008)


http://www.sheepusa.org/index.phtml?page=site/get_file&print=1&file_id=f4d1b2a4739ab82d903cf042526bd9ee


http://www.eradicatescrapie.org/Educational%20Resources/PDFs%20&%20PPTs/Genotyping%20PPT/Slide%20Notes%20for%20Genotyping.pdf


http://www.eradicatescrapie.org/Educational%20Resources/PDFs%20&%20PPTs/Genotyping%20PPT/Genotyping%20A%20Tool%20for%20Controlling%20Classical%20Scrapie.ppt


P7.12 Co-existence of classical and atypical scrapie strains in a sheep from an Italian outbreak

Results: IHC revealed the simultaneous presence in the brain of pathological features characteristic of Nor98 and classical scrapie. This was confirmed by WB: PrPres fragments characteristic of Nor98 and scrapie were simultaneously present in all areas investigated, although in different proportions, with Nor98 being more abundant in the cortex and classical scrapie in the brainstem. The lymph node showed the presence of PrPsc with a molecular pattern referable only to classical scrapie, while the tongue resulted negative. Genetic analysis showed the following genotype: A136L141R154Q171/A136F141R154Q171.

Discussion: Our results suggest either the co-existence of Nor98 and classical scrapie in this sheep or the presence of a new scrapie phenotype. The presence of classical scrapie in the outbreak and the genotype of the animal support the first event, which might be explained by the different genetic and cellular targets of the two strains. The bioassay analysis in bank voles and Tg338 mice, at the moment in progress, will help to confirm this hypothesis.


also see ;

P9.18 First Report of Classical Scrapie in Portugal, including co-existence with atypical scrapie in the same flock


also see ;

P9.37 Sporadic Creutzfeldt-Jakob Disease in the Basque Country: Coexistence of prion protein strains in the same and different brain regions

Discussion: The Basque Country presents one of the highest annual incidences of confirmed sCJD in Spain and in Europe (1.87 per million inhabitants per annum). It also suffers the highest frequency of FFI (43.12%). Regarding regional pattern distribution, our results show a predominance of PrPsc type 1 in sCJD. Surprisingly, a high frequency of cases with both types of prion protein was observed, indicating that the coexistence of two different molecular patterns in the same individual is more common than expected.

http://www.neuroprion.org/resources/pdf_docs/conferences/prion2008/abstract-book-prion2008.pdf

Saturday, May 2, 2009

APHIS AND WHO PLAN TO EXEMPT THE ATYPICAL SCRAPIE NOR-98 FROM REGULATIONS AT MEETING THIS MONTH


http://nor-98.blogspot.com/2009/05/aphis-and-who-plan-to-exempt-atypical.html

2.1. European dimension of the problem.

Sheep and goats have suffered from scrapie for centuries in the EU and world wide but the current distribution and incidence of scrapie is largely unknown due to the inadequacy of the epidemiosurveillance networks in different Member States. Until the beginning of the BSE epidemic in the UK in the 1980s there was no particular concern about the national incidence of scrapie (or other TSEs in sheep). One reason was because it is generally accepted that there was no epidemiological relationship between the occurrence of scrapie in sheep and the incidence of Creutzfeldt-Jakob disease (CJD) in humans over a period of over 250 years. However, attention was drawn by the Scientific Veterinary Committee to the high incidence of familial CJD in the Orava region of Slovakia at a level of about 30 times the normal level of 1 per million adults per year. This form of familial CJD is attributed to mutations in the PrP gene at codon 200. This particular mutation is not fully penetrant and some of the workers in Slovakia have suggested that exposure to sheep scrapie may also be necessary for disease to result. This is partly because scrapie had been identified in sheep in the region following a period since the late XIXth century when scrapie was thought to be absent from the country.

http://ec.europa.eu/food/fs/sc/ssc/out48_en.html

http://www.springerlink.com/content/l564271136v7626t/

http://www.springerlink.com/content/v847132717217040/

1: Cent Eur J Public Health 2003 Mar;11(1):19-22

Analysis of unusual accumulation of Creutzfeldt-Jakob disease cases in Orava and Liptov regions (northern Slovak focus) 1983-2000.

