Volume 24, Number 12—December 2018
Research
Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway
Laura Pirisinu, Linh Tran, Barbara Chiappini, Ilaria Vanni, Michele A. Di Bari, Gabriele Vaccari, Turid Vikøren, Knut Ivar Madslien, Jørn Våge, Terry Spraker, Gordon Mitchell, Aru Balachandran, Thierry Baron, Cristina Casalone, Christer M. Rolandsen, Knut H. Røed, Umberto Agrimi, Romolo Nonno, and Sylvie L. BenestadComments to Author
Author affiliations: Istituto Superiore di Sanità, Rome, Italy (L. Pirisinu, B. Chiappini, I. Vanni, M.A. Di Bari, G. Vaccari, U. Agrimi, R. Nonno); Norwegian Veterinary Institute, Oslo, Norway (L. Tran, T. Vikøren, K. Madslien, J. Våge, S.L. Benestad); Colorado State University, Fort Collins, Colorado, USA (T. Spraker); Canadian Food Inspection Agency, Ottawa, Ontario, Canada (G. Mitchell, A. Balachandran); Anses Lyon Unité “Maladies Neuro-Dégénératives”, Lyon, France (T. Baron); Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Torino, Italy (C. Casalone); Norwegian Institute for Nature Research, Trondheim, Norway (C.M. Rolandsen); Norwegian University of Life Sciences, Faculty of Veterinary Science, Oslo (K.H. Røed) Suggested citation for this article
Abstract
Chronic wasting disease (CWD) persists in cervid populations of North America and in 2016 was detected for the first time in Europe in a wild reindeer in Norway. We report the detection of CWD in 3 moose (Alces alces) in Norway, identified through a largescale surveillance program. The cases occurred in 13–14-year-old female moose, and we detected an abnormal form of prion protein (PrPSc) in the brain but not in lymphoid tissues. Immunohistochemistry revealed that the moose shared the same neuropathologic phenotype, characterized by mostly intraneuronal deposition of PrPSc. This pattern differed from that observed in reindeer and has not been previously reported in CWD-infected cervids. Moreover, Western blot revealed a PrPSc type distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. These findings suggest that these cases in moose represent a novel type of CWD.
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Results
CWD was diagnosed in 2 moose in May 2016 in Norway’s Selbu municipality and in 1 moose in October 2017 in Lierne municipality. Selbu and Lierne are respectively located ≈300 and ≈450 km northeast of Nordfjella, where CWD in reindeer was detected in 2016. Norway is populated by several species of wild cervids with varying degrees of overlapping range. Seasonal migrations are common and distances might exceed 150 km (13–15). However, studies tracking global positioning satellite–collared moose have not documented regular seasonal migrations between Selbu and Lierne municipalities, suggesting that these can be considered different moose subpopulations.
We initially detected PrPSc in brain samples by using a rapid test and then confirmed by WB (data not shown) and IHC. Sequencing analysis of the entire PrP coding sequence revealed that the 3 moose had the wild type PrP genotype, homozygous for lysine at codon 109 and for methionine at codon 209 (KK109MM209) (GenBank accession no. MH230115).
Discriminatory PrPSc Immunohistochemistry Show Differences between Reindeer and Moose Thumbnail of Immunohistochemical detection of disease-associated prion protein in brain sections at the level of the obex in cervids with chronic wasting disease, Norway. A–E) Reindeer; F–J) moose. mAbs used were 12B2 (A, F), 9A2 (B, G), L42 (C, H), SAF 84 (D, I), and F99/97.6 (E, J). Staining obtained in the reindeer tissues are similar regardless of mAbs used (A–E). Conversely, for moose tissues, the staining was primarily observed intraneuronally with L42, SAF84, and F99/97.6 (H–J) but was no Figure 1. Immunohistochemical detection of disease-associated prion protein in brain sections at the level of the obex in cervids with chronic wasting disease, Norway. A–E) Reindeer; F–J) moose. mAbs used were 12B2 (A,...
The distribution of PrPSc staining was examined by IHC and compared in the tissues of the 3 moose and the reindeer by using 5 different antibodies (Figure 1). No staining was observed in CWD-negative reindeer and moose independently of the antibody used. The distribution of PrPSc in the reindeer was identical for each of the 5 antibodies and did not differ from the description of PrPSc distribution in North America cervids (16–18). The labeling was most consistent within the gray matter of the medulla oblongata, particularly in the dorsal motor of the vagus nerve (7). The thalamic and brain stem regions of the brain were most affected, with a minimal amount of PrPSc identified dorsal to the corpus callosum.
PrPSc labeling in the moose brains (Figure 1, panels F–J) was clearly different from that of the reindeer (Figure 1, panels A–E). In the moose, after staining with F99/97.6 and L42, PrPSc was almost exclusively observed as intraneuronal aggregates, although intraastrocytic type (multiple small granules scattered in the cytoplasm of astrocyte-resembling cells) and intramicroglial type (1 as single or a few large granules in close proximity to microglia-like nuclei) were also observed in the cerebral cortices and olfactory bulb (Technical Appendix Figure 1). The degree of PrPSc staining was more intensive and appeared more widespread in the neuropil using SAF84.
