Ju¨ rgen A. Richt1¤*, S. Mark Hall2 1 National Animal Disease Center, United States Department of Agriculture, Agriculture Research Service, Ames, Iowa, United States of America, 2 National Veterinary Services Laboratories, Pathobiology Laboratory, Animal and Plant Health Inspection Service, United States Department of Agriculture, Ames, Iowa, United States of America
Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle.
Citation: Richt JA, Hall SM (2008) BSE Case Associated with Prion Protein Gene Mutation. PLoS Pathog 4(9): e1000156. doi:10.1371/journal.ppat.1000156 Editor: David Westaway, University of Alberta, Canada
Received June 5, 2008; Accepted August 15, 2008; Published September 12, 2008
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by the USDA-ARS-National Animal Disease Center (NADC) and USDA-APHIS-National Veterinary Services Laboratories (NVSL) and by the NIAID-NIH PO1 AI 77774-01 ‘‘Pathogenesis, Transmission and Detection of Zoonotic Prion Diseases’’.
Competing Interests: Patent pending: Dr. Ju¨ rgen A. Richt submitted a patent application entitled ‘‘Novel Polymorphism in Bovine Prion Protein Gene Sequence’’ (Docket Number 0078.06; Serial No. 11/787,784) on April 18, 2006.
* E-mail: firstname.lastname@example.org
¤ Current address: Kansas State University, College of Veterinary Medicine, DM/P, Manhattan, Kansas, United States of America
Bovine spongiform encephalopathy (BSE or Mad Cow Disease), a transmissible spongiform encephalopathy (TSE) or prion disease of cattle, was first discovered in the United Kingdom in 1986. BSE is most likely the cause of a human prion disease known as variant Creutzfeldt Jakob Disease (vCJD). In this study, we identified a novel mutation in the bovine prion protein gene (Prnp), called E211K, of a confirmed BSE positive cow from Alabama, United States of America. This mutation is identical to the E200K pathogenic mutation found in humans with a genetic form of CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in ‘‘the approximately 10-year-old cow’’ carrying the E221K mutation.
Transmissible spongiform encephalopathy (TSE) agents induce fatal neurodegenerative diseases in humans and in some mammalian species . According to the prion-only hypothesis, infectious prions are composed of an abnormal isoform of a hostencoded glycoprotein, called prion protein (PrPc). The diseaseassociated form, PrPd, is derived from PrPc by a post-translational mechanism that involves conformational change . Human TSEs include Creutzfeldt–Jakob disease (CJD), Gerstmann– Stra¨ussler–Scheinker syndrome, Kuru and Fatal Familial Insomnia . In animals, several distinct TSE diseases are recognized: Scrapie in sheep and goats, transmissible mink encephalopathy in mink, chronic wasting disease in cervids, and bovine spongiform encephalopathy (BSE) in cattle . BSE was first detected in 1986 in the United Kingdom and is the most likely cause of variant CJD (vCJD) in humans. The origin of the original case(s) of BSE still remains an enigma. Hypotheses include (i) sheep- or goat-derived scrapie-infected tissues included in meat and bone meal fed to cattle, (ii) a previously undetected sporadic or genetic bovine TSE contaminating cattle feed or (iii) origination from a human TSE through animal feed contaminated with human remains . This study will provide support to the hypothesis that BSE originated from a previously undetected genetic bovine TSE contaminating cattle feed in the U.K.
Here we report a case of bovine BSE associated with a mutation within the prion protein gene (Prnp) sequence, not previously described for the bovine Prnp. The animal (called ‘‘U.S. BSE Alabama’’) was an approximately 10 year-old red crossbred (Bos indicus6Bos taurus) hybrid beef cow from Alabama (see Materials and Methods). The ELISA-based BSE test (see Materials and Methods) on brainstem from this animal was repeated five times and revealed a strongly positive reaction with mean optical density (OD) value of 2.4060.57, whereas the OD value of bovine control obex was ,0.04.
Confirmatory BSE tests employing Western Blot (WB) and immunohistochemical (IHC) analyses for presence of PrPd were subsequently performed (see Materials and Methods). Immunoblots (Figure 1A) revealed (i) presence of PrPd and (ii) intensity of reaction with antibody P4 similar as with antibody 6H4 at identical milligram equivalent amounts. This reaction pattern was described as being unusual or atypical for BSE . Molecular weight analysis revealed unglycosylated and monoglycosylated isoforms of PrPd migrated with an apparent molecular weight higher than respective isoforms of classical or C-type BSE isolates (Figure 1A). A similar migration pattern was observed for the U.S. BSE 2004 isolate, an H-type BSE isolate . Obvious lesions of spongiform encephalopathy diagnostic for BSE were not present in the brainstem, however it was positive for the presence of PrPd by IHC (Figure 1B). Distribution of PrPd in the brainstem of this animal was not as uniform or as intense as seen with the C-type U.S. BSE case from 2003 (Figure 1C) . We concluded from these studies, that this animal contracted an H-type BSE phenotype. Recent work on the molecular characterization of cattle PrPd has allowed to define criteria for the identification of atypical BSE cases in cattle, which showed molecular features of the PrPd distinct from the majority of cattle with C-type BSE [4– 6]. There have been two molecular types of unusual BSE isolates described in the literature [5,6]: (i) a type with a lower molecular mass of the unglycosylated isoform (L-type) and (ii) a type with higher molecular mass of the unglycosylated isoform (H-type) when compared to C-type of BSE.
In order to confirm the specimen from this case was of cattle origin and to determine whether the case was associated with a Prnp mutation, the full coding sequence from exon 3 of the Prnp was amplified from DNA isolated from fresh brainstem material (see Materials and Methods) and aligned with known Prnp sequences from cattle, sheep and cervids (Figure 2A,B). The Prnp DNA sequence of this animal is of bovine origin, different from sheep and cervid Prnp sequences (Figure 2A). The sequence was heterozygous on two positions: a synonymous polymorphism on codon 78 (CAA/CAG; Q78Q) and a non-synonymous polymorphism on codon 211 (GAA/AAA; E211K; Figure 2A,B; Table 1). The animal had six copies of the octapeptide repeat region on both of its Prnp alleles (Figure 2A,B). The finding of the E211K mutation is of significant interest because an identical mutation, E200K, at the homologous codon 200 position in human Prnp (Figure 1D) has been described as the most common mutation in humans with genetic CJD .
