Kathy said:
Jpn J Infect Dis. 2007 Sep;60(5):317-20. Links
Experimental Transmission of Two Young and One Suspended Bovine Spongiform Encephalopathy (BSE) Cases to Bovinized Transgenic Mice.
Yokoyama T, Masujin K, Yamakawa Y, Sata T, Murayama Y, Shu Y, Okada H, Mohri S, Shinagawa M.
Prion Disease Research Center, National Institute of Animal Health, Ibaraki 305-0856, Japan.
[email protected]
Bovine spongiform encephalopathy (BSE) is caused by a prion that primarily consists of an abnormal isoform of the prion protein (PrP(Sc)). Since PrP(Sc) is partially resistant to proteolytic digestion, the routine diagnosis of BSE is based on the immunological detection of the proteinase K (PK)-resistant moiety of PrP(Sc) (PrP(core)). However, transmission studies are indispensable in order to demonstrate prion infectivity and to analyze prion characteristics. Transmission experiments were accordingly performed on 2 young BSE cases (BSE/JP8, BSE/JP9) and 1 suspected BSE case (Suspended-1) that were detected by the BSE screening program in Japan. In this study, we attempted to transmit the prion from these 3 animals by using transgenic mice overexpressing bovine PrP (TgBoPrP). In spite of the use of BSE-sensitive transgenic mice, none of the mice developed neurological signs nor accumulated PrP(Sc) in their brains for more than 600 days post-inoculation, even with subsequent blind passages. The results of a dilution experiment using the classical BSE prion indicated that prion infectivity in these 3 cattle was below the detection limit of 10(3.0) LD(50)/g.
PMID: 17881878
Jpn. J. Infect. Dis., 60 (5), 305-308, 2007
Short Communication
Accumulation of Mono-Glycosylated Form-Rich, Plaque-Forming PrPSc in the
Second Atypical Bovine Spongiform Encephalopathy Case in Japan
Ken'ichi Hagiwara1,3*, Yoshio Yamakawa1,3, Yuko Sato2, Yuko Nakamura1,
Minoru Tobiume2, Morikazu Shinagawa3 and Tetsutaro Sata2,3
1Department of Biochemistry and Cell Biology and 2Department of Pathology,
National Institute of Infectious Diseases, Tokyo 162-8640, and 3The Expert
Committee for BSE Diagnosis, Ministry of Health, Labour and Welfare, Tokyo
100-8916, Japan
(Received March 23, 2007. Accepted June 1, 2007)
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*Corresponding author: Mailing address: Department of Biochemistry and Cell
Biology, National Institute of Infectious Diseases, Toyama 1-23-1,
Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-5285-1111, Fax:
+81-3-5285-1157, E-mail:
[email protected]
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SUMMARY: The recent identification of several atypical cases of bovine
spongiform encephalopathy (BSE) has raised the possibility of the existence
of distinct strains of BSE agents, arguing against the previous notion that
BSE is caused by a single strain. To date, at least, two atypical types (L
and H) of agent have been reported based on the molecular sizes of the
proteinase K-resistant forms of prion protein (PrPSc). These atypical agents
were identified first in Japan, Italy, France, and Germany, and later in
other European countries. Here, we have identified a case of BSE in a
169-month-old cow (designated as BSE/JP24), in which predominant deposition
of the mono-glycosylated form of PrPSc was observed by Western-blot
analysis, and plaques of PrPSc were detected in the brain by
immunohistochemical analysis. The glycoform ratio of PrPSc was different
from that of the typical BSE agent, in which the di-glycosylated form is
dominant; instead, the ratio resembled that of type-2 human sporadic
Creutzfeldt-Jakob disease and that reported for the L-type BSE. The
characteristic glycoform ratio and plaques of PrPSc suggested that the agent
in BSE/JP24 was relevant, if not identical, to the agent in bovine
amyloidotic spongiform encephalopathy (BASE), an L-type BSE identified in
Italy. It was of interest that at the level of the obex, the medulla
oblongata was devoid of plaques of PrPSc, and a pathological phenotype
similar to that of typical BSE specimens with vacuolations and coarse
granular/linear deposition of PrPSc were observed.
http://www.nih.go.jp/JJID/60/305.html
FULL TEXT ;
http://www.nih.go.jp/JJID/60/305.pdf
P02.35
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.