At the level of the obex, we found stained neurons in all nuclei, whereas the dorsal motor of the vagus nerve was not remarkably stained, as observed in reindeer. The intensity of labeling varied between the 3 moose; no. 2 displayed sparse labeling, no. 3 widespread and abundant labeling, and no. 1 intermediate labeling intensity. We observed PrPSc in all parts of the brain investigated except the cerebellums of moose nos. 1 and 2. A diffuse or discrete punctate staining was observed in the granular layer of the cerebellum of moose no. 3, with stronger staining in some Golgi neurons (Technical Appendix Figure 1, panel B). In all 3 moose, the cortical regions showed laminar staining of neurons in all the cell layers, especially in fusiform-shaped neurons. The neurons of the olfactory tubercle from all 3 also stained strongly, and some glia-associated staining could be observed.
In contrast to the reindeer, the downstream flexible tail mAbs 12B2 and 9A2 did not stain in the moose (Figure 1, panels F and G), suggesting that the moose PrPSc was truncated by endogenous proteases further upstream in the N terminus than was reindeer PrPSc. Contrary to previous findings in reindeer, PrPSc was not detected in the Ln from moose no. 1 or in the Ln and tonsils from moose no. 3 (lymphoid tissues were not available in moose no. 2) by either IHC or ELISA.
PrPSc from Norway Moose Compared with Other CWD Isolates from Canada and Norway Thumbnail of Western blot analysis of PrPres in brains of chronic wasting disease-affected cervids from Norway and Canada. A) Western blot analysis PrPres in brains of moose and reindeer from Norway. Membrane was probed with L42 monoclonal antibodies. Molecular weights (kDa) are indicated on the right. Tissue equivalent loaded per lane was 1 mg. B) Western blot analysis of PrPres from moose isolates from Norway (lanes 6–7) compared with PrPres from chronic wasting disease–affected elk or wapiti Figure 2. Western blot analysis of PrPres in brains of chronic wasting disease-affected cervids from Norway and Canada. A) Western blot analysis PrPres in brains of moose and reindeer from Norway. Membrane was...
We compared the PrPSc features in moose from Norway with those of other CWD isolates from Norway and Canada by discriminatory WB, which enabled comparison of PrPres by epitope mapping with different antibodies. Norway moose PrPres had a lower apparent molecular weight (MW) than PrPres from Norway reindeer (Figure 2, panel A) or from Canada isolates (Figure 2, panel B). This lower MW was explained by the occurrence of more C-terminal cleavage of PrPSc by protease K, as confirmed by the partial loss of the 12B2 epitope (Figure 2, panel B).
Thumbnail of Characterization of PrPres fragments from moose (Alces alces) in Europe by epitope mapping. Mapping with mAbs spanning the whole prion protein enabled the analysis of PrPres in moose samples before (PNGase F–) and after deglycosylation (PNGase F+), based on presence or absence of the epitopes and apparent molecular weight. Solid arrowheads indicate C-terminal fragment of ≈13 kDa fragment (present in both samples and detected with SAF84 mAbs). Open arrowheads indicate C-terminal frag Figure 3. Characterization of PrPres fragments from moose (Alces alces) in Europe by epitope mapping. Mapping with mAbs spanning the whole prion protein enabled the analysis of PrPres in moose samples before (PNGase...
Given the unusual pattern observed in moose isolates from Norway, we further investigated their biochemical characteristics with additional mAbs and by enzymatic deglycosylation (Figure 3). Moose samples showed a main C-terminal fragment of ≈17 kDa, detected with SAF84, L42, and 9A2, and an additional glycosylated C-terminal fragment of ≈13 kDa (CTF13) detected only with SAF84. The N terminal 12B2 epitope was mainly lost, although a small amount of PrPres was still detectable in moose no. 1 (Figure 3) and no. 3 (Technical Appendix Figure 2) with this antibody.
In moose nos. 2 and 3, an additional glycosylated C-terminal fragment of ≈16 kDa (CTF16) was detected by SAF84 and L42 mAbs (Figure 3; Technical Appendix Figure 2). We cannot exclude that a small amount of CTF16 was also present in moose no. 1, given that a weak PrPres fragment of ≈16 kDa was detectable upon deglycosylation and long exposure of blots (Figure 3; Technical Appendix Figure 3). Moose nos. 1 and 3 also had a nonglycosylated internal fragment of ≈10 kDa, cleaved at both N and C termini of PrPSc, which was recognized by using mAbs 9A2 (Figure 3). Moreover, the analysis of PrPres from different neuroanatomic regions showed that the slight differences observed among the 3 moose were not dependent on the area analyzed (Technical Appendix Figure 2).
Comparison of the PrPSc Features of the Norway Moose with Sheep and Cattle Prion Strains from Europe Thumbnail of Bar graph of antibody-signal ratios (y-axis) showing discrimination of the ovine, bovine, moose and reindeer samples (x-axis) analyzed in a study characterizing chronic wasting disease in moose (Alces alces), Norway. The antibody ratio is the L42/12B2 ratio of the chemiluminescence signal relative to the L42/12B2 ratio of the control scrapie loaded in each blot. Bars represent median values of >3 independent determinations; error bars represent the range of observed values. Bars Figure 4. Bar graph of antibody-signal ratios (y-axis) showing discrimination of the ovine, bovine, moose and reindeer samples (x-axis) analyzed in a study characterizing chronic wasting disease in moose (Alces alces), Norway. The...