Our results demonstrate for the first time a potential pathogenic mutation (E211K) within the Prnp gene of a bovine with an H-type BSE phenotype at a position representing the most common mutation in humans (E200K) associated with genetic TSEs . This mutation was not found in the Prnp gene of other North American (1 H-type U.S.; 1 H-type and 1 L-type Canadian) and European (7 H-type and 3 L-type cases) cattle  and a miniature zebu (H-type)  with atypical BSE phenotypes. The functional significance of this finding, however, remains unknown. Importantly, the penetrance of the E200K mutation in humans is very high [7,10]. The origin of atypical BSE cases still remains unexplained. Several hypotheses have been considered including the existence of a previously unrecognized ‘‘sporadic’’ form of a TSE in this species. The detection of the E211K Prnp mutation, known to be pathogenic in humans, in a 10 year old hybrid cow (Bos indicus6Bos taurus) with H-type BSE could provide additional support to the following hypotheses: (i) that U.K. BSE has been acquired from a genetic case or cases of cattle BSE, (ii) that all three etiological forms of human TSEs (sporadic, genetic and infectious) are also present in cattle, and (iii) that BSE started on the Indian subcontinent. However, more data are required to support these hypotheses. It is well known, that large amounts of mammalian protein material were imported from India to the U.K. during the relevant time period (late 1970s and early 1980s) . Therefore it could be speculated that one possible route of contamination of U.K. cattle with BSE was through animal feed containing imported meat and bone meal material contaminated with a case or cases of genetic BSE.
Epidemiological investigations conducted by USDA personnel failed to reveal any evidence of a feed source contaminated with TSE material fed to this animal (http://www.aphis.usda.gov/newsroom/ hot_issues/bse/downloads/EPI_Final5-2-06.pdf). There are several possibilities for the origin of the Prnp K211 allele in animal B14842: (i) it arose de novo in a germ line cell from the U.S. BSE Alabama animal or one of its parents; (ii) it arose as a somatic mutation in the U.S. BSE Alabama animal (rather unlikely), and (iii) it is present in a cattle population or breed yet to be found. Recently, it was determined that the 2-year-old heifer offspring of the U.S. BSE Alabama cow also carries the E211K polymorphism, indicating that the allele is heritable and may persist within the cattle population (11) In a recent epidemiological study which included 6062 cattle from 5 commercial beef processing plants (3892 carcasses) and 2170 registered cattle from 42 breeds., the K211 allele was not detected using a newly developed mass spectrometry assay specific for the E211K variant . These data indicate a rather low prevalence of the E211K variant of less than 1 in 2000 cattle when using Bayesian analysis . This newly developed assay system for K211  will offer the possibility for genetic surveillance of cattle for rare pathogenic mutations that may be associated with BSE.
Materials and Methods
The animal B14842 (called U.S. BSE Alabama case), approximately 10 years old as determined by dentition, was a red crossbred (Bos indicus6Bos taurus) hybrid beef cow found in lateral recumbency on a farm in Alabama. She was euthanized by the attending veterinarian, the brainstem removed for BSE testing and shipped to the Georgia Veterinary Medical Diagnostic Laboratory, where it was found to be positive by a rapid enzyme-linked immunosorbent assay (ELISA)-based BSE test. The brainstem was then forwarded to the National Veterinary Services Laboratories in Ames, IA, USA, for confirmatory testing. The classical BSE case was reported previously .
The rapid BSE test used in the U.S. is the Platelia/TeSeETM ELISA BSE test (Bio-Rad, Hercules, CA, USA). Fresh samples from the brainstem were used for the analysis and the samples were treated with proteinase K (PK) in order to digest the PKsensitive normal prion protein, PrPc.
Brain homogenates from the U.S. BSE Alabama case (animal B14842) were prepared from 1.1 gram of brainstem material and analyzed using the PrionicsH-Check Western Kit (Prionics, Schlieren, Switzerland) with modifications and the OIE-recommended Scrapie Associated Fibril (SAF)-Immunoblot method (http://www. oie.int/eng/normes/mmanual/A_summry.htm). Preparation and analysis of brainstem homogenate using both methods has been described previously . Please note that the brain homogenates were treated with PK (2 U/ml) for 60 minutes at 37uC before Western Blot analysis in order to digest the PK-sensitive PrPc. As positive control samples, BSE-positive brain material from the U.S. BSE cases 2003 and 2004 as well as a sheep scrapie isolate were used. As negative controls, brain material from a BSE-negative cow was used.
Brain tissue was placed in 10% buffered formalin and after a minimum of 4 days of fixation appropriate sections of brainstem in the obex region were put in cassettes and kept in fresh formalin until they were processed for routine paraffin embedding. The procedure was described in detail previously . The IHC results were interpreted as follows: (i) positive for PrPd: pink to red and (ii) background and negative for PrPd: only blue background. As positive controls, slides from the brainstem of a BSE-positive cow, obtained from the United Kingdom and from the U.S. BSE Case 2003 were used. As negative controls, slides from brainstem material of BSE-negative cattle and scrapie-negative sheep were used.
DNA isolation, PCR amplification and sequence analysis Genomic DNA was extracted from 200 ml of a 10% brain homogenate as described previously . The fragment was sequenced in duplicate using the original two primers and two internal primers (available upon request) for a total of 8 reactions. Databases were searched using standard nucleotide-nucleotide BLAST at the National Center for Biotechnology Information Web Site (http://www.ncbi.nlm.nih.gov). The database is a collection of sequences from several sources, including GenBank and Reference Sequence. The nucleotide sequences of the Prnp gene of the U.S. BSE Alabama case was aligned using both CLUSTAL V and CLUSTAL W with the following GENBANK accession numbers: AY335912 (bovine), AY367641 (bovine), AF016227(elk), AY275712 (white-tailed deer), AF166334 (sheep), AJ567986 (sheep), and the Canadian BSE case  using Lasergene version 5.07 software (DNASTAR-Madison WI). Accession number
The GenBank accession number for the Prnp gene of U.S. BSE Alabama case is EU809428.