FC5.5.1
BASE Transmitted to Primates and MV2 sCJD Subtype Share PrP27-30 and PrPSc
C-terminal Truncated Fragments
Zanusso, G1; Commoy, E2; Fasoli, E3; Fiorini, M3; Lescoutra, N4; Ruchoux,
MM4; Casalone, C5; Caramelli, M5; Ferrari, S3; Lasmezas, C6; Deslys, J-P4;
Monaco, S3 1University of Verona, of Neurological and Visual Sciences,
Italy; 2CEA, IMETI/SEPIA, France; 3University of Verona, Neurological and Visual
Sciences, Italy; 4IMETI/SEPIA, France; 5IZSPLVA, Italy; 6The Scripps Research Insitute, USA
The etiology of sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human
prion disease, remains still unknown. The marked disease phenotype heterogeneity
observed in sCJD is thought to be influenced by the type of proteinase K-resistant
prion protein, or PrPSc (type 1 or type 2 according to the electrophoretic mobility of
the unglycosylated backbone), and by the host polymorphic Methionine/Valine (M/V)
codon 129 of the PRNP. By using a two-dimensional gel electrophoresis (2D-PAGE)
and imunoblotting we previously showed that in sCJD, in addition to the PrPSc type,
distinct PrPSc C-terminal truncated fragments (CTFs) correlated with different sCJD
subtypes. Based on the combination of CTFs and PrPSc type, we distinguished three
PrPSc patterns: (i) the first was observed in sCJD with PrPSc type 1 of all genotypes,;
(ii) the second was found in M/M-2 (cortical form); (iii) the third in amyloidogenic M/V-
2 and V/V-2 subtypes (Zanusso et al., JBC 2004) . Recently, we showed that sCJD
subtype M/V-2 shared molecular and pathological features with an atypical form of
BSE, named BASE, thus suggesting a potential link between the two conditions. This
connection was further confirmed after 2D-PAGE analysis, which showed an
identical PrPSc signature, including the biochemical pattern of CTFs. To pursue this
issue, we obtained brain homogenates from Cynomolgus macaques intracerebrally
inoculated with brain homogenates from BASE. Samples were separated by using
a twodimensional electrophoresis (2D-PAGE) followed by immunoblotting. We here
show that the PrPSc pattern obtained in infected primates is identical to BASE
and sCJD MV-2 subtype. These data strongly support the link, or at least a common
ancestry, between a sCJD subtype and BASE. This work was supported by Neuroprion
(FOOD-CT-2004-506579)
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USA MAD COW CASES IN ALABAMA AND TEXAS
***PLEASE NOTE***
USA BASE CASE, (ATYPICAL BSE), AND OR TSE (whatever they are calling it
today), please note that both the ALABAMA COW, AND THE TEXAS COW, both were
''H-TYPE'', personal communication Detwiler et al Wednesday, August 22, 2007
11:52 PM. ...TSS
http://lists.ifas.ufl.edu/cgi-bin/wa.exe?A2=ind0708&L=sanet-mg&T=0&P=19779
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FC5.5.2 Transmission of Italian BSE and BASE Isolates in Cattle Results into
a
Typical BSE Phenotype and a Muscle Wasting Disease
Zanusso, G1; Lombardi, G2; Casalone, C3; D’Angelo, A4; Gelmetti, D2; Torcoli, G2;
Barbieri, I2; Corona, C3; Fasoli, E1; Farinazzo, A1; Fiorini, M1; Gelati, M1; Iulini, B3;
Tagliavini, F5; Ferrari, S1; Monaco, S1; Caramelli, M3; Capucci, L2
1University of Verona, Neurological and Visual Sciences, Italy; 2IZSLER, Italy; 3IZSPLVA,
Italy; 4University of Turin, Animal Pathology, Italy; 5Isituto Carlo Besta, Italy
The clinical phenotype of bovine spongiform encephalopathy has been extensively
reported in early accounts of the disorder. Following the introduction of statutory active
surveillance, almost all BSE cases have been diagnosed on a pathological/molecular
basis, in a pre-symptomatic clinical stage. In recent years, the active surveillance
system has uncovered atypical BSE cases, which are characterized by distinct