Mad'ar R, Maslenova D, Ranostajova K, Straka S, Baska T.

Institute of Epidemiology, Jessenius Faculty of Medicine, Comenius University, Sklabinska 26, Martin, 037 53 Slovakia. MADAR@jfmed.uniba.sk

While familial cases of Creutzfeldt-Jakob disease are extremely rare all over the world, 3 familial clusters were observed between 1983-2000 in a relatively small area situated in the North of Slovakia. Prevalence of CJD in this area exceeded the overall prevalence in Slovakia more than 8 times. The majority of CJD patients admitted consuming sheep brain. Most patients lived in small secluded villages with rather common familial intermarriage. CJD affected both sexes equally. All patients were prior to the disease mentally normal individuals. Shortly after the onset of CJD their mental status deteriorated remarkably with an average survival rate of 3.6 months.

PMID: 12690798

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12690798&dopt=Abstract

------------------------------------------------------------------------

1: Eur J Epidemiol 1991 Sep;7(5):520-3

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=PubMed&cmd=Display&dopt=pubmed_pubmed&from_uid=1761109

"Clusters" of CJD in Slovakia: the first laboratory evidence of scrapie.

Mitrova E, Huncaga S, Hocman G, Nyitrayova O, Tatara M.

Institute of Preventive and Clinical Medicine, Bratislava.

Epidemic-like occurrence of Creutzfeldt-Jakob disease was observed in 1987 in Slovakia (Orava). Search for the cause of CJD focus indicated a coincidence of genetic and environmental risks in clustering patients. Since Spongiform Encephalopathies might be transmitted orally, (Bovine Spongiform Encephalopathy), the possibility of zoonotic source of CJD cases in Orava was also considered. A deficient knowledge about the occurrence of scrapie in Slovakia stimulated an examination of sheep with signs of CNS disorders in two flocks of Valasky breed in Orava. In one flock, neurohistopathological examination revealed in sheep brains lesions characteristic for scrapie. Frozen brain tissue of these animals were used for the detection of scrapie associated fibrils. They were found in 2 animals from the same flock. This is the first laboratory confirmation of scrapie in Czecho-Slovakia. The possible epidemiological and economical implications are emphasized.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1761109&dopt=Abstract

STATEMENT OF DR HELEN GRANT MD FRCP ISSUED 13/05/1999

BSE INQUIRY

http://www.bseinquiry.gov.uk/files/ws/s410.pdf

http://www.bseinquiry.gov.uk/files/ws/s410x.pdf

http://www.bseinquiry.gov.uk/evidence/ws/ws8.htm

CWD to CJD in humans (why not?), as easy as BSE/Scrapie;

The EMBO Journal, Vol. 19, No. 17 pp. 4425-4430, 2000 © European Molecular Biology Organization

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease

G.J. Raymond1, A. Bossers2, L.D. Raymond1, K.I. O?Rourke3, L.E. McHolland4, P.K. Bryant III4, M.W. Miller5, E.S. Williams6, M. Smits2 and B. Caughey1,7

1NIAID/NIH Rocky Mountain Laboratories, Hamilton, MT 59840, 3USDA/ARS/ADRU, Pullman, WA 99164-7030, 4USDA/ARS/ABADRL, Laramie, WY 82071, 5Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526-2097, 6Department of Veterinary Sciences, University of Wyoming, Laramie, WY 82070, USA and 2ID-Lelystad, Institute for Animal Science and Health, Lelystad, The Netherlands 7Corresponding author e-mail: bcaughey@nih.gov Received June 7, 2000; revised July 3, 2000; accepted July 5, 2000.