The authors would like to thank D. Clouser and K. Hassall for their excellent technical support and Dr. M. Kehrli for his encouragement and support. We thank Drs. T. Baron, S. Czub and T. Seuberlich for sharing DNA from atypical BSE cases. The authors also wish to thank the seven State/University Veterinary Diagnostic Laboratories for their efforts in screening U.S. cattle for the presence of BSE that enabled detection of this new allele.
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 U.S. Department of Agriculture. Author Contributions
Conceived and designed the experiments: JAR SMH. Performed the experiments: JAR SMH. Analyzed the data: JAR SMH. Contributed reagents/materials/analysis tools: JAR. Wrote the paper: JAR SMH. References
1. Prusiner SB (1998) The prion diseases. Brain Pathol 8: 499–513.
2. Wells GA, Wilesmith JW (1995) The neuropathology and epidemiology of bovine spongiform encephalopathy. Brain Pathol 1995 5: 91–103.
3. Colchester AC, Colchester NT (2005) The origin of bovine spongiform encephalopathy: the human prion disease hypothesis. Lancet 366: 856–61.
4. Richt JA, Kunkle RA, Alt D, Nicholson EM, Hamir AN, et al. (2007) Identification and characterization of two bovine spongiform encephalopathy cases diagnosed in the United States. J Vet Diagn Invest 19: 142–154.
5. Jacobs JG, Langeveld JP, Biacabe AG, Acutis PL, Polak MP, et al. (2007) Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe. J Clin Microbiol 45: 1821–1829.
6. Baron T, Biacabe AG, Arsac JN, Benestad S, Groschup MH (2007) Atypical transmissible spongiform encephalopathies (TSEs) in ruminants. Vaccine 25: 5625–5630.
7. Kovacs GG, Puopolo M, Ladogana A, Pocchiari M, Budka H, et al. (2005) Genetic prion disease: the EUROCJD experience. Hum Genet 118: 166–174.
8. Clawson ML, Richt JA, Baron T, Biacabe AG, Czub S, et al. (2008) Association of a bovine prion gene haplotype with atypical BSE. PLoS ONE 3: e1830. doi:10.1371/journal.pone.0001830.
9. Seuberlich T, Botteron C, Wenker C, Cafe´-Marcal VA, Oevermann A, et al. (2006) Spongiform encephalopathy in a miniature zebu. Emerg Infect Dis 12: 1950–1953.
10. Kong Q, Surewicz WK, Peterson RB, Zou W, Chen SG, et al. (2004) Inherited Prion Diseases. In: Prusiner SB, ed. Prion Biology and Diseases. New York: Cold Spring Harbor Monograph Series 41. 1050 p.
11. Nicholson EN, Brunelle BW, Richt JA, Kehrli Jr ME, Greenlee JJ (2008) Identification of a heritable polymorphism in bovine PRNP associated with genetic transmissible spongiform encephalopathy: evidence of heritable BSE. PLoS ONE 3: e2912. doi:10.1371/journal.pone.0002912.
12. Heaton MP, Keele JW, Harhay GP, Richt JA, Koohmaraie M, et al. (2008) Prevalence of the prion gene E211K variant in U.S. cattle. BMC Vet Res 4: 25.
13. Coulthart MB, Mogk R, Rancourt JM, Godal DL, Czub S (2003) Prion protein gene sequence of Canada’s first non-imported case of bovine spongiform encephalopathy (BSE). Genome 46: 1005–1009.
Table 1. DNA sequence analysis of codon 211 of the bovine Prnp and codon 200 of the human Prnp. Nucleotide Amino Acid Bovine majority G A A/G A A E211/E211 U.S. 2006 BSE case G A A/A A A E211/K211 Human majority G A G/G A G E200/E200 Human genetic CJD G A G/A A G E200/K200 doi:10.1371/journal.ppat.1000156.t001 Novel Bovine Prion Protein Gene Mutation PLoS
Figure 1. Analysis of brainstem samples from BSE-infected animals employing various methods. (A) Hybrid Immunoblot Analysis using enriched samples: Lanes 1–5: monoclonal antibody 6H4 (raised against human PrP residues 144–152), lanes 8–12 monoclonal antibody P4 (raised against ovine PrP residues 89–104): 1 = sheep scrapie control, 2 mg; 2 = classical BSE (2003 U.S. BSE case), 2 mg; 3 = H-type BSE case (2004 U.S. BSE case), 2 mg; 4 = U.S. BSE Alabama case, 1 mg; 5 = U.S. BSE Alabama case, 2.5 mg; 6,7 = protein weight maker; 8 = U.S. BSE Alabama case, 2.5 mg; 9 = U.S. BSE Alabama case, 1 mg; 10 = H-type BSE case (2004 U.S. BSE case), 2 mg; 11 = classical BSE (2003 U.S. BSE case), 2 mg; 12 = sheep scrapie control, 2 mg. (B) Immunohistochemistry of the U.S. BSE Alabama case (H-type BSE) using PrP-specific monoclonal antibody F99/97.6.1. Brainstem at the level of obex was examined. Bar = 35 mm. (C) Immunohistochemistry of a classical BSE case  using PrP-specific monoclonal antibody F99/97.6.1. Brainstem at the level of obex was examined. Spongiform changes are found in the area with highly PrPd-positive cells. Bar = 90 mm. doi:10.1371/journal.ppat.1000156.g001
Figure 2. Alignment of bovine, ovine, cervid and human Prnp sequences. (A) Nucleotide sequences. Standard single letter codes are used for nucleotides. Y = C or T; R = A or G; K =G or T; W= A or T. Boxed area indicates the 6th octapeptide-repeat of the bovine protein (U.S. BSE Alabama case and sequence AY335912). Additional Prnp sequences are as follows: AJ567986 (sheep), AF016227 (elk), Hsap M13899 (human, normal) and Hsap PRNPvar [human, variant; see ]. (B) Amino acid sequences. Standard IUPAC single letter codes are used for amino acids. Codon numbering refers to the most common six-copy octapeptide repeat allele for Bos Taurus. Boxed area indicates the 6th octapeptide repeat of the bovine protein [animals B14842  and AY335912]. AJ567986 (sheep), AF016227 (elk), Hsap M13899 (human, normal) and Hsap PRNPvar [human, variant; see ] each contain a 5 octapeptide repeat region in the protein. doi:10.1371/journal.ppat.1000156.g002
BANNED MAD COW FEED IN COMMERCE IN ALABAMA
Date: September 6, 2006 at 7:58 am PST PRODUCT
a) EVSRC Custom dairy feed, Recall # V-130-6;
b) Performance Chick Starter, Recall # V-131-6;
c) Performance Quail Grower, Recall # V-132-6;
d) Performance Pheasant Finisher, Recall # V-133-6.