Thumbnail of Comparison of protease-resistant PrPres from moose (Alces alces) with chronic wasting disease and from sheep with scrapie, Europe. Representative blots show epitope mapping analysis ofPrPres (lane 4, CH1641; lane 5, moose no. 1; lane 6, moose no. 2) in comparison with different ovine transmissible spongiform encephalopathy isolates (lane 1, atypical/Nor98; lane 2, classical scrapie; and lane 3, CH1641). A chronic wasting disease isolate from Canada was loaded as control (lane 7). Th Figure 5. Comparison of protease-resistant PrPres from moose (Alces alces) with chronic wasting disease and from sheep with scrapie, Europe. Representative blots show epitope mapping analysis ofPrPres (lane 4, CH1641; lane 5, moose...
Comparison with ovine and bovine prions was performed to determine the N terminal cleavage of the main PrPres fragment by analyzing the different PrPres fragments in each sample, the MW of these fragments, and the L42/12B2 antibody ratio (Figure 4; Technical Appendix Table). Among ovine prions, classical scrapie and atypical/Nor98 were easily discriminated from moose isolates (Figure 5). Classical scrapie PrPres had a higher MW than moose PrPres, as confirmed by the preservation of 12B2 epitope. As previously observed (19), Nor98 PrPres was cleaved at both the N and C termini, and the characteristic 11–12 kDa band was detected by L42, 9A2, and 12B2 mAbs (Figure 5). In contrast, CH1641 samples showed molecular features partially overlapping with the moose (Figure 5). CH1641 samples showed a PrPres of ≈17 kDa and were accompanied by an additional C-terminal fragment of 13–14 kDa detected by using SAF84 mAbs (20). However, CTF16 and the internal PrPres fragment of 10 kDa could not be detected in CH1641 samples.
Thumbnail of Comparison of protease-resistant core of abnormal form of prion protein from moose (Alces alces) in Europe with chronic wasting disease and from cattle with BSE. Representative blots show epitope mapping analysis of protease-resistant core of abnormal form of prion protein in moose (lane 5, moose no. 1; lane 6, moose no. 2) in comparison with different BSE isolates (lane 2, classical BSE; lane 3, H-type BSE; and lane 4, L-type BSE). A sheep scrapie isolate was loaded as control (lan Figure 6. Comparison of protease-resistant core of abnormal form of prion protein from moose (Alces alces) in Europe with chronic wasting disease and from cattle with BSE. Representative blots show epitope mapping analysis...
Moose PrPSc did not overlap with any type of bovine PrPSc. The lack of the 12B2 epitope in moose PrPres was similar to C-type and L-type atypical BSE, but the 2 bovine prions had neither CTF13, CTF16, nor the internal fragment (Figure 6). H-type atypical BSE showed the CTF13 and the internal fragment similar to moose PrPres, but the main PrPres fragment showed a higher MW and preserved the 12B2 epitope (Figure 6).
The ratio of reactivity obtained with L42 and 12B2 antibodies reflected the N terminal cleavage of the main fragment of PrPSc, enabling confirmation that the differences observed in MW of PrPres actually depend on different N terminal proteinase K cleavage, irrespective of the host species (Figure 4). Values >2 are indicative of BSE-like cleavage, whereas values <1 12b2="" a="" behavior="" better="" bse-like="" compared="" epitope="" in="" indicate="" moose="" of="" preservation="" prpres="" ratio="" respect="" scrapie.="" the="" this="" was="" with="">2). However, moose no. 1 had a ratio lower than moose nos. 2 and 3. The CH1641-like field sample was similar to moose no. 1 in this respect, whereas CH1641 was similar to moose nos. 2 and 3. Finally, the value <1 and="" appendix="" atypical="" bse="" canada="" classical="" compared="" cwd="" div="" for="" from="" h-type="" higher="" in="" isolates="" mw="" observed="" online="" prpres="" reflected="" reindeer="" scrapie="" table="" technical="" their="" with="">
Discussion
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The phenotype variant found in moose from Norway could be hypothetically attributed to host species factors. To address this issue, we directly compared PrPSc characteristics in the Norway moose with those in a Canada moose with CWD. In agreement with the available evidence, we found that the Canada moose PrPSc had features different from Norway moose PrPSc and were indistinguishable from other cervids with classical CWD. This finding suggests that the variant PrPSc type observed in Norway moose could not simply reflect a host species factor. Notably, in both natural and experimental conditions, CWD-affected moose in North America have been reported to display disease features indistinguishable from CWD in other cervids and had detectable PrPSc in lymphoid tissues (21,27).
