“Prion 2005: Between fundamentals and society’s needs”
Tuesday, 18.October 2005
snip...
GEN-12
A special form of PrPSc in muscles of a BSE-infected cow
I Schiller1, J Duss1, F Kuhn1, J Schmid1, M Glatzel2, F Ehrensperger2, M Hilbe2, K Zlinszky2, B
Stierstorfer3, B Oesch1, A.J. Raeber1
1 Prionics AG, Switzerland; 2 Universitiy of Zurich, Switzerland; 3 Ludwig-Maximilians-Universitaet Muenchen,
Germany
PrPSc, the abnormal form of the prion protein, has been detected in muscles of patients with
sporadic Creutzfeldt-Jakob disease, in natural and experimental scrapie in sheep and in ro-dent
adapted scrapie but not in cattle with bovine spongiform encephalopathy (BSE).
In this study we have analyzed 60 muscle samples originating from 7 Swiss and 6 Bavarian cows
with confirmed BSE by 3 different methods: (1) phosphotungstic acid precipitation fol-lowed by
proteinase K digestion, (2) phosphotungstic acid precipitation followed by immuno-precipitation
with the PrPSc conformation specific antibody 15B3, and (3) ultracentrifugation followed by
immunoprecipitation with the antibody 15B3. Analysis of PrP was performed by Western
blotting with the antibody 6H4. Here we report for the first time the detection of a special form of
PrPSc in muscles of a clinical BSE case. This form of PrPSc is protease-resistant yet the
protease-resistant fragment detected on Western blot differs from that typi-cally found in BSE
positive brains. In 1 of 7 Swiss BSE cases, we detected PrPSc in sem-itendinous muscle, triceps
muscle, sternocephalicus muscle and in the tongue but not in two other muscles analysed. Our
findings show that low levels of a special form of PrPSc are present in a small percentage of
muscles from BSE affected cattle. Whether this type of PrPSc in muscle of cattle with BSE is
associated with infectivity remains to be established.
snip...page 227 of 411 pages...tss
PATH-26
Pathological Prion Protein in Muscles of Rodents Infected with BSE or vCJD
Achim Thomzig1, Franco Cardone2, Dominique Krüger1, Maurizio Pocchiari2, Paul Brown3,
Michael Beekes1
1 Robert Koch-Institut, P24; 2 Instituto Superiore di Sanità, Depatment of Cell Biology and Neurosciences, Rome, Italy;
3 Bethesda, Maryland, USA
Recently, pathological prion protein PrPTSE was detected in muscles from sheep infected with
scrapie, the archetype of transmissible spongiform encephalopathies (TSEs). This finding has
highlighted the question of whether mammalian muscle may potentially also provide a reservoir
for TSE agents related to Bovine Spongiform Encephalopathy (BSE) and variant Creutzfeldt-
Jakob Disease (vCJD). We here report results from studies in hamsters and mice which provide
direct experimental evidence, for the first time, of BSE- and vCJD-associated PrPTSE deposition
in muscles. Our findings emphasize the need for further assessment of possible public health
risks from TSE involvement of skeletal muscle.
snip...page 311...tss
also, see ;
HUMAN-14
Transmission of BSE to cynomolgus macaques
J Montag1, G Hunsmann1, W Schul-Schaeffer2, D Motzkus1
1 German Primate Centre, Department of immunology and virology, Göttingen, Germany; 2 Georg-August-Universität
Göttingen, Dept. of Neurology, Robert-Koch-Str. 40, 37075 Göttingen, Germany
The risk of transmitting transmissible spongiform encephalopathies (TSEs) from animal to
humans is still an important question in TSE research. There are scientific indications that bovine
spongiform encephalopathy (BSE) is transmitted to humans causing variant Creutzfeldt Jakob
disease (vCJD). Previous studies have shown that infectious material from cattle can cause TSEs
in cynomolgus macaques (M. fascicularis) resembling the lesion profile of vCJD. Recently, oral
infection using macaque-adapted brain homogenate was successful in the same animal model.
In cooperation with five European partners a quantitative study for the transmission of the BSE
agent to M. fascicularis was initiated to assess the risk of vCJD infection in humans through
contaminated of BSE-infected material. Infection was performed orally and, as a control,
intracerebrally with non adapted BSE material. As the first result we report the successful
intracerebral transmission of BSE to M. fascicularis. 6/6 have died of TSE. The analysis of one
animal is exemplified here. By immunohistology, amyloid plaques with PrP depositions were
detected in different brain sections. The Western Blot analysis revealed PrP-specific bands
between 26 and 39 kDa. After PK-digestion of the infected macaque brain homogenate this
pattern shifted to three bands between 17 - 36 kDa. The bandshift was also detected after PNGase
digestion. These data confirm the clinical diagnosis of vCJD.