CODE None RECALLING FIRM/MANUFACTURER Donaldson & Hasenbein/dba J&R Feed Service, Inc., Cullman, AL, by telephone on June 23, 2006 and by letter dated July 19, 2006. Firm initiated recall is complete.
Dairy and poultry feeds were possibly contaminated with ruminant based protein.
VOLUME OF PRODUCT IN COMMERCE 477.72 tons
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CODE All products manufactured from 02/01/2005 until 06/20/2006 RECALLING FIRM/MANUFACTURER Recalling Firm: Tucker Milling LLC, Guntersville, AL, by telephone and visit on June 20, 2006, and by letter on June 23, 2006. Manufacturer: H. J. Baker and Brothers Inc., Stamford, CT. Firm initiated recall is ongoing.
REASON Poultry and fish feeds which were possibly contaminated with ruminant based protein were not labeled as "Do not feed to ruminants".
VOLUME OF PRODUCT IN COMMERCE 7,541-50 lb bags
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RECALLING FIRM/MANUFACTURER Alabama Farmers Cooperative, Inc., Decatur, AL, by telephone, fax, email and visit on June 9, 2006. FDA initiated recall is complete.
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VOLUME OF PRODUCT IN COMMERCE 125 tons
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MAD COW FEED RECALL USA EQUALS 10,878.06 TONS NATIONWIDE Sun Jul 16, 2006 09:22 220.127.116.11
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RECALLING FIRM/MANUFACTURER H. J. Baker & Bro., Inc., Albertville, AL, by telephone on June 15, 2006 and by press release on June 16, 2006. Firm initiated recall is ongoing.
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10,000,000+ LBS. of PROHIBITED BANNED MAD COW FEED I.E. BLOOD LACED MBM IN COMMERCE USA 2007
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Pfeiffer, Arno, Inc, Greenbush, WI. by conversation on February 5, 2007.
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Rangen, Inc, Buhl, ID, by letters on February 13 and 14, 2007. Firm initiated recall is complete.
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Tuesday, March 2, 2010
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ANIMAL PROTEIN I.E. MAD COW FEED IN COMMERCE A REVIEW 2010
Terry S. Singeltary Sr. (Submitted question): Monday, April 5, 2010
Update on Feed Enforcement Activities to Limit the Spread of BSE April 5, 2010
Friday, April 23, 2010
Upcoming BSE Webinar on Thursday, April 22, 2010 a review
Molecular characterization of BSE in Canada
Jianmin Yang1, Sandor Dudas2, Catherine Graham2, Markus Czub3, Tim McAllister1, Stefanie Czub1 1Agriculture and Agri-Food Canada Research Centre, Canada; 2National and OIE BSE Reference Laboratory, Canada; 3University of Calgary, Canada
Background: Three BSE types (classical and two atypical) have been identified on the basis of molecular characteristics of the misfolded protein associated with the disease. To date, each of these three types have been detected in Canadian cattle.
Objectives: This study was conducted to further characterize the 16 Canadian BSE cases based on the biochemical properties of there associated PrPres. Methods: Immuno-reactivity, molecular weight, glycoform profiles and relative proteinase K sensitivity of the PrPres from each of the 16 confirmed Canadian BSE cases was determined using modified Western blot analysis.
Results: Fourteen of the 16 Canadian BSE cases were C type, 1 was H type and 1 was L type. The Canadian H and L-type BSE cases exhibited size shifts and changes in glycosylation similar to other atypical BSE cases. PK digestion under mild and stringent conditions revealed a reduced protease resistance of the atypical cases compared to the C-type cases. N terminal- specific antibodies bound to PrPres from H type but not from C or L type. The C-terminal-specific antibodies resulted in a shift in the glycoform profile and detected a fourth band in the Canadian H-type BSE.
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.
Parallels between different forms of sheep scrapie and types of Creutzfeldt-Jakob disease (CJD)
Wiebke M. Wemheuer1, Sylvie L. Benestad2, Arne Wrede1, Wilhelm E. Wemheuer3, Tatjana Pfander1, Bjørn Bratberg2, Bertram Brenig3,Walter J. Schulz-Schaeffer1 1University Medical Center Goettingen, Germany; 2Institute of Veterinary Medicine Oslo, Norway; 3Institute of Veterinary Medicine Goettingen, Germany
Background: Scrapie in sheep and goats is often regarded as the archetype of prion diseases. In 1998, a new form of scrapie – atypical/Nor98 scrapie – was described that differed from classical scrapie in terms of epidemiology, Western blot profile, the distribution of pathological prion protein (PrPSc) in the body and its stability against proteinase K. In a similar way, distinct disease types exist in sporadic Creutzfeldt-Jakob disease (CJD). They differ with regard to their clinical outcome, Western blot profile and PrPSc deposition pattern in the central nervous system (CNS). Objectives: The comparison of PrPSc deposits in sheep scrapie and human sporadic CJD.
Methods: Tissues of the CNS of sheep with classical scrapie, sheep with atypical/Nor98 scrapie and 20 patients with sporadic CJD were examined using the sensitive Paraffin Embedded Tissue (PET) blot method. The results were compared with those obtained by immunohistochemistry. With the objective of gaining information on the protein conformation, the PrPSc of classical and atypical/Nor98 sheep scrapie and sporadic CJD was tested for its stability against denaturation with guanidine hydrochloride (GdnHCl) using a Membrane Adsorption Assay.
Results: The PrPSc of atypical/Nor98 scrapie cases and of CJD prion type 1 patients exhibits a mainly reticular/synaptic deposition pattern in the brain and is relatively sensitive to denaturation with GdnHCl. In contrast classical scrapie cases and CJD prion type 2 patients have a more complex PrPSc deposition pattern in common that consists of larger PrPSc aggregates and the PrPSc itself is comparatively stable against denaturation.
Discussion: The similarity between CJD types and scrapie types indicates that at least two comparable forms of the misfolded prion protein exist beyond species barriers and can elicit prion diseases. It seems therefore reasonable to classify classical and atypical/Nor98 scrapie – in analogy to the existing CJD types – as different scrapie types.