conformers of the PrPSc, named high-type (BSE-H) and low-type (BSE-L), whose
clinicopathological phenotypes remain unknown. We recently reported two
Italian atypical cases with a PrPSc type similar to BSE-L, pathologically
characterized by PrP amyloid plaques. Experimental transmission to TgBov mice has recently
disclosed that BASE is caused by a distinct prion strain which is extremely virulent. A
major limitation of transmission studies to mice is the lack of reliable information on
clinical phenotype of BASE in its natural host. In the present study, we experimentally
infected Fresian/Holstein and Alpine/Brown cattle with Italian BSE and BASE isolates
by i.c. route. BASE infected cattle showed survival times significantly
shorter than BSE, a finding more readily evident in Fresian/Holstein, and in keeping with
previous observations in TgBov mice. Clinically, BSE-infected cattle developed a
disease phenotype highly comparable with that described in field BSE cases
and in experimentally challenged cattle. On the contrary, BASE-inoculated cattle
developed an amyotrophic disorder accompanied by mental dullness.
The molecular and neuropathological profiles, including PrP deposition
pattern, closely matched those observed in the original cases. This study further confirms
that BASE is caused by a distinct prion isolate and discloses a novel disease
phenotype in cattle, closely resembling the phenotype previous reported in scrapie-inoculated
cattle and in some subtypes of inherited and sporadic Creutzfeldt-Jakob disease.
Oral Abstracts
14
FC5.1.1
Transmission Results in Squirrel Monkeys Inoculated with Human sCJD, vCJD,
and GSS Blood Specimens: the Baxter Study
Brown, P1; Gibson, S2; Williams, L3; Ironside, J4; Will, R4; Kreil, T5;
Abee, C3 1Fondation Alliance BioSecure, France; 2University of South Alabama, USA;
3University of Texas MD Anderson Cancer Center, USA; 4Western General
Hospital, UK; 5Baxter BioSience, Austria
Background: Rodent and sheep models of Transmissible Spongiform Encephalopathy
(TSE) have documented blood infectivity in both the pre-clinical and clinical phases of
disease. Results in a (presumably more appropriate) non-human primate model have not been reported.
Objective: To determine if blood components (red cells, white cells, platelets, and
plasma) from various forms of human TSE are infectious.
Methods: Blood components were inoculated intra-cerebrally (0.1 ml) and
intravenously (0.5 ml) into squirrel monkeys from 2 patients with sporadic
Creutzfeldt- Jakob disease (sCJD) and 3 patients with variant Creutzfeldt-Jakob disease
(vCJD). Additional monkeys were inoculated with buffy coat or plasma samples from
chimpanzees infected with either sCJD or Gerstmann-Sträussler-Scheinker
disease (GSS). Animals were monitored for a period of 5 years, and all dying
or sacrificed animals had post-mortem neuropathological examinations and
Western blots to determine the presence or absence of the misfolded ‘prion’ protein (PrPTSE).
Results: No transmissions occurred in any of the animals inoculated with
blood components from patients with sporadic or variant CJD. All donor
chimpanzees
(sCJD and GSS) became symptomatic within 6 weeks of their pre-clinical phase
plasmapheresis, several months earlier than the expected onset of illness.
One monkey inoculated with purified leukocytes from a pre-clinical GSS
chimpanzee developed disease after 36 months.
Conclusion: No infectivity was found in small volumes of blood components
from 4 patients with sporadic CJD and 3 patients with variant CJD.
***However, a single transmission from a chimpanzee-passaged strain of GSS
shows that infectivity may be present in leukocytes, and the ‘shock’ of
general anaesthesia and plasmspheresis appears to have triggered the onset of illness in
pre-clinical donor chimpanzees.