Species-specific amino acid polymorphisms in the cervid PrP are associated with CWD susceptibility, incubation time, and pathology (28–30). In transmission experiments, atypical features were reported in elk or wapiti and mule deer with genotypes associated with a relative resistance to disease, extension of the incubation period, or both (31,32). Moose PrP is polymorphic at codon 109 (K/Q) and 209 (M/I), combined in 3 alleles: K109M209 (observed in Europe and North America), Q109M209 (observed in Europe), and K109I209 (observed in North America) (33,34). The 3 moose with CWD from Norway had the KK109MM209 genotype, whereas the moose case from Canada used for comparison had KK109II209 genotype. Thus, we cannot exclude that the differences observed between Norway and Canada moose in our study are dependent on differences in PrP genotype. However, a classical CWD phenotype has been reported in naturally (21) and experimentally infected (27) moose with the KK109MM209 genotype, suggesting that a difference at PrP codon 209 is probably not the cause of the variant phenotype observed in moose in Norway. All of these findings suggest that neither the species nor the individual PrP genotypes are likely to have caused the variant phenotypes observed and imply that this variant phenotype could represent a novel CWD strain.
CWD is known to be a highly contagious disease in North America; however, data relating to the disease in moose is sparse and insufficient to understand the epidemiology and the implications of CWD in this species. The apparent low CWD prevalence reported for moose in North America compared with other cervid species might be attributable to the individual social behavior of moose and the minimal habitat overlap between moose and other cervids in areas with CWD. Additionally, surveillance program design, disease variability, and host genetics might influence the prevalence of the disease. Based on the epizootic dynamics in North America, CWD plausibly could have become established in reindeer in Norway more than a decade ago (35). In this scenario, the disease in moose could possibly be linked to the disease observed in reindeer, with strain mutation or phenotype shift putatively caused by interspecific transmission. However, a main cause of strain mutation after interspecies transmission (i.e., PrP amino acid differences between the donor and host species) is not relevant in this case because reindeer and moose share the same PrP primary sequence. An alternative hypothesis could be that moose have a prion disease which is independent of the reindeer epidemic, being either specific to the Norwegian moose or acquired by species other than the reindeer.
The 3 moose were 13, 14, and 13 years of age. Although moose can reach ages beyond 20 years, we consider these moose as old because female moose >10–12 years of age start to show signs of senescence and declining survival and reproduction rates (36,37). The old age of the moose, the absence of lymphoid tissue involvement, and the low disease prevalence observed so far (3 of 10,531 moose tested) could suggest that CWD in moose is less contagious than classical CWD or could represent a spontaneous TSE. The finding that the affected moose were from the same geographic area does not seem to support a spontaneous origin of the disease; however, the actual evidence for geographic clustering could have been biased by oversampling in Trøndelag County, where the first positive moose was detected. Lack of detailed data on the ages of the moose tested so far in different geographic areas prevents any definitive conclusion. Still, the recent detection of a positive moose in Finland, several hundred kilometers from Trøndelag County, might indicate that the disease is not restricted to Norway (38). The ongoing intensive surveillance in Norway and several European Union countries with large moose populations will help to better clarify the actual geographic distribution and prevalence and will be critical for understanding the contagious or spontaneous nature of the disease.
The 3 moose analyzed shared a distinctive IHC pattern, mainly characterized by intraneuronal accumulation of PrPSc, and common PrPSc features, such as the proteinase K N-terminal cleavage and the presence of an additional CTF13 fragment. However, we also observed unexpected differences among the 3 moose. By WB, the CTF16 fragment was observed in moose nos. 2 and 3 but not in moose no. 1, whereas the nonglycosylated internal fragment of 10 kDa was evident in moose nos. 1 and 3 but could not be detected in moose no. 2. Furthermore, we also showed that these differences did not depend on the brain area investigated. We cannot rule out that these slight differences might depend on technical issues rather than represent actual PrPSc variations. The outcome of the ongoing bioassay experiments will help to clarify the meaning of the observed variations.
By comparing the moose PrPSc features with other animal TSEs circulating in Europe, we found no evidence of similarities with bovine and ovine prions. Minimal similarities were observed with CH1641 samples; however, CH1641 cases have not yet been detected in Norway. Bioassay in a large spectrum of rodent models will assist in determining whether these molecular similarities imply biologic association between the atypical CWD in moose and small ruminant CH1641. Transmission studies in several rodent models are underway and will help to clarify whether the different phenotype observed (designated Nor16CWD) could reflect the presence of a new cervid prion strain in moose from Norway.
Dr. Pirisinu is a researcher at the Istituto Superiore di Sanità in Rome, Italy. Her primary research interests include the prion strain characterization and zoonotic potential of animal prion diseases.