Monday, December 14, 2009
Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types
Monday, December 14, 2009
Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types
hmmm, this is getting interesting now...
> Sporadic CJD type 1 and atypical/ Nor98 scrapie are characterized by fine (reticular) deposits,
see also ;
> All of the Heidenhain variants were of the methionine/ methionine type 1 molecular subtype.
see full text ;
Monday, December 14, 2009
Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types
Epidemiology of Scrapie in the United States 1977
Tuesday, April 28, 2009
Nor98-like Scrapie in the United States of America
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.
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 (email@example.com); 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.
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.
Monday, December 1, 2008
When Atypical Scrapie cross species barriers
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.
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.
Transmission of atypical BSE in humanized mouse models
Liuting Qing1, Wenquan Zou1, Cristina Casalone2, Martin Groschup3, Miroslaw Polak4, Maria Caramelli2, Pierluigi Gambetti1, Juergen Richt5, Qingzhong Kong1 1Case Western Reserve University, USA; 2Instituto Zooprofilattico Sperimentale, Italy; 3Friedrich-Loeffler-Institut, Germany; 4National Veterinary Research Institute, Poland; 5Kansas State University (Previously at USDA National Animal Disease Center), USA
Background: Classical BSE is a world-wide prion disease in cattle, and the classical BSE strain (BSE-C) has led to over 200 cases of clinical human infection (variant CJD). Atypical BSE cases have been discovered in three continents since 2004; they include the L-type (also named BASE), the H-type, and the first reported case of naturally occurring BSE with mutated bovine PRNP (termed BSE-M). The public health risks posed by atypical BSE were largely undefined.
Objectives: To investigate these atypical BSE types in terms of their transmissibility and phenotypes in humanized mice. Methods: Transgenic mice expressing human PrP were inoculated with several classical (C-type) and atypical (L-, H-, or Mtype) BSE isolates, and the transmission rate, incubation time, characteristics and distribution of PrPSc, symptoms, and histopathology were or will be examined and compared.
Results: Sixty percent of BASE-inoculated humanized mice became infected with minimal spongiosis and an average incubation time of 20-22 months, whereas only one of the C-type BSE-inoculated mice developed prion disease after more than 2 years. Protease-resistant PrPSc in BASE-infected humanized Tg mouse brains was biochemically different from bovine BASE or sCJD. PrPSc was also detected in the spleen of 22% of BASE-infected humanized mice, but not in those infected with sCJD. Secondary transmission of BASE in the humanized mice led to a small reduction in incubation time. The atypical BSE-H strain is also transmissible with distinct phenotypes in the humanized mice, but no BSE-M transmission has been observed so far.
Discussion: Our results demonstrate that BASE is more virulent than classical BSE, has a lymphotropic phenotype, and displays a modest transmission barrier in our humanized mice.
BSE-H is also transmissible in our humanized Tg mice.
The possibility of more than two atypical BSE strains will be discussed.
Supported by NINDS NS052319, NIA AG14359, and NIH AI 77774.
PrPSc distribution pattern in cattle experimentally challenged with H-type and L-type atypical BSE
Anne Buschmann1, Ute Ziegler1, Leila McIntyre2, Markus Keller1, Ron Rogers3, Bob Hills3, Martin H. Groschup1 1Friedrich-Loeffler-Institut, INEID, Germany; 2Faculty of Veterinary Medicine, University of Calgary, Canada; 3Health Canada, Ottawa, Canada
Background: After the detection of two novel BSE forms designated H-type and L-type BSE, the question of the pathogenesis and the agent distribution in cattle affected with these forms was fully open. From initial studies, it was already known that the PrPSc distribution in L-type BSE affected cattle differed from that known for classical BSE (C-type) where the obex region always displays the highest PrPSc concentrations. In contrast in L-type BSE cases, the thalamus and frontal cortex regions showed the highest levels of the pathological prion protein, while the obex region was only weakly involved. No information was available on the distribution pattern in H-type BSE.
Objectives: To analyse the PrPSc and infectivity distribution in cattle experimentally challenged with H-type and L-type BSE.
Methods: We analysed CNS and peripheral tissue samples collected from cattle that were intracranially challenged with Htype (five animals) and L-type (six animals) using a commercial BSE rapid test (IDEXX HerdChek), immunohistochemistry (IHC) and a highly sensitive Western blot protocol including a phosphotungstic acid precipitation of PrPSc (PTA-WB). Samples collected during the preclinical and the clinical stages of the disease were examined. For the detection of BSE infectivity, selected samples were also inoculated into highly sensitive Tgbov XV mice overexpressing bovine prion protein (PrPC).
Results: Analysis of a collection of fifty samples from the peripheral nervous, lymphoreticular, digestive, reproductive, respiratory and musculo-skeletal systems by PTA-WB, IDEXXHerdChek BSE EIA and IHC revealed a general restriction of the PrPSc accumulation to the central nervous system.
Discussion: Our results on the PrPSc distribution in peripheral tissues of cattle affected with H-type and L-type BSE are generally in accordance with what has been known for C-type BSE. Bioassays are ongoing in highly sensitive transgenic mice in order to reveal infectivity.
Infectivity in skeletal muscle of BASE-infected cattle
Silvia Suardi1, Chiara Vimercati1, Fabio Moda1, Ruggerone Margherita1, Ilaria Campagnani1, Guerino Lombardi2, Daniela Gelmetti2, Martin H. Groschup3, Anne Buschmann3, Cristina Casalone4, Maria Caramelli4, Salvatore Monaco5, Gianluigi Zanusso5, Fabrizio Tagliavini1 1Carlo Besta” Neurological Institute,Italy; 2IZS Brescia, Italy; 33FLI Insel Riems, D, Germany; 4CEA-IZS Torino, Italy; 5University of Verona, Italy
Background: BASE is an atypical form of bovine spongiform encephalopathy caused by a prion strain distinct from that of BSE. Upon experimental transmission to cattle, BASE induces a previously unrecognized disease phenotype marked by mental dullness and progressive atrophy of hind limb musculature. Whether affected muscles contain infectivity is unknown. This is a critical issue since the BASE strain is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible.