FC5.1.2
Interim Transmission Results in Cynomolgus Macaques Inoculated with BSE and
vCJD Blood Specimens
Lasmezas, C1; Lescoutra, N2; Comoy, E2; Holznagel, E3; Loewer, J3; Motzkus,
D4; Hunsmann, G4; Ingrosso, L5; Bierke, P6; Pocchiari, M5; Ironside, J7; Will,
R7; Deslys, JP2 1Scripps Florida, Infectology, USA; 2CEA, France; 3PEI,
Germany; 4DPZ, Germany; 5Istituto Superiore di Sanita, Italy; 6SMI, Sweden; 7CJD
Surveillance Unit, UK
BSE and vCJD transmitted to cynomolgus macaques reproduce many features of
human vCJD, including clinical symptoms, neuropathological hallmarks of vCJD,
PrPres electrophoretical pattern and, most importantly, the wide distribution of
infectivity in peripheral organs. The latter characteristic distinguishes vCJD from sCJD
in both humans and cynomolgus macaques, and prompted us to use this non-human
primate model for further investigations of vCJD and its risk for human health. The
occurrence of four vCJD infections in humans transfused with blood from patients who
later developed vCJD has raised concern about blood transfusion safety in countries
with vCJD.
In this collaborative European study, we investigated the infectivity of
blood components and whole blood administered by intracerebral (ic) and
intravenous (iv) routes. Buffy-coat and whole blood was inoculated by ic and
iv route, respectively, from two vCJD patients and from two clinical vCJD-inoculated
macaques. Transfusions were also performed from whole blood and blood leucodepleted
according to hospital practice standards from two clinical BSE inoculated
macaques. Blood infectivity during the preclinical phase is being examined in orally
infected macaques. Whole blood was collected and transfused from one such animal two
years after oral challenge, whereas buffy-coat and plasma from two animals
at 2 and 4.5 years post-challenge, respectively, have been inoculated by the ic
route.
This is an ongoing study in which recipient animals continue to be observed
at various times post-inoculation. So far, we have had one positive transmission in one
animal transfused 65 months earlier with 40 ml of whole blood from a vCJD macaque
(the characteristics of the disease in this animal will be shown in a separate poster by E.
Comoy). This positive transmission reproduces transfusion transmission of vCJD in
humans, with an incubation of 5.5 years compatible with incubation periods
observed in humans.
FC5.3
Assessing the Risk of vCJD Transmission by Dentistry; Distribution of
Infectivity in Oral Tissues of VM Mice after Simulated Oral Feeding of
BSE-301V
Sutton, JM1; Kirby, E1; Dickinson, J1; Dennis, M1; Cornwall, M1; Vassey,
MJ1; Smith, A2; Marsh, PD3; Walker, JT1; Raven, NDH1
1Health Protection Agency, Centre for Emergency Preparedness and Response,,
TSE Research group, UK; 2University of Glasgow, Dental School, UK; 3Health
Protection Agency, Centre for Emergency Preparedness and Response,, UK
Background: Ongoing concerns about the prevalence of variant Creutzfeldt Jakob
Disease (vCJD) in the UK population has heightened concerns about the risks
of iatrogenic transmission of the disease. Although there have been no cases
to date of transmission by surgery there have been 4 cases involving blood
transfusion. This study aims to assess the potential of transmission of the disease by
dental procedures. Whilst the risks are undoubtably low the very large numbers of
procedures carried out annually have the potential to amplify the risks considerably
and there is very little data in this area to form the basis for accurate risk
assessments. Aim(s)/Objective(s): To assess the relative levels of infectivity in oral
tissues from a murine model following exposure to BSE-301V through the small intestine.
Methods. The study uses a BSE-301V, VM mouse model as a clinically relevant model for
assessing iatrogenic vCJD transmission between humans. Infectious mouse
brain homogenate was prepared and inoculated into a loop of the duodenum, to
prevent direct contamination of the oral tissues. Mice were sacrificed at 3-weekly
intervals and at appearance of clinical symptoms. A range of oral tissues,
including dental pulp, gingival margin, salivary gland, saliva, lingual tonsil and
trigeminal ganglia, together with brain and spleen tissues were removed, processed as
homogenates and reinoculated intracranially (ic.) into indicator mice.