page 259...tss
PATH-39
Adaptation of the Bovine Spongiform Encephalopathy to the Primate Microcebus
murinus by Oral Route
N MESTRE-FRANCES1, B EL MATHARI1, C CROZET2, V PASCAL1, S ROULAND1, S
LEHMANN2, S PICAUD3, JL BERGE-LEFRANC4, JM VERDIER1
1 INSERM U710-EPHE-University Montpellier 2, France; 2 CNRS UPR1142, IGH, Montpellier, France; 3 INSERM
U592, Paris, FRANCE; 4 EA1784-IFR PMSE 112, University Aix -Marseille, France
Experimental transmission of BSE through oral route to mice or to non-human primates such as
Cynomolgus macaca have been reported in several studies. Here, we report on the investigation
of oral transmission of BSE to a small non-human primate, the lesser-mouse lemur (Microcebus
murinus). Seven microcebes were contaminated by bovine brain or bovine-adapted macaca brain.
Only the three microcebes that received BSE-macaca brain develop a neurological disease after
long incubation periods (44 months). The duration of clinical stage was 2 months. The symptoms
begin by nervousness evolving rapidly to agressivity, visual troubles, imbalance then
incoordination of movements, myoclonic jerks and later on, the microcebes became ataxic,
turning round on itself.
The spongiform extent was related with the duration of illness. It ranged from small discrete
vacuoles randomly dispersed throughout the neuropil to large confluent cystic spaces traversed
by thin septae. Spongiform changes were most pronounced in the thalamus, the basal ganglia, the
hypothalamus and the brainstem. The neocortex was relatively spared by spongiosis: small sparse
vacuoles were seen in the frontal and occipital lobes whereas the hippocampus showed larger
vacuoles. Spongiform changes were accompanied by important astrocytic gliosis.
Proteinase K resistant-prion protein was detected by western blot as well as
immunocytochemistry. PrPres accumulation was observed in all the microcebes presenting
clinical signs. The prion immunostaining was more intense in thalamus, basal ganglia and
brainstem. Focal deposits or plaques were seen in the cortex. In the cerebellum, some plaques
were evidenced in the molecular layer.
In conclusion, BSE agent does not seem to be directly pathogenic to microcebe by oral route, but
necessitates an adaptation of the strain to macaca. ...page 324....tss
snip...
http://www.tse-forum.de/tse_forum/deutsch/oeffentlich/bilder/Abstract_BookFINAL_nov2.pdf
TSS
Tuesday, 18.October 2005
snip...
GEN-12
A special form of PrPSc in muscles of a BSE-infected cow
I Schiller1, J Duss1, F Kuhn1, J Schmid1, M Glatzel2, F Ehrensperger2, M Hilbe2, K Zlinszky2, B
Stierstorfer3, B Oesch1, A.J. Raeber1
1 Prionics AG, Switzerland; 2 Universitiy of Zurich, Switzerland; 3 Ludwig-Maximilians-Universitaet Muenchen,
Germany
PrPSc, the abnormal form of the prion protein, has been detected in muscles of patients with
sporadic Creutzfeldt-Jakob disease, in natural and experimental scrapie in sheep and in ro-dent
adapted scrapie but not in cattle with bovine spongiform encephalopathy (BSE).
In this study we have analyzed 60 muscle samples originating from 7 Swiss and 6 Bavarian cows
with confirmed BSE by 3 different methods: (1) phosphotungstic acid precipitation fol-lowed by
proteinase K digestion, (2) phosphotungstic acid precipitation followed by immuno-precipitation
with the PrPSc conformation specific antibody 15B3, and (3) ultracentrifugation followed by
immunoprecipitation with the antibody 15B3. Analysis of PrP was performed by Western
blotting with the antibody 6H4. Here we report for the first time the detection of a special form of
PrPSc in muscles of a clinical BSE case. This form of PrPSc is protease-resistant yet the
protease-resistant fragment detected on Western blot differs from that typi-cally found in BSE
positive brains. In 1 of 7 Swiss BSE cases, we detected PrPSc in sem-itendinous muscle, triceps
muscle, sternocephalicus muscle and in the tongue but not in two other muscles analysed. Our
findings show that low levels of a special form of PrPSc are present in a small percentage of
muscles from BSE affected cattle. Whether this type of PrPSc in muscle of cattle with BSE is
associated with infectivity remains to be established.