Objectives: To investigate the distribution of infectivity in peripheral tissues of cattle experimentally infected with BASE. Methods: Groups of Tg mice expressing bovine PrP (Tgbov XV, n= 7-15/group) were inoculated both i.c. and i.p. with 10% homogenates of a variety of tissues including brain, spleen, cervical lymph node, kidney and skeletal muscle (m. longissimus dorsi) from cattle intracerebrally infected with BASE. No PrPres was detectable in the peripheral tissues used for inoculation either by immunohistochemistry or Western blot.
Results: Mice inoculated with BASE-brain homogenates showed clinical signs of disease with incubation and survival times of 175±15 and 207±12 days. Five out of seven mice challenged with skeletal muscle developed a similar neurological disorder, with incubation and survival times of 380±11 and 410±12 days. At present (700 days after inoculation) mice challenged with the other peripheral tissues are still healthy. The neuropathological phenotype and PrPres type of the affected mice inoculated either with brain or muscle were indistinguishable and matched those of Tgbov XV mice infected with natural BASE.
Discussion: Our data indicate that the skeletal muscle of cattle experimentally infected with BASE contains significant amount of infectivity, at variance with BSE-affected cattle, raising the issue of intraspecies transmission and the potential risk for humans. Experiments are in progress to assess the presence of infectivity in skeletal muscles of natural BASE.
Differences in the expression levels of selected genes in the brain tissue of cattle naturally infected with classical and atypical BSE.
Magdalena Larska1, Miroslaw P. Polak1, Jan F. Zmudzinski1, Juan M. Torres2 1National Veterinary Institute, Poland; 2CISA/INIA
Background: Recently cases of BSE in older cattle named BSE type L and type H were distinguished on the basis of atypical glycoprofiles of PrPres. The nature of those strains is still not fully understood but it is suspected that the atypical BSE cases are sporadic. Hitherto most BSE cases were studied in respect to the features of PrPSc. Here we propose gene expression profiling as a method to characterize and distinguish BSE strains.
Objectives: The aim of the study was to compare the activities of some factors which are known to play a role in TSE’s pathogenesis in order to distinguish the differences/similarities between all BSE types.
Methods: 10 % homogenate of brain stem tissue collected from obex region of medulla oblongata from 20 naturally infected BSE cows (8 assigned as classical BSE, other 8 and 4 infected with atypical BSE L type and H type respectively) was used in the study. As negative control animals we’ve used 8 animals in the age between 2.5 and 13 years. The genes were relatively quantified using SYBR Green real time RT-PCR. Raw data of Ct values was transformed into normalized relative quantities using Qbase Plus®. Results and
Discussion: In most of the tested genes significant differences in the expression levels between the brain stem of healthy cattle and animals infected with different BSE types were observed. In c-type BSE in comparison to healthy and atypical BSE the overexpression of the gene of bcl-2, caspase 3, 14-3-3 and tylosine kinase Fyn was significant.
Simultaneously in atypical BSEs type-L and type-H the levels of prion protein, Bax and LPR gene was elevated in comparison to c-BSE. Additionally L-BSE was characterized by the overexpression of STI1 and SOD genes compared to the other of BSE types. The downregulation of the gene encoding NCAM1 was observed in all BSE types in comparison to healthy cows. Different gene expression profiles of bovine brains infected with classical and atypical BSE indicates possible different pathogenesis or source of the disease.
Transmission of uncommon forms of bovine prions to transgenic mice expressing human PrP: questions and progress
Vincent Béringue, Hubert Laude INRA, UR 892, Virologie Immunologie Moléculaires, France
The active, large-scale testing of livestock nervous tissues for the presence of protease-resistant prion protein (PrPres) has led to the recognition of 2 uncommon PrPres molecular signatures, termed H-type and L-type BSE. Their experimental transmission to various transgenic and inbred mouse lines unambiguously demonstrated the infectious nature of such cases and the existence of distinct prion strains in cattle. Like the classical BSE agent, H- and L-type (or BASE) prions can propagate in heterologous species. In addition L-type prions acquire molecular and neuropathologic phenotypic traits undistinguishable from BSE or BSE-related agents upon transmission to transgenic mice expressing ovine PrP (VRQ allele) or wild-type mice. An understanding of the transmission properties of these newly recognized prions when confronted with human PrP sequence was therefore needed. Toward this end, we inoculated mice expressing human PrP Met129 with several field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. In contrast, we repeatedly failed to infect them with Htype prions. Ongoing investigations aim to extend the knowledge on these uncommon strains: are these agents able to colonize lymphoid tissue, a potential key factor for successful transmission by peripheral route; is there any relationship between these assumedly sporadic forms of TSE in cattle and some sporadic forms of human CJD are among the issues that need to be addressed for a careful assessment of the risk for cattle-to-human transmission of H- and L-type prions.
Molecular Features of the Protease-resistant Prion Protein (PrPres) in H-type BSE
Biacabe, A-G1; Jacobs, JG2; Gavier-Widén, D3; Vulin, J1; Langeveld, JPM2; Baron, TGM1 1AFSSA, France; 2CIDC-Lelystad, Netherlands; 3SVA, Sweden
Western blot analyses of PrPres accumulating in the brain of BSE-infected cattle have demonstrated 3 different molecular phenotypes regarding to the apparent molecular masses and glycoform ratios of PrPres bands. We initially described isolates (H-type BSE) essentially characterized by higher PrPres molecular mass and decreased levels of the diglycosylated PrPres band, in contrast to the classical type of BSE. This type is also distinct from another BSE phenotype named L-type BSE, or also BASE (for Bovine Amyloid Spongiform Encephalopathy), mainly characterized by a low representation of the diglycosylated PrPres band as well as a lower PrPres molecular mass. Retrospective molecular studies in France of all available BSE cases older than 8 years old and of part of the other cases identified since the beginning of the exhaustive surveillance of the disease in 20001 allowed to identify 7 H-type BSE cases, among 594 BSE cases that could be classified as classical, L- or H-type BSE. By Western blot analysis of H-type PrPres, we described a remarkable specific feature with antibodies raised against the C-terminal region of PrP that demonstrated the existence of a more C-terminal cleaved form of PrPres (named PrPres#2 ), in addition to the usual PrPres form (PrPres #1). In the unglycosylated form, PrPres #2 migrates at about 14 kDa, compared to 20 kDa for PrPres #1. The proportion of the PrPres#2 in cattle seems to by higher compared to the PrPres#1. Furthermore another PK-resistant fragment at about 7 kDa was detected by some more N-terminal antibodies and presumed to be the result of cleavages of both N- and C-terminal parts of PrP. These singular features were maintained after transmission of the disease to C57Bl/6 mice. The identification of these two additional PrPres fragments (PrPres #2 and 7kDa band) reminds features reported respectively in sporadic Creutzfeldt-Jakob disease and in Gerstmann-Sträussler-Scheinker (GSS) syndrome in humans.