Acknowledgments
TUESDAY, JUNE 13, 2017
PRION 2017 CONFERENCE
ABSTRACT
Chronic Wasting Disease in European moose is associated with PrPSc features different from North American CWD
P178 Chronic Wasting Disease in European moose is associated with PrPSc features different from North American CWD
Dr Laura Pirisinu1, Dr Linh Tran2, Dr Gordon Mitchell3, Dr Aru Balachandran3, Dr Thierry Baron4, Dr Cristina Casalone5, Dr Michele Di Bari1, Dr Umberto Agrimi1. Dr Romolo Nonno1, Dr Sylvie Benestad2
1Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita, Rome, Italy, 2Norwegian Veterinary Institute, Oslo, Norway, 3Canadian Food Inspection Agency, National and OlE Reference Laboratory for Scrapie and CWD, Ottawa Laboratory Fallowfield, Ottawa, Canada, 4Neurodeqenerative Diseases Unit, ANSES - French Agency for Food, Environmental and Occupational Health & Safety, Lyon, France, 5Istituto Zooprofilattico Sperimentale del Pietnonte, Liguria e Valle d'Aosta, Turin, Italy
Aims: In 2016, Chronic Wasting Disease (CWD) was detected for the first time in Europe in three wild Norwegian reindeer (Rangifer tarandus tarandus) and in two moose (Alces alces). The biochemical analysis and the immunohistochemical distribution of PrPSc from Norwegian reindeer revealed a pattern similar to North American (NA) isolates1. In this study, we studied the biochemical features of PrPSc from the two CWD cases in Norwegian moose.
Methods: Western blot (WB) analysis of PK-treated PrPSc (PrPres) from Norwegian moose and reindeer isolates was performed according to the ISS discriminatory WB protocol (used in BSE and scrapie Italian surveillance). PrPres fragments were determined by epitope mapping (SAF84, L42, 9A2, 12B2 mAbs), before and after deglycosylation. CWD isolates from Canadian cervids (wapiti, moose and white tailed deer) and a panel of small ruminant and bovine prion strains circulating in Europe were also analysed.
Results: WB analysis with different mAbs showed that PrPres from both Norwegian moose samples was different from that usually associated with CWD in cervids. Indeed, their main C-terminal fragment had a MW lower than the other CWD isolates, and could be discriminated by the absence of the 12B2 epitope. Furthermore, while NA CWD PrPSc is composed of a single PrPres fragment, Norwegian moose samples had an additional C-terminal PrPres fragment of ~13 kDa (CTF13). Among ovine TSEs, classical scrapie and Nor98 were discriminated from both Norwegian moose isolates, while CH1641 samples had molecular features partially overlapping with the moose, i.e. a low MW PrPres and the presence of CTF13. In contrast, moose PrPSc did not overlap with any bovine PrPSc. Indeed, the MW of moose PrPres was lower than H-BSE and similar to C-BSE and L-BSE PrPres, but the two bovine prions lacked additional PrPres fragments.
Conclusions: Unexpectedly, PrPSc from Norwegian moose revealed features substantially different from all other CWD isolates. The PrPSc pattern of Norwegian moose was also different from Canadian moose, suggesting that the variant PrPSc type observed does not simply reflect a host factor and could represent a new CWD strain. Furthermore, PrPSc of Norwegian moose can be easily discriminated from all BSE types, classical scrapie and Nor98, while showing significant overlapping only with CH1641. Bioassay in voles will help to clarify whether the different PrPSc types observed reflect the presence of a new CWD strain in Norwegian moose, and its relationships with known animal TSEs.
References: 1Benestad et al, Vet Res (2016}47:88
PRION 2017 DECIPHERING NEURODEGENERATIVE DISORDERS
please see;
***Our transmission study demonstrates that CH 1641-like scrapie is likely to be more virulent than classical scrapie in cattle.
In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) and the disease phenotype is similar to that seen with experimental strain CH1641.
***Our transmission study demonstrates that CH 1641-like scrapie is likely to be more virulent than classical scrapie in cattle.
P-088 Transmission of experimental CH1641-like scrapie to bovine PrP overexpression mice
Kohtaro Miyazawa1, Kentaro Masujin1, Hiroyuki Okada1, Yuichi Matsuura1, Takashi Yokoyama2
1Influenza and Prion Disease Research Center, National Institute of Animal Health, NARO, Japan; 2Department of Planning and General Administration, National Institute of Animal Health, NARO
Introduction: Scrapie is a prion disease in sheep and goats. CH1641-lke scrapie is characterized by a lower molecular mass of the unglycosylated form of abnormal prion protein (PrpSc) compared to that of classical scrapie. It is worthy of attention because of the biochemical similarities of the Prpsc from CH1641-like and BSE affected sheep. We have reported that experimental CH1641-like scrapie is transmissible to bovine PrP overexpression (TgBoPrP) mice (Yokoyama et al. 2010). We report here the further details of this transmission study and compare the biological and biochemical properties to those of classical scrapie affected TgBoPrP mice.
Methods: The details of sheep brain homogenates used in this study are described in our previous report (Yokoyama et al. 2010). TgBoPrP mice were intracerebrally inoculated with a 10% brain homogenate of each scrapie strain. The brains of mice were subjected to histopathological and biochemical analyses.
Results: Prpsc banding pattern of CH1641-like scrapie affected TgBoPrP mice was similar to that of classical scrapie affected mice. Mean survival period of CH1641-like scrapie affected TgBoPrP mice was 170 days at the 3rd passage and it was significantly shorter than that of classical scrapie affected mice (439 days). Lesion profiles and Prpsc distributions in the brains also differed between CH1641-like and classical scrapie affected mice.
Conclusion: We succeeded in stable transmission of CH1641-like scrapie to TgBoPrP mice. Our transmission study demonstrates that CH 1641-like scrapie is likely to be more virulent than classical scrapie in cattle.