Results: The primary challenge proved to be a very efficient route of
infection with a 100% attack rate and a mean incubation to clinical disease
of 157 ± 17 days (compared to 120 days for the same titre inoculum ic.). Infectivity was
observed in all oral and control tissues with varying time-courses and titres estimated from
incubation period.
Discussion: The results throw new light on the potential routes of
dissemination and spread of infectivity from the small intestine to the oral cavity and its
implications for possible iatrogenic transmission of vCJD via dental, endoscopic or other
forms of surgery.
Conclusion: The data generated from the study provides support for ongoing
risk assessments to look at the potential for vCJD transmission via dental
procedures alongside other elements of studies looking at effectiveness of
decontamination and re-use of dental instruments.
P02.15
Beyond the PrPres Type1/ Type2 dichotomy in Creutzfeldt-Jakob Disease
Cassard, H1; Uro-Coste, E2; Simon, S3; Bilheude, JM4; Perret-Liaudet, A5;
Ironside, J6; Haik, S7; Basset-Leobon, C2; Lacroux, C1; Peoch’, K8; Stressenberger, N9;
Langeveld, J10; Head, M11; Hauw, JJ12; Schecher, F1; Delisle, MB13; Andreoletti, O1
1Ecole Natinale Vétérinaire de Toulouse, France; 22Service d’Anatomie Pathologique
and INSERM U466 R, France; 3DRM, CEA/Saclay, France; 4 Bio-Rad, R&D, France;
5 Hôpital Neurologique Service de Neurochimie, France; 6School of Molecular &
Clinical Medicine (Pathology),, National Creutzfeldt-Jakob Disease Surveillance Un,
UK; 7Pitié Salpetriere Universitary Hospital, France; 8Hôpital Lariboisière, Service de
Biochimie et Biologie Moléculaire,, France; 9Hôpital Neurologique -Service de
Neurochimie, France; 10CIDC-Lelystad, Netherlands; 11University of Edinburgh, Western General
Hospital, UK; 12Pitié Salpetriere Universitary Hospital,, Laboratoire de Neuropathologie, France;
13Rangueil Universitary Hospital, Service d’Anatomie Pathologique, France
Creutzfeldt-Jakob disease (CJD) cases are currently classified according to
established diagnostic criteria and by the genotype at codon 129 of the PRNP
gene and the Western blotting of the proteinase K digested abnormal prion protein
that distinguishes a type 1 and a type 2 profile. These biochemically distinct
PrPres types have been proposed to represent distinct prion strains. However, since the
cooccurence of type 1 and type 2 PrPres in the same patient is common, the rationale of
this classification and strain concept as applied to CJD are currently under discussion.
Five different brain areas from of 40 sporadic CJD and 11 iatrogenic CJD (both dura
matter-, and growth hormone-associated) cases, originating from UK and France, were
systematically investigated, using Western blotting typing, and by two others
biochemical assays that depend on the behaviour of PrPSc in variable PK digestion
conditions. As described previously, co-occurrence of type 1 and type 2 PrPres was
found in 30% of the CJD patients examined. However, our novel PK concentration
dependent assays identified a single uniform PrP type in cases where both type 1 and
type 2 were present. Moreover, in sCJD four distinct biochemical PrPSc
signatures were identified by the PK concentration dependent assays and these
correlated to the current genotype/clinico-pathological sCJD groups. In iCJD the four similar
biochemical signatures were observed, but were not correlated to particular PRNP 129
polymorphism or Western Blot PrPres patterns. Moreover notable differences were
observed between PrPSc biochemical properties of French and UK GH-CJD cases,
which could reflect, as already suspected, differences in the causative agents.
Identification, in sCJD and iCJD, of four different PrPSc phenotypes
irrespective of patients PRNP polymorphism at codon 129 and Western blot profile provides
new insights into human prion disease aetiology and could reflects an unsuspected
diversity of TSE agents in human disease. Further investigations are currently
underway using animal transmission to correlate agent strain with our new
discriminant biochemical assays.
see full text 143 pages ;
http://www.prion2007.com/pdf/Prion%20Book%20of%20Abstracts.pdf
TSS