Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected
Sheep
Oliver Bannach1#*, Eva Birkmann1,2#, Elke Reinartz1, Karl-Erich Jaeger3,
Jan P. M. Langeveld4, Robert G. Rohwer5, Luisa Gregori5,7¤, Linda A. Terry6,
Dieter Willbold1,2, Detlev Riesner1
1 Institute of Physical Biology, Heinrich-Heine-University Düsseldorf,
Düsseldorf, Germany, 2 Institute of Complex Systems (ICS-6), Research Center
Jülich, Jülich, Germany, 3 Institute of Molecular Enzyme Technology,
Heinrich-Heine-University Düsseldorf, Research Center Jülich, Jülich, Germany, 4
Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The Netherlands,
5 VA Maryland Health Care System, Molecular Neurovirology Laboratory, Medical
Research Service 151, VA Medical Center, Baltimore, Maryland, United States of
America, 6 Animal Health and Veterinary Laboratories Agency, New Haw,
Addlestone, Surrey, United Kingdom, 7 Department of Neurology, University of
Maryland at Baltimore, Baltimore, Maryland, United States of America
Abstract Top
Prion diseases are transmissible neurodegenerative diseases affecting
humans and animals. The agent of the disease is the prion consisting mainly, if
not solely, of a misfolded and aggregated isoform of the host-encoded prion
protein (PrP). Transmission of prions can occur naturally but also accidentally,
e.g. by blood transfusion, which has raised serious concerns about blood product
safety and emphasized the need for a reliable diagnostic test. In this report we
present a method based on surface-FIDA (fluorescence intensity distribution
analysis), that exploits the high state of molecular aggregation of PrP as an
unequivocal diagnostic marker of the disease, and show that it can detect
infection in blood. To prepare PrP aggregates from blood plasma we introduced a
detergent and lipase treatment to separate PrP from blood lipophilic components.
Prion protein aggregates were subsequently precipitated by phosphotungstic acid,
immobilized on a glass surface by covalently bound capture antibodies, and
finally labeled with fluorescent antibody probes. Individual PrP aggregates were
visualized by laser scanning microscopy where signal intensity was proportional
to aggregate size. After signal processing to remove the background from low
fluorescence particles, fluorescence intensities of all remaining PrP particles
were summed. We detected PrP aggregates in plasma samples from six out of ten
scrapie-positive sheep with no false positives from uninfected sheep. Applying
simultaneous intensity and size discrimination, ten out of ten samples from
scrapie sheep could be differentiated from uninfected sheep. The implications
for ante mortem diagnosis of prion diseases are discussed.
snip...
As for any transmissible disease, sensitive and reliable diagnostic
procedures are obvious prerequisites to the control of transmission. To control
the BSE epidemic not only in Europe, but also in Japan and Canada, an effective
strategy of active monitoring is being carried out through post mortem testing
on cattle brain tissue. All of these tests are based on detection of the
PK-resistant forms of PrPSc except for a single test that detects PrPSc
aggregates captured by an aggregate-specific ligand without PK digestion [5].
The BSE epidemic is now largely contained. Approximately 200 cases of variant
CJD (vCJD) have shown, however, that BSE can cross the species barrier to human.
Unresolved problems include the lack of sensitive live tests, incomplete
knowledge of sources and routes of exposure and transmission, and means to
assess, monitor and manage the public health risks from infected blood.
Transmission via blood has been shown in experimental rodents like hamster
[6], [7] as well as in species naturally susceptible to prion diseases like
sheep and deer [8], [9]. Moreover, some cases of secondary variant Creutzfeldt
Jakob disease (svCJD) have been reported that were caused by blood transfusion
from presymptomatic vCJD patients [10], [11]. Transfusion transmission occurs
despite the low concentration of prion infectivity in blood, ~10 infectious
doses/ml in clinically affected rodents, or 7 to 9 orders of magnitude less than
the concentration in the brains of symptomatic mice or hamsters [7]. Post mortem
tests on brain samples can be carried out with high sensitivity and reliability,
whereas qualitatively similar tests based on body fluids of afflicted humans or
animals have yet to be developed. Blood tests are, however, highly desired for
pathogenesis studies, blood transfusion safety and CJD-therapy assessment.
In recent years significant progress has been made in the field of prion
diagnostics with the development of prion seeded amplification technologies like
protein misfolding cyclic amplification (PMCA, [12]), quaking induced conversion
(QuIC, [13]), and amyloid seeding assay (ASA, [14]). QuIC was successfully
applied to cerebrospinal fluid samples from sporadic CJD patients [15], [16] and
rodent blood [17]. Using PMCA, it has been possible to detect PrPSc in blood
from prion-infected hamsters, sheep and deer [18]–[23]. At present, however,
PMCA is carried out reliably, i.e. without false positives, only in highly
specialized laboratories. In another development, PrPSc was detected in the
peripheral mononuclear blood cells (PBMC) of scrapie-afflicted sheep [24], and
in blood samples of variant CJD cases by an improved immune detection method of
surface-captured prions that did not require the use of in vitro amplification
and protease digestion [25].