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RESEARCH ARTICLE
Phenotype Shift from Atypical Scrapie to CH1641 following Experimental Transmission in Sheep
Marion M. Simmons*, S. Jo Moore¤a, Richard Lockey¤b, Melanie J. Chaplin, Timm Konold, Christopher Vickery, John Spiropoulos
Animal and Plant Health Agency—Weybridge, Woodham Lane, Addlestone, Surrey, KT15 3NB, United Kingdom
¤a Current address: School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia, 6150, Australia
¤b Current address: University of Southampton, Southampton, SO17 1BJ, United Kingdom * marion.simmons@apha.gsi.gov.uk
Abstract
The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein (PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.
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Despite naturally-occurring atypical scrapie being observed in a range of genotypes, successful experimental transmissions of clinical disease have so far only been reported within a particular homologous donor-recipient genotype model using sheep which are AHQ/AHQ homozygous [8,15,16]. These published transmissions represent part of a large study at APHA which has been running since 2004, investigating the potential transmissibility of atypical scrapie in a range of both homologous and cross-genotype combinations. Here we describe an unexpected and interesting finding from that study where one experimental challenge in which atypical scrapie from an ARR/ARR donor was inoculated intracerebrally into two AHQ/AHQ recipient sheep, and in one of them the resulting disease had a phenotype that was indistinguishable from CH1641 [29], a classical scrapie strain which has some BSE-like Western blot properties.
*** One of these isolates (TR316211) behaved like the CH1641 isolate, with PrPres features in mice similar to those in the sheep brain. From two other isolates (O100 and O104), two distinct PrPres phenotypes were identified in mouse brains, with either high (h-type) or low (l-type) apparent molecular masses of unglycosylated PrPres, the latter being similar to that observed with CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions in the brains of the individual mice. In contrast with BSE, l-type PrPres from "CH1641-like" isolates showed lower levels of diglycosylated PrPres. From one of these cases (O104), a second passage in mice was performed for two mice with distinct PrPres profiles. This showed a partial selection of the l-type phenotype in mice infected with a mouse brain with predominant l-type PrPres, and it was accompanied by a significant increase in the proportions of the diglycosylated band. These results are discussed in relation to the diversity of scrapie and BSE strains.
Subject: more on scrapie/BSE strain CH1641
From: tom
Reply-To: Bovine Spongiform Encephalopathy
Date: Sun, 10 Jan 1999 21:52:05 -0800
Content-Type: text/plain
Parts/Attachments: Parts/Attachments text/plain (37 lines) Reply Reply
Recall a forthcoming J Gen Virol Jan 1999 v80:1 - 4 says there are similarities between BSE and an experimental isolate of natural scrapie, CH1641. This might then be the long-sought missing scrapie strain that could have given rise to the BSE epidemic. It would raise additional questions about the harmlessness to humans of scrapie.
On the other hand, CH1641 happened to be one of the scrapie strains studied very recently by Collinge's group, Neurosci Lett. 1998 Oct 23;255(3):159-62. It did not have the prp-sc type identical to BSE passaged in sheep.
The CH1641 strain is mentioned only twice before in Medline abstracts (though there could be many fulltext mentions), one of these being the original naming of the strain in 1988:
The unusual properties of CH1641, a sheep-passaged isolate of scrapie.
Foster JD, Dickinson AG Vet Rec 1988 Jul 2;123(1):5-8
An isolate of scrapie designated CH1641 was identified from a natural case of scrapie in a Cheviot sheep by passage in sheep and goats. It has not been possible to transmit scrapie to mice from this source. The Sip gene which controls the incubation periods of experimental scrapie in Cheviot sheep has two alleles; sA which shortens and pA which lengthens the incubation periods of most strains of scrapie after the first experimental injection in sheep (the A group of strains). The CH1641 isolate differs from them in that the alleles of Sip act in the opposite way, with incubation being shorter in the pA homozygotes. There is some evidence that one or more genes, in addition to Sip, may be implicated in the control of scrapie incubation in sheep and the possibility of a carrier infection with CH1641 is also discussed.
Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period.
J Gen Virol 1996 Nov;77 ( Pt 11):2885-91 Published erratum appears in J Gen Virol 1997 Mar;78(Pt 3):697 Goldmann W, Martin T, Foster J, Hughes S, Smith G, Hughes K, Dawson M, Hunter N
Age at disease onset and rate of progression of transmissible spongiform encephalopathies in man, sheep and mice are modulated by the host genome, in particular by the PrP gene and its allelic forms. Analysis of the caprine PrP gene revealed several different alleles. Four PrP protein variants were found, three of which were goat specific with single amino acid changes at codons 142, 143 and 240. The fourth was identical to the most common sheep PrP protein variant (Ala136-Arg154-Gln171). The dimorphism at codon 142 (Ile --> Met) appeared to be associated with differing disease incubation periods in goats experimentally infected with isolates of bovine spongiform encephalopathy, sheep scrapie CH1641 or sheep-passaged ME7 scrapie.