Abstract

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease-resistant PrP (PrP-res) of one species induces the in vitro conversion of the normal PrP (PrP-sen) of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents. Here we show that the CWD-associated PrP-res (PrPCWD) of cervids readily induces the conversion of recombinant cervid PrP-sen molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrPCWD-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD.

snip...

Clearly, it is premature to draw firm conclusions about CWD passing naturally into humans, cattle and sheep, but the present results suggest that CWD transmissions to humans would be as limited by PrP incompatibility as transmissions of BSE or sheep scrapie to humans. Although there is no evidence that sheep scrapie has affected humans, it is likely that BSE has caused variant CJD in 74 people (definite and probable variant CJD cases to date according to the UK CJD Surveillance Unit). Given the presumably large number of people exposed to BSE infectivity, the susceptibility of humans may still be very low compared with cattle, which would be consistent with the relatively inefficient conversion of human PrP-sen by PrPBSE. Nonetheless, since humans have apparently been infected by BSE, it would seem prudent to take reasonable measures to limit exposure of humans (as well as sheep and cattle) to CWD infectivity as has been recommended for other animal TSEs.

snip...


http://www.emboj.org/current.shtml


Scrapie to Humans USA?

1: Neuroepidemiology. 1985;4(4):240-9.

Sheep consumption: a possible source of spongiform encephalopathy in humans.

Davanipour Z, Alter M, ***el E, Callahan M.

A fatal spongiform encephalopathy of sheep and goats (scrapie) shares many characteristics with Creutzfeldt-Jakob disease (CJD), a similar dementing illness of humans. To investigate the possibility that CJD is acquired by ingestion of contaminated sheep products, we collected information on production, slaughtering practices, and marketing of sheep in Pennsylvania. The study revealed that sheep were usually marketed before central nervous system signs of scrapie are expected to appear; breeds known to be susceptible to the disease were the most common breeds raised in the area; sheep were imported from other states including those with a high frequency of scrapie; use of veterinary services on the sheep farms investigated and, hence, opportunities to detect the disease were limited; sheep producers in the area knew little about scrapie despite the fact that the disease has been reported in the area, and animal organs including sheep organs were sometimes included in processed food. Therefore, it was concluded that in Pennsylvania there are some 'weak links' through which scrapie-infected animals could contaminate human food, and that consumption of these foods could perhaps account for spongiform encephalopathy in humans. The weak links observed are probably not unique to Pennsylvania.

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3915057&dopt=Abstract

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.

PMID: 6997404

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

Libyan Jews

A countrywide search for CJD in Israel uncovered 29 cases with onset between 1963-72.25 The incidence in various ethnic groups varied from 0.4 to 1.9 per million, except among Jewish immigrants from Libya, among whom the incidence was 31.3 per million. The eating of lightly cooked sheep brain, a delicacy amongst Mediterranean Jews, was at first postulated to be responsible, but the incidence of CJD amongst other ethnic groups with the same dietary habits was only 1-2 cases per million of the population.

The Chilean cluster

A cluster in Chile, involving four cases of histologically-proven CJD in three farmers and one housewife, occurred in 1982-3. The four lived in small rural communities situated within a 20-km radius of the town of Chillian. They were unrelated and did not know each other. Their life long dietary habits are of interest, but uncertain relevance. They all consumed uncooked sheep blood and poorly-cooked sheep blood and brain.

http://qjmed.oxfordjournals.org/cgi/content/full/93/9/617

Mutation of the prion protein in Libyan Jews with Creutzfeldt-Jakob disease Article Abstract:

Creutzfeldt-Jakob disease (CJD) is a neurodegenerative disease of a class referred to as the spongiform encephalopathies. The disease can be transmitted experimentally and has also been transmitted accidentally. The causative agent in these transmissions is unlike any well characterized infectious agent and is referred to by many as a prion. Creutzfeldt-Jakob disease may occur in families, but is usually sporadic. The incidence of Creutzfeldt-Jakob disease is about one or two cases per million people. However, among Jews from Libya, the incidence is 100 times higher. Many possible explanations have been put forward to account for this unusually high incidence, but none has sustained any scrutiny. One of the more popular notions was that the eating of sheep brains, a popular delicacy in the region, infected people with scrapie, a prion disease of sheep similar to CJD. However, the eating of sheep brain is popular throughout the Mediterranean, and cannot explain the specificity of the increased incidence to Libyan Jews. Mediterranean sheep, if anything, have a lower rate of scrapie than other areas of Europe and North America. A study was undertaken to determine if the increased incidence of CJD among these people might be accounted for by genetic factors. The prion protein genes were analyzed in 11 Libyan Jews with Creutzfeldt-Jakob disease. Investigation of one patient revealed that a mutation had occurred in the 200th codon of the gene, that is, the 200th set of three DNA bases. The net result of this change would be to substitute a lysine for glutamine in the resulting protein. After this mutation was identified, it was confirmed in the other 10 Libyan patients. It is interesting to note that the mutation was not present in a Moroccan Jew with CJD. The results suggest that the increased incidence of Creutzfeldt-Jakob disease in this population is the result of a gene carried by this group. In eight of the present cases, a family history of CJD could be confirmed. Although not all families were cooperative in providing information of the ancestral heritage, all the families for which such information was available could be traced to Djerba, which is an island off the coast of Tunisia. (Consumer Summary produced by Reliance Medical Information, Inc.)

author: Scarlato, Guglielmo, Prusiner, Stanley B., Hsiao, Karen, Meiner, Zeev, Kahana, Esther, Cass, Carin, Kahana, Irit, Avrahami, Dana, Abramsky, Oded, Gabizon, Ruth Publisher: Massachusetts Medical Society Publication Name: The New England Journal of Medicine Subject: Health ISSN: 0028-4793 Year: 1991

http://www.faqs.org/abstracts/Health/Mutation-of-the-prion-protein-in-Libyan-Jews-with-Creutzfeldt-Jakob-disease.html

Friday, August 22, 2008 Creutzfeldt Jakob Disease and Veterans and how they are treated at death

They worry that he may have gotten it from eating sheep brains locals served to soldiers as an honor in Oman two years ago.

http://creutzfeldt-jakob-disease.blogspot.com/2008/08/creutzfeldt-jakob-disease-and-veterans.html


SCRAPIE DETECTED IN ANOTHER GOAT USA UPDATE MARCH 2009

http://scrapie-usa.blogspot.com/2009/04/scrapie-detected-in-another-goat-usa.html

Full Scientific Reports

Experimental oral transmission of United States origin scrapie to neonatal sheep

Amir N. Hamir1, Robert A. Kunkle, Justin J. Greenlee and Juergen A. Richt Correspondence: 1Corresponding Author: Amir N. Hamir, National Animal Disease Center, ARS, USDA, 2300 Dayton Avenue, PO Box 70, Ames, IA 50010. amir.hamir@ars.usda.gov

Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. The current study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrPSc) by immunohistochemistry and Western blot in tissues of genetically susceptible and resistant neonatal lambs inoculated with pooled brain homogenates from 13 U.S. origin scrapie-affected ewes. Nine Suffolk lambs with genotypes AA/RR/QQ (n = 5) and AA/RR/QR (n = 4) at codons 136, 154, and 171, respectively) were orally inoculated, within 12 hr of birth, with 1 ml of a 10% (w/v) brain homogenate prepared from scrapie-affected sheep brains. Inoculated animals were euthanized when advanced clinical signs of scrapie were observed. All QQ sheep developed clinical signs of scrapie, with a mean survival time of 24 months. Spongiform lesions in the brains and PrPSc deposits in the central nervous system and lymphoid tissues were present in these sheep. None of the QR sheep succumbed to the disease. A previous study that used a larger volume (30 ml of 10% brain suspension) of the same inoculum in 4-month-old Suffolk lambs of susceptible genotype documented longer survival periods (average 32 months), and only 5 of 9 inoculated sheep developed scrapie. Findings of this study suggest that orally exposed neonatal lambs of a susceptible (QQ) genotype exhibit a higher attack rate and shorter incubation period than older (4-month-old) lambs exposed to a larger dose (30x) of the same inoculum.

