Download TSE diagnostics 4

IDEXX
ELISA
Western Blot
I) SDS-PAGE
II) Blotting
III) Immunodetection
and revelation
III) Immunodetection and revelation
linked protein.
weight marker
Secondary
linked to
causes theAbprecipitation
of an
peroxidase
insoluble (luminol
product andinoxigen
the
peroxide),
causing
the emission
nitrocellulose
membrane.
of
able to impress
a
Thelightquantity
of products
is
photographic
plate
proportional to
the amount of
Molecular
1)
Blocking: ofsaturation
of
2)
Incubation
the
3)
Incubation
of
the
aspecific
link
sites
(non-fat
dry
nitrocellulose
membrane
4a) Signal detection
nitrocellulose
membrane
milk)
with
aa primary
Detection
systemAb
based on a
with
enzyme-conjugated
secondary
linked
to an
4b) SignalAb
detection
secondary
Ab
(i.e peroxidase,
enzyme that acts on its
alkaline
phosphatase).
Detection
system based on
substrate
(DAB);
chemiluminiscence.
The enzyme-substrate reaction
PRIONICS®-CHECK WESTERN SR
1) Preparation of the brain sample and isolation of the scrapie
prion protein
The test sample is a small piece of obex, a defined region of the
brain stem.
The homogenized brain sample is incubated with an optimized
reagent mixture consisting of digestion enzymes and a buffer
solution (I).
This solution degrades the normal prion protein. Only the TSEspecific prion protein remains in the test sample.
2) Separation of proteins by gel electrophoresis and immunological
detection of scrapie prion protein
The proteins in the sample are then separated according to size
by gel electrophoresis (II) and transferred to a special blot
membrane for detection (III).
The scrapie-specific prion protein on the membrane is detected
with specific antibodies and is visualized on a digital file or on a
film.
It is especially designed
for the detection of
scrapie in sheep and
goats.
saPMCA
Detection of prions in blood
Joaquin Castilla, Paula Saa, Claudio Soto
Protein Misfolding Cyclic Amplification (PMCA) for the
presymtomatic detection of PrPsc in blood of hamster
experimentally infected with scrapie
Nature Medicine (Agosto 2005)
Confirmatory tests after rapid test
 Histology
 Immunohistochemistry
 Western blot
Atypical TSEs
 Atypical Scrapie
 BASE (Bovine Amyloidotic Spongiform Encephalopaty)
 BSE in Sheep/goat
Atypical TSEs
Atypical Scrapie
Atypical scrapie
 Atypical scrapie was first recorded in Norway (Benestad and others
2003) and reports of other `atypical' TSE phenotypes, including
those that are similar to or the same as Nor98 have now been
published, from France (Buschmann and others 2004b), Germany
(Buschmann and others 2004a, b), Sweden (Gavier-Widen and
others 2004), Ireland (Onnasch and others 2004), Portugal (Orge
and others 2004), Belgium (De Bosschere and others 2004) and
the UK (Everest and others 2005; M. M. Simmons, J. Spiropoulos,
H. Elliott, P. Webb, J. Nash, J. Ryan, Y. I. Spencer, personal
communication).
 Atypical scrapie can occur in countries with little or no classical
scrapie (for example, Norway and Portugal).
Atypical Scrapie in different European Countries
Nor98
 Not always detected with
rapid tests
 Characteristic neuropathologic
pattern (cerebellum, cortex)
 Not detected in VRQ,
particularly in AHQ … other
polimorphisms
 Single case/outbreak
 No involvement of LRS
 Characteristic molecular
pattern of PrPSc
M
Atypical
Scrapie
58.1
39.8
M
Atypical
scrapie
Classical
scrapie
29.0
20.1
14.3
(A)
Western blot detection of PrPres in sheep with atypical (A) or
classical (B) scrapie.
Note the complex electrophoretic pattern in atypical scrapie
with at least five major bands, including a band at ≈10–
12 kDa, while three bands between 18 and 30 kDa are
present in classical scrapie.