TSE PRION UPDATE USA 2012
re-BSE in goats can be mistaken for scrapie
Wednesday, January 18, 2012
BSE IN GOATS CAN BE MISTAKEN FOR SCRAPIE
February 1, 2012
Wednesday, January 18, 2012
Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie
Journal of Neuropathology & Experimental Neurology:
February 2012 - Volume 71 - Issue 2 - p 140–147
Monday, March 21, 2011
Sheep and Goat BSE Propagate More Efficiently than Cattle BSE in Human PrP Transgenic Mice
snip...
On the other hand, this component would not be distinguishable from bovine-passaged BSE prions due to the current limits of the standard biological methods and/or the molecular tools employed here to characterize prion strains. Whatever the mechanism, the notion that a passage through an intermediate species can profoundly alter prion virulence for the human species has important public-health issues, regarding emerging and/or expanding TSEs, like atypical scrapie or CWD.
snip...
Taken all together, our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, which has important implications on public and animal health policies. On one hand, although the exact magnitude and characteristic of the vCJD epidemic is still unclear, its link with cattle BSE is supported by strong epidemiological ground and several experimental data. On the other hand, the molecular typing performed in our studies, indicates that the biochemical characteristics of the PrPres detected in brains of our sheep and goat BSE-inoculated mice seem to be indistinguishable from that observed in vCJD. Considering the similarity in clinical manifestation of BSE- and scrapie-affected sheep [48], a masker effect of scrapie over BSE, as well as a potential adaptation of the BSE agent through subsequent passages, could not be ruled out. As BSE infected sheep PrPSc have been detected in many peripheral organs, small ruminant-passaged BSE prions might be a more widespread source of BSE infectivity compared to cattle [19], [49], [50].
*** This fact is even more worrying since our transmission studies suggest that apparently Met129 human PrP favours a BSE agent with ovine rather than a bovine sequence. Finally, it is evident that, although few natural cases have been described and so far we cannot draw any definitive conclusion about the origin of vCJD, we can not underestimate the risk of a potential goat and/or sheep BSE agent.
snip...
Technical Abstract:
Prion strains may vary in their ability to transmit to humans and animals. Few experimental studies have been done to provide evidence of differences between U.S. strains of scrapie, which can be distinguished by incubation times in inbred mice, microscopic lesions, immunoreactivity to various antibodies, or molecular profile (electrophoretic mobility and glycoform ratio). Recent work on two U.S. isolates of sheep scrapie supports that at least two distinct strains exist based on differences in incubation time and genotype of sheep affected. One isolate (No. 13-7) inoculated intracerebrally caused scrapie in sheep AA at codon 136 (AA136) and QQ at codon 171 (QQ171) of the prion protein in an average of 19 months post-inoculation (PI) whereas a second isolate (No. x124) caused disease in less than 12 months after oral inoculation in AV136/QQ171 sheep. Striking differences were evident when further strain analysis was done in R111, VM, C57Bl6, and C57Bl6xVM (F1) mice. No. 13-7 did not induce disease in any mouse strain at any time post-inoculation (PI) nor were brain tissues positive by western blot (WB). Positive WB results were obtained from mice inoculated with isolate No. x124 starting at day 380 PI. Incubation times averaged 508, 559, 601, and 633 days PI for RIII, C57Bl6, VM, and F1 mice, respectively. Further passage will be required to characterize these scrapie strains in mice. This work provides evidence that multiple scrapie strains exist in U.S. sheep.
One of these isolates (TR316211) behaved like the CH1641 isolate, with PrPres features in mice similar to those in the sheep brain. From two other isolates (O100 and O104), two distinct PrPres phenotypes were identified in mouse brains, with either high (h-type) or low (l-type) apparent molecular masses of unglycosylated PrPres, the latter being similar to that observed with CH1641, TR316211, or BSE. Both phenotypes could be found in variable proportions in the brains of the individual mice. In contrast with BSE, l-type PrPres from "CH1641-like" isolates showed lower levels of diglycosylated PrPres. From one of these cases (O104), a second passage in mice was performed for two mice with distinct PrPres profiles. This showed a partial selection of the l-type phenotype in mice infected with a mouse brain with predominant l-type PrPres, and it was accompanied by a significant increase in the proportions of the diglycosylated band. These results are discussed in relation to the diversity of scrapie and BSE strains.
In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) and the disease phenotype is similar to that seen with experimental strain CH1641.
snip...see ;
Thursday, July 14, 2011
Histopathological Studies of "CH1641-Like" Scrapie Sources Versus Classical Scrapie and BSE Transmitted to Ovine Transgenic Mice (TgOvPrP4)
SHEEP AND BSE
PERSONAL AND CONFIDENTIAL
SHEEP AND BSE
A. The experimental transmission of BSE to sheep.
Studies have shown that the ''negative'' line NPU flock of Cheviots can be experimentally infected with BSE by intracerebral (ic) or oral challenge (the latter being equivalent to 0.5 gram of a pool of four cow brains from animals confirmed to have BSE).
RB264
BSE - TRANSMISSION STUDIES
Wednesday, January 18, 2012
Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie
Journal of Neuropathology & Experimental Neurology:
February 2012 - Volume 71 - Issue 2 - p 140–147
***however in 1 C-type challenged animal, Prion 2015 Poster Abstracts
S67 PrPsc was not detected using rapid tests for BSE.