snip...page 227 of 411 pages...tss
PATH-26
Pathological Prion Protein in Muscles of Rodents Infected with BSE or vCJD
Achim Thomzig1, Franco Cardone2, Dominique Krüger1, Maurizio Pocchiari2, Paul Brown3,
Michael Beekes1
1 Robert Koch-Institut, P24; 2 Instituto Superiore di Sanità, Depatment of Cell Biology and Neurosciences, Rome, Italy;
3 Bethesda, Maryland, USA
Recently, pathological prion protein PrPTSE was detected in muscles from sheep infected with
scrapie, the archetype of transmissible spongiform encephalopathies (TSEs). This finding has
highlighted the question of whether mammalian muscle may potentially also provide a reservoir
for TSE agents related to Bovine Spongiform Encephalopathy (BSE) and variant Creutzfeldt-
Jakob Disease (vCJD). We here report results from studies in hamsters and mice which provide
direct experimental evidence, for the first time, of BSE- and vCJD-associated PrPTSE deposition
in muscles. Our findings emphasize the need for further assessment of possible public health
risks from TSE involvement of skeletal muscle.
snip...page 311...tss
also, see ;
HUMAN-14
Transmission of BSE to cynomolgus macaques
J Montag1, G Hunsmann1, W Schul-Schaeffer2, D Motzkus1
1 German Primate Centre, Department of immunology and virology, Göttingen, Germany; 2 Georg-August-Universität
Göttingen, Dept. of Neurology, Robert-Koch-Str. 40, 37075 Göttingen, Germany
The risk of transmitting transmissible spongiform encephalopathies (TSEs) from animal to
humans is still an important question in TSE research. There are scientific indications that bovine
spongiform encephalopathy (BSE) is transmitted to humans causing variant Creutzfeldt Jakob
disease (vCJD). Previous studies have shown that infectious material from cattle can cause TSEs
in cynomolgus macaques (M. fascicularis) resembling the lesion profile of vCJD. Recently, oral
infection using macaque-adapted brain homogenate was successful in the same animal model.
In cooperation with five European partners a quantitative study for the transmission of the BSE
agent to M. fascicularis was initiated to assess the risk of vCJD infection in humans through
contaminated of BSE-infected material. Infection was performed orally and, as a control,
intracerebrally with non adapted BSE material. As the first result we report the successful
intracerebral transmission of BSE to M. fascicularis. 6/6 have died of TSE. The analysis of one
animal is exemplified here. By immunohistology, amyloid plaques with PrP depositions were
detected in different brain sections. The Western Blot analysis revealed PrP-specific bands
between 26 and 39 kDa. After PK-digestion of the infected macaque brain homogenate this
pattern shifted to three bands between 17 - 36 kDa. The bandshift was also detected after PNGase
digestion. These data confirm the clinical diagnosis of vCJD.
page 259...tss
PATH-39
Adaptation of the Bovine Spongiform Encephalopathy to the Primate Microcebus
murinus by Oral Route
N MESTRE-FRANCES1, B EL MATHARI1, C CROZET2, V PASCAL1, S ROULAND1, S
LEHMANN2, S PICAUD3, JL BERGE-LEFRANC4, JM VERDIER1
1 INSERM U710-EPHE-University Montpellier 2, France; 2 CNRS UPR1142, IGH, Montpellier, France; 3 INSERM
U592, Paris, FRANCE; 4 EA1784-IFR PMSE 112, University Aix -Marseille, France
Experimental transmission of BSE through oral route to mice or to non-human primates such as
Cynomolgus macaca have been reported in several studies. Here, we report on the investigation
of oral transmission of BSE to a small non-human primate, the lesser-mouse lemur (Microcebus
murinus). Seven microcebes were contaminated by bovine brain or bovine-adapted macaca brain.
Only the three microcebes that received BSE-macaca brain develop a neurological disease after
long incubation periods (44 months). The duration of clinical stage was 2 months. The symptoms
begin by nervousness evolving rapidly to agressivity, visual troubles, imbalance then
incoordination of movements, myoclonic jerks and later on, the microcebes became ataxic,
turning round on itself.
The spongiform extent was related with the duration of illness. It ranged from small discrete
vacuoles randomly dispersed throughout the neuropil to large confluent cystic spaces traversed
by thin septae. Spongiform changes were most pronounced in the thalamus, the basal ganglia, the
hypothalamus and the brainstem. The neocortex was relatively spared by spongiosis: small sparse
vacuoles were seen in the frontal and occipital lobes whereas the hippocampus showed larger
vacuoles. Spongiform changes were accompanied by important astrocytic gliosis.
Proteinase K resistant-prion protein was detected by western blot as well as
immunocytochemistry. PrPres accumulation was observed in all the microcebes presenting
clinical signs. The prion immunostaining was more intense in thalamus, basal ganglia and
brainstem. Focal deposits or plaques were seen in the cortex. In the cerebellum, some plaques
were evidenced in the molecular layer.
In conclusion, BSE agent does not seem to be directly pathogenic to microcebe by oral route, but
necessitates an adaptation of the strain to macaca. ...page 324....tss
snip...
http://www.tse-forum.de/tse_forum/deutsch/oeffentlich/bilder/Abstract_BookFINAL_nov2.pdf
TSS