Baron T et al Vaccine 2007;25:5625-5630
TSEs typing
Titolo presentazione
Typing of TSEs strains
Lack of appropriate definition of strain
“Phenotypic” definition
Biological typing – specific, but too long (1-2 yrs)
Molecular typing (!?) – rapid, but with limited
specificity
Tests and laboratories approved for TSEs discrimination
VLA (UK)
WB M. Stack
IHC M. Jeffrey
AFFSA (Francia)
WB T. Baron
CEA (Francia)
WB J.P. Deslys
ELISA J. Grassi
FLI (Germania)
WB M. Groschup
CIDC (Olanda)
WB J. Langeveld
ISS (Italia)
WB U. Agrimi, R. Nonno
Typing of TSEs in conventional mice
• Disease characteristics in a panel of
inbred mice:



RIII, C57BL (PrP-a)
VM (PrP-b)
C57BLxVM (PrP-ab)
• Mean incubation period (Dickinson et al.,1968)
• Distribution of vacuolation in brain: “lesion
profile” (Fraser & Dickinson, 1968)
Biological characteristics of different strains
Natural
scrapie
Lines of mice
BSE
FSE
vCJD
Tempi di incubazione (in giorni)
days
(Bruce et al., 1997)
Discriminatory WB: validation ring trial
SAF84
25 kDa
20 kDa
P4
25 kDa
20 kDa
Discriminatory WB: mw & glicotype
Molecular weight
scrapie
Glicotype
BSE Sh
Atypical TSEs
BASE
(Bovine Amyloidotic Spongiform Encephalopaty)
 presence of PrP-immunopositive amyloid plaques, as opposed to
the lack of amyloid deposition in typical BSE cases
 different pattern of regional distribution and topology of brain
PrP(Sc) accumulation
 Western blot analysis showed a PrP(Sc) type with predominance
of the low molecular mass glycoform and a protease-resistant
fragment of lower molecular mass than BSE-PrP(Sc)
 the molecular signature of this previously undescribed bovine
PrP(Sc) was similar to that encountered in a distinct subtype of
sporadic Creutzfeldt-Jakob disease
 presence of PrP-immunopositive amyloid plaques, as opposed to the
lack of amyloid deposition in typical BSE cases
 different pattern of regional
distribution and topology of
brain PrP(Sc) accumulation
 Western blot analysis showed a PrP(Sc) type with predominance of
the low molecular mass glycoform and a protease-resistant fragment of
lower molecular mass than BSE-PrP(Sc)
 the molecular signature of this previously undescribed bovine
PrP(Sc) was similar to that encountered in a distinct subtype of
sporadic Creutzfeldt-Jakob disease
Western blot detection of
PrPres in cattle with atypical
forms of BSE, including the
H-type (lanes 5–6) and
L-type
(lane
3),
in
comparison with typical
BSE (panel A, lanes 2 and 4)
BSE
L-type BSE H-type H-type
29.0
20.1
14.3
Production percentages of
di-glycosylated (d)
mono-glycosylated (m)
un-glycosylated (u)
PrPres glycoforms, showing
the decreased levels of diglycosylated PrP in atypical
cases of BSE, especially in
the L-type, compared to
typical BSE
Baron T et al Vaccine 2007;25:5625-5630
Transmission of new bovine prion to mice
Baron et al. (2006) Emerging Infect Diseases 12, 11251128,
A recent study indicated that the type of BSE in older cattle
in the US (and potentially in Canada) are the same type as
the atypical BSE that has been identified in France.
Atypical TSEs
BSE in goat/sheep
BSE in goat/sheep
 There are only two confirmed cases of BSE in a sheep or a
goat that were not infected experimentally.
 These were in a French goat that died in 2002 and a
Scottish goat that died in 1990.
BSE in goat/sheep
Fig. 1. Western blot detection of PrPres in BSE (arrow) compared to three
scrapie sources, in C57Bl/6 mice (panel A) or in sheep (panel B).
The lower band corresponding to the unglycosylated PrPres shows a lower
apparent molecular mass in BSE-infected animals.
Baron T et al Vaccine 2007;25:5625-5630
Differential Diagnosis of Infections with the Bovine
Spongiform Encephalopathy (BSE) and Scrapie Agents
in Sheep
M. Jeffrey et al.
J. Comp. Pathol. 125, 271-284, 2001
LRS tissues
BSE
Scrapie
BSE infected sheep
CNS
BSE
Scrapie
BSE
Scrapie
Scrapie
BSE
Thank you for your attention
Franco Mutinelli
Istituto Zooprofilattico Sperimentale delle Venezie
e-mail: [email protected]