***Subsequent testing resulted in the detection of pathologic lesion in unusual brain location and PrPsc detection by PMCA only.
*** IBNC Tauopathy or TSE Prion disease, it appears, no one is sure ***
Posted by Terry S. Singeltary Sr. on 03 Jul 2015 at 16:53 GMT
Discussion: The C, L and H type BSE cases in Canada exhibit molecular characteristics similar to those described for classical and atypical BSE cases from Europe and Japan.
*** This supports the theory that the importation of BSE contaminated feedstuff is the source of C-type BSE in Canada.
*** It also suggests a similar cause or source for atypical BSE in these countries. ***
see page 176 of 201 pages...tss
*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply;
FINLAND MOOSE FOUND DEAD IN FOREST WITH CHRONIC WASTING DISEASE 8.3.2018 12:56
The chronic wasting disease (CWD) has been found in a moose or European elk (Alces alces) for the first time ever in Finland. The disease was diagnosed in Kuhmo in a 15-year old moose that had died naturally. The results of the analyses carried out by Finnish Food Safety Authority Evira have been verified by a EU reference laboratory. Species of the deer family, known as “cervids”, can suffer from the chronic wasting disease, and it is always fatal. The disease is not known to have been contracted by people.
Norway was before this case the only European country where CWD has been diagnosed. The monitoring of the occurrence of the disease was intensified from the beginning of 2018 in Finland and five other EU Member States.
In Finland, the occurrence of the disease has been studied already since 2003. None of the ca. 2 500 samples analysed so far had tested positive for the disease. The monitoring of the disease will now be further intensified in the Kuhmo and Kainuu region. Hunters are going to be provided with more instructions before the start of the next hunting season, if appropriate.
The chronic wasting disease is not known to have been contracted by people. Moose meat is safe to eat and no restrictions are imposed on the sales and exportation of meat of animals of the deer family. As a precautionary measure the export of live animals of the deer family to other countries will be discontinued for now.
CWD is a slowly progressing disease of deer, elk, reindeer, and moose which always leads to death. The chronic wasting disease is a prion disease and related to the BSE (bovine spongiform encephalopathy) and other TSE diseases (transmissible spongiform encephalopathy). The disease is common in North America. The moose found in Kuhmo did not suffer from the North American, highly contagious form of the chronic wasting disease. The disease seems to resemble most the form of cervid TSE diagnosed in Norway, which appears to be found incidentally in individual animals of the deer family.
For more information, please contact:
Leena Räsänen, Director, tel. +358 50 388 6518 (Food Safety)
Terhi Laaksonen, Head of Unit, tel. +358 40 159 5812 (Control of Animal Diseases)
Sirkka-Liisa Korpenfelt, Senior Resarcher, tel. + 358 50 351 0308 (Laboratory Analyses)
Antti Oksanen, Research Professor, tel. +358 44 561 6491 (Wild Animal Diseases)
Kajsa Hakulin, Ministerial Advisor, Ministry of Agriculture and Forestry, tel. +358 295 162361 (National and EU Legislation)
SATURDAY, MARCH 10, 2018
Chronic Wasting Disease CWD TSE Prion Goes Global Finland Falls, Behind Norway and S. Korea
FINLAND REPORTS FIRST CASE OF CHRONIC WASTING DISEASE CWD TSE PRION IN A moose or European elk (Alces alces)
THURSDAY, OCTOBER 25, 2018
***> Norway New additional requirements for imports of hay and straw for animal feed from countries outside the EEA due to CWD TSE Prion
SATURDAY, NOVEMBER 10, 2018
Canada Saskatchewan New Case Of CWD TSE PRION Detected Near Melfort Released on November 7, 2018
SUNDAY, OCTOBER 28, 2018
Alberta, Canada 2017 Fall CWD TSE Prion Surveillance Results
TUESDAY, OCTOBER 30, 2018
Québec federal officials found third case Chronic Wasting Disease CWD domestic red deer on a farm reported Boileau
SATURDAY, NOVEMBER 10, 2018
***> cwd, bse, scrapie, cjd, tse prion updated November 10 2018
USA MAD COW CASE 2018 FLORIDA
WEDNESDAY, SEPTEMBER 26, 2018
JAVMA In Short Update USDA announces detection of atypical BSE
WEDNESDAY, OCTOBER 24, 2018
Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy
TUESDAY, AUGUST 07, 2018
Passage of scrapie to deer results in a new phenotype upon return passage to sheep
TUESDAY, APRIL 24, 2018
ARS Research atypical Nor98 and Michigan Scrapie, CWD, CJD and mad cow feed
Research Project: Genetic Impact and Improved Diagnostics for Sheep and Goat Transmissible Spongiform Encephalopathies
THURSDAY, NOVEMBER 01, 2018
National Scrapie Eradication Program September 2018 Monthly Report Fiscal Year 2018 October 15, 2018
THURSDAY, OCTOBER 04, 2018
Cervid to human prion transmission 5R01NS088604-04 Update
Terry S. Singeltary Sr.
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