TRANSMISSION OF ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY TO MICROCEBUS MURINUS, A NON-HUMAN PRIMATE. DEVELOPMENT OF CLINICAL SYMPTOMS AND TISSUE DISTRIBUTION OF PRPRES
Nadine Mestre-Frances1, Anne-Gaelle Biacabe2, Sylvie Rouland1, Thierry Baron2, Jean-Michel Verdier1, 1INSERM U710, Montpellier, France; 2AFSSA, Lyon, France. Contact e-mail: nfrances@univmontp2. fr
Background: Atypical BSE cases have been observed in Europe, Japan and North America. They differ in their PrPres profiles from those found in classical BSE. These atypical cases fall into 2 types, depending on the molecular mass of the unglycosylated PrPres band observed by Western blot: the L-type (lower molecular mass than the typical BSE cases) and H-type (higher molecular mass than the typical BSE cases).
Methods: Height animals (4 males and 4 females) were intracerebrally inoculated with 50 l of a 10% brain homogenates of atypical (L and H-type) French BSE cases.
Results: Only one of the four lemurs challenge with H-type BSE died without clinical signs after 19 months post inoculation (mpi), the 4 animals inoculated with L-type BSE died at 19 mpi (2 males) and 22 mpi (2 females). Three months before their sacrifice, they developed blindness, tremor, abnormal posture, incoordinated movements, balance loss. Symptoms get worse according to the disease progression, until severe ataxia. The brain tissue were biochemically and immunocytochemically investigated for PrPres. For the H-types, spongiform changes without PrPres accumulation were observed in the brainstem. Western blot analysis confirmed that no PrPres was detected into the brain. For the L-types, severe spongiosis was evidenced into the thalamus, the striatum, the mesencephalon, and the brainstem, whereas into the cortex the spongiosis was evidenced, but the vacuolisation was weaker. Strong deposits of PrPres was detected by western blot, PET-blot and immunocytochemistry in the CNS: dense accumulation was observed into the thalamus, the striatum, and the hippocampus whereas in the cerebral cortex, PrPres was prominently accumulated in plaques. Western blot analysis confirmed the presence of protease-resistant prion protein.
Conclusions: L-type infected lemurs showed survival times considerably shorter than for classical BSE strain, indicating that the disease is caused by a very virulent distinct prion strain.
Atypical BSE (BASE) Transmitted from Asymptomatic Aging Cattle to a Primate
Emmanuel E. Comoy1*, Cristina Casalone2, Nathalie Lescoutra-Etchegaray1, Gianluigi Zanusso3, Sophie Freire1, Dominique Marcé1, Frédéric Auvré1, Marie-Magdeleine Ruchoux1, Sergio Ferrari3, Salvatore Monaco3, Nicole Salès4, Maria Caramelli2, Philippe Leboulch1,5, Paul Brown1, Corinne I. Lasmézas4, Jean-Philippe Deslys1
1 Institute of Emerging Diseases and Innovative Therapies, CEA, Fontenay-aux-Roses, France, 2 Istituto Zooprofilattico Sperimentale del Piemonte, Turin, Italy, 3 Policlinico G.B. Rossi, Verona, Italy, 4 Scripps Florida, Jupiter, Florida, United States of America, 5 Genetics Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
Abstract Top Background Human variant Creutzfeldt-Jakob Disease (vCJD) results from foodborne transmission of prions from slaughtered cattle with classical Bovine Spongiform Encephalopathy (cBSE). Atypical forms of BSE, which remain mostly asymptomatic in aging cattle, were recently identified at slaughterhouses throughout Europe and North America, raising a question about human susceptibility to these new prion strains.
Methodology/Principal Findings Brain homogenates from cattle with classical BSE and atypical (BASE) infections were inoculated intracerebrally into cynomolgus monkeys (Macacca fascicularis), a non-human primate model previously demonstrated to be susceptible to the original strain of cBSE. The resulting diseases were compared in terms of clinical signs, histology and biochemistry of the abnormal prion protein (PrPres). The single monkey infected with BASE had a shorter survival, and a different clinical evolution, histopathology, and prion protein (PrPres) pattern than was observed for either classical BSE or vCJD-inoculated animals. Also, the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine.
Conclusion/Significance Our results point to a possibly higher degree of pathogenicity of BASE than classical BSE in primates and also raise a question about a possible link to one uncommon subset of cases of apparently sporadic CJD. Thus, despite the waning epidemic of classical BSE, the occurrence of atypical strains should temper the urge to relax measures currently in place to protect public health from accidental contamination by BSE-contaminated products.
Citation: Comoy EE, Casalone C, Lescoutra-Etchegaray N, Zanusso G, Freire S, et al. (2008) Atypical BSE (BASE) Transmitted from Asymptomatic Aging Cattle to a Primate. PLoS ONE 3(8): e3017. doi:10.1371/journal.pone.0003017
Editor: Neil Mabbott, University of Edinburgh, United Kingdom
Received: April 24, 2008; Accepted: August 1, 2008; Published: August 20, 2008
Copyright: © 2008 Comoy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by the Network of Excellence NeuroPrion.
Competing interests: CEA owns a patent covering the BSE diagnostic tests commercialized by the company Bio-Rad.