Key Words: Immunohistochemistry . neonatal sheep . scrapie . spongiform encephalopathy . Western blot

http://jvdi.org/cgi/content/abstract/21/1/64?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=prion&searchid=1&FIRSTINDEX=0&volume=21&issue=1&resourcetype=HWCIT

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://www.bseinquiry.gov.uk/files/yb/1994/02/07002001.pdf

Title: Characterization of a U.S. Sheep Scrapie Isolate with Short Incubation Time

Authors

Hamir, Amirali Richt, Juergen Kunkle, Robert Greenlee, Justin Bulgin, M - UNIVERSITY OF IDAHO Gregori, L - VA MEDICAL CENTER, MD Rohwer, R - VA MEDICAL CENTER, MD

Submitted to: Veterinary Pathology Publication Type: Peer Reviewed Journal Publication Acceptance Date: April 16, 2009 Publication Date: N/A

Interpretive Summary: Scrapie is a naturally occurring fatal disease of sheep and goats. In a previous study it was shown that sheep inoculated with US scrapie inoculum (No. 13-7) induced terminal disease within an average of 19 months. We have since produced an inoculum, No. X124 from pooled brains of US origin sheep scrapie, that results in incubations nearly 3 fold shorter. The present study documents laboratory findings in tissues of sheep inoculated with No. X124. All inoculated sheep developed clinical disease and were euthanized within an average of 7.7 months post inoculation (MPI). Sheep that were genetically susceptible developed the disease faster (within 6 months). Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was supposed to be highly resistant to scrapie. This indicates that inoculum No. X124 appears to be more virulent than inoculum No. 13-7. Importantly this strain of scrapie represents a significant development in that it provides a natural model that requires less than 25 percent of the time for the disease to develop, thus enabling a faster pace for research investigating prion disease pathogenesis and inactivation. Technical Abstract: Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US origin sheep scrapie, that results in incubations nearly 3 fold shorter. The present study documents clinicopathological findings and the distribution of abnormal prion proteins (PrP**Sc) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanized within an average of 7.7 months post-inoculation (MPI). Sheep that had VV or AV at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=230885

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.

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

snip...

A The Present Position with respect to Scrapie

A1 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.

snip...

76/10.12/4.6

http://www.bseinquiry.gov.uk/files/yb/1976/10/12004001.pdf

Epidemiology of Scrapie in the United States 1977

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

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

Like lambs to the slaughter 31 March 2001 by Debora MacKenzie Magazine issue 2284

FOUR years ago, Terry Singeltary watched his mother die horribly from a degenerative brain disease. Doctors told him it was Alzheimer's, but Singeltary was suspicious. The diagnosis didn't fit her violent symptoms, and he demanded an autopsy. It showed she had died of sporadic Creutzfeldt-Jakob disease.

Most doctors believe that sCJD is caused by a prion protein deforming by chance into a killer. But Singeltary thinks otherwise. He is one of a number of campaigners who say that some sCJD, like the variant CJD related to BSE, is caused by eating meat from infected animals. Their suspicions have focused on sheep carrying scrapie, a BSE-like disease that is widespread in flocks across Europe and North America.

Now scientists in France have stumbled across new evidence that adds weight to the campaigners' fears. To their complete surprise, the researchers found that one strain of scrapie causes the same brain damage in ...

http://www.newscientist.com/article/mg16922840.300-like-lambs-to-the-slaughter.html

http://nor-98.blogspot.com/

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


Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518

Wednesday, July 1, 2009

Nor98 scrapie identified in the United States J Vet Diagn Invest 21:454-463 (2009)

http://nor-98.blogspot.com/2009/07/nor98-scrapie-identified-in-united.html


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