* E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000110/!x-usc:mailto:firstname.lastname@example.org
Saturday, December 01, 2007
Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model
Volume 13, Number 12–December 2007 Research
Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model
Thierry Baron,* Anna Bencsik,* Anne-Gaëlle Biacabe,* Eric Morignat,* andRichard A. Bessen†*Agence Française de Sécurité Sanitaire des Aliments–Lyon, Lyon, France; and†Montana State University, Bozeman, Montana, USA
Transmissible mink encepholapathy (TME) is a foodborne transmissible spongiform encephalopathy (TSE) of ranch-raised mink; infection with a ruminant TSE has been proposed as the cause, but the precise origin of TME is unknown. To compare the phenotypes of each TSE, bovine-passaged TME isolate and 3 distinct natural bovine spongiform encephalopathy (BSE) agents (typical BSE, H-type BSE, and L-type BSE) were inoculated into an ovine transgenic mouse line (TgOvPrP4). Transgenic mice were susceptible to infection with bovine-passaged TME, typical BSE, and L-type BSE but not to H-type BSE. Based on survival periods, brain lesions profiles, disease-associated prion protein brain distribution, and biochemical properties of protease-resistant prion protein, typical BSE had a distint phenotype in ovine transgenic mice compared to L-type BSE and bovine TME.The similar phenotypic properties of L-type BSE and bovine TME in TgOvPrP4 mice suggest that L-type BSE is a much more likely candidate for the origin of TME than is typical BSE.
These studies provide experimental evidence that the Stetsonville TME agent is distinct from typical BSE but has phenotypic similarities to L-type BSE in TgOvPrP4 mice. Our conclusion is that L-type BSE is a more likely candidate for a bovine source of TME infection than typical BSE. In the scenario that a ruminant TSE is the source for TME infection in mink, this would be a second example of transmission of a TSE from ruminants to non-ruminants under natural conditions or farming practices in addition to transmission of typical BSE to humans, domestic cats, and exotic zoo animals(37). The potential importance of this finding is relevant to L-type BSE, which based on experimental transmission into humanized PrP transgenic mice and macaques, suggests that L-type BSE is more pathogenic for humans than typical BSE (24,38).
Saturday, February 28, 2009NEW RESULTS ON IDIOPATHIC BRAINSTEM NEURONAL CHROMATOLYSIS TYPE BSE
"All of the 15 cattle tested showed that the brains had abnormally accumulated PrP" 2009 SEAC 102/2
Wednesday, October 08, 2008
Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?
>>>Conclusions: L-type infected lemurs showed survival times considerably shorter than for classical BSE strain, indicating that the disease is caused by a very virulent distinct prion strain. >>>
seems the survival time was the same for the h-type BSE and the l-type BSE i.e. 19 months post inoculation (mpi), interesting. ...TSS
Wednesday, March 31, 2010
Atypical BSE in Cattle / position: Post Doctoral Fellow
Wednesday, February 24, 2010
Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America 14th
ICID International Scientific Exchange Brochure -
National Prion Disease Pathology Surveillance Center Cases Examined1 (July 31, 2010)
(please see video at the bottom of this url...tss)
Tuesday, August 03, 2010
Variably protease-sensitive prionopathy: A new sporadic disease of the prion protein
Monday, August 9, 2010
Variably protease-sensitive prionopathy: A new sporadic disease of the prion protein or just more Prionbaloney ?
Wednesday, July 28, 2010
re-Freedom of Information Act Project Number 3625-32000-086-05, Study of Atypical BSE UPDATE July 28, 2010
Wednesday, July 28, 2010
Atypical prion proteins and IBNC in cattle DEFRA project code SE1796 FOIA Final report
Thursday, June 24, 2010
Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues
Volume 16, Number 7–July 2010
To date the OIE/WAHO assumes that the human and animal health standards set out in the BSE chapter for classical BSE (C-Type) applies to all forms of BSE which include the H-type and L-type atypical forms. This assumption is scientifically not completely justified and accumulating evidence suggests that this may in fact not be the case. Molecular characterization and the spatial distribution pattern of histopathologic lesions and immunohistochemistry (IHC) signals are used to identify and characterize atypical BSE. Both the L-type and H-type atypical cases display significant differences in the conformation and spatial accumulation of the disease associated prion protein (PrPSc) in brains of afflicted cattle. Transmission studies in bovine transgenic and wild type mouse models support that the atypical BSE types might be unique strains because they have different incubation times and lesion profiles when compared to C-type BSE. When L-type BSE was inoculated into ovine transgenic mice and Syrian hamster the resulting molecular fingerprint had changed, either in the first or a subsequent passage, from L-type into C-type BSE. In addition, non-human primates are specifically susceptible for atypical BSE as demonstrated by an approximately 50% shortened incubation time for L-type BSE as compared to C-type. Considering the current scientific information available, it cannot be assumed that these different BSE types pose the same human health risks as C-type BSE or that these risks are mitigated by the same protective measures.
Wednesday, March 31, 2010
Atypical BSE in Cattle
Let's look at some sound science on atypical Nor-98 Scrapie, shall we ;
[Although atypical scrapie is not yet ruled out, it is important to realize this is a type of scrapie that thus far has only tended to appear as a sporadic condition in older animals. Currently it has not been shown to follow the same genetic tendencies for propagation as the usual scrapie.
However, the atypical phenotypic appearance has been shown to be preserved on experimental passage.
Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries, as Australia may join that list. It is likely that this case will be sent to the UK for definitive conformation.
[Ref: M Simmons, T Konold, L Thurston, et al. BMC Veterinary Research 2010, 6:14 [provisional abstract available at
"Background ----------- "Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. [Many of the neurological diseases can be transmitted by intracerebral inoculation, which causes this moderator to approach intracerebral studies as a tool for study, but not necessarily as a direct indication of transmissibility of natural diseases. - Mod.TG]
"The 1st successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.
"Results -------- "This study demonstrates that atypical scrapie has distinct clinical, pathological, and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.
"Conclusions ------------ Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage."
Lastly, this moderator wishes to thank Terry Singletary for some of his behind the scenes work of providing citations and references for this posting. - Mod.TG]
Saturday, June 5, 2010
Research Project: Transmissible Spongiform Encephalopathies: Identification of atypical scrapie in Canadian sheep
Sunday, April 18, 2010
SCRAPIE AND ATYPICAL SCRAPIE TRANSMISSION STUDIES A REVIEW 2010
If the scrapie agent is generated from ovine DNA and thence causes disease in other species, then perhaps, bearing in mind the possible role of scrapie in CJD of humans (Davinpour et al, 1985), scrapie and not BSE should be the notifiable disease. ...
Tuesday, July 27, 2010
Spontaneous generation of mammalian prions
Thursday, August 12, 2010
Seven main threats for the future linked to prions
Terry S. Singeltary Sr.
P.O. Box 42
Bacliff, Texas USA 77518