In this study we have investigated PrP aggregates, PK-resistant as well as
PK-sensitive forms, in blood plasma of scrapie-infected sheep. We have adapted
our previously developed fluorescence intensity distribution analysis
(surface-FIDA) technique [26], [27] for analysis of blood samples of sheep. PrP
aggregates are partially purified from blood plasma, captured on a surface by
covalently bound antibodies and made visible by fluorophore-labeled detection
antibodies. The fluorescence emitted in response to a scanning laser beam is
transformed into an image of the PrP fluorescence intensities on the surface.
Several features of the method, e.g. sample preparation, detection, and data
processing, guarantee that PrP aggregates can be differentiated safely from
PrPC. We show that PrP aggregates are detectable in blood of scrapie-infected
sheep and that their presence indicates scrapie infection.
snip...
In an earlier study, we described the detection of PrP aggregates with high
sensitivity in brain homogenate of BSE cattle, and in a small number of
cerebrospinal fluid samples from BSE cattle [26]. According to the literature,
infectivity in blood - even in symptomatic experimental hamsters - is as low as
10 infectious units per ml [33], [34]. In BSE-afflicted cattle infectivity is
absent from the lymphatic system and has never been reported in blood [35],
[36].
However, seeding activity was demonstrated in a small number of BSE serum
samples [37].
Considerable effort was spent not only in improving the sensitivity of the
assay but also in optimizing the preparation of PrP aggregates from blood
plasma. Though it is not certain that the PrP aggregates we analyzed are indeed
the carriers of infectivity in blood, they are a consistent marker of infection.
The direct determination of infectivity in these PrP aggregates from blood
remains to be established.
MAD COW USDA ATYPICAL L-TYPE BASE BSE, the rest of the story...
***Oral Transmission of L-type Bovine Spongiform Encephalopathy in Primate
Model
***Infectivity in skeletal muscle of BASE-infected cattle
***feedstuffs- It also suggests a similar cause or source for atypical BSE
in these countries.
***Also, a link is suspected between atypical BSE and some apparently
sporadic cases of Creutzfeldt-Jakob disease in humans.
full text ;
atypical L-type BASE BSE
Tuesday, May 1, 2012 BSE MAD COW LETTERS TO USDA (Tom Vilsack, Secretary
of Agriculture) and FDA (Magaret Hamburg, Commissioner of FDA) May 1, 2012
Wednesday, May 2, 2012
ARS FLIP FLOPS ON SRM REMOVAL FOR ATYPICAL L-TYPE BASE BSE RISK HUMAN AND
ANIMAL HEALTH
Friday, May 4, 2012
May 2, 2012: Update from APHIS Regarding a Detection of Bovine Spongiform
Encephalopathy (BSE) in the United States
Sunday, March 11, 2012
APHIS Proposes New Bovine Spongiform Encephalopathy Import Regulations in
Line with International Animal Health Standards Proposal Aims to Ensure Health
of the U.S. Beef Herd, Assist in Negotiations
Wednesday, April 4, 2012
Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine
Products APHIS-2008-0010-0008 RIN:0579-AC68
Sunday, May 6, 2012
Bovine Spongiform Encephalopathy Mad Cow Disease, BSE May 2, 2012 IOWA
State University OIE
Sunday, February 12, 2012
National Prion Disease Pathology Surveillance Center Cases Examined1
(August 19, 2011) including Texas
Subject: Prion diseases are efficiently transmitted by blood transfusion in
sheep Date: July 26, 2008 at 8:55 am PST
-------------------- BSE-L@LISTS.AEGEE.ORG --------------------
Submitted April 18, 2008 Accepted June 28, 2008
Prion diseases are efficiently transmitted by blood transfusion in sheep
Fiona Houston*, Sandra McCutcheon, Wilfred Goldmann, Angela Chong, James
Foster, Silvia Siso, Lorenzo Gonzalez, Martin Jeffrey, and Nora Hunter Division
of Animal Production and Public Health, Faculty of Veterinary Medicine,
University of Glasgow, Glasgow, United Kingdom Neuropathogenesis Division,
Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom Veterinary
Laboratories Agency, Lasswade Laboratory, Edinburgh, United Kingdom
* Corresponding author; email: f.houston@vet.gla.ac.uk.
The emergence of variant Creutzfeld-Jakob disease (vCJD), following on from
the bovine spongiform encephalopathy (BSE) epidemic, led to concerns about the
potential risk of iatrogenic transmission of disease by blood transfusion and
the introduction of costly control measures to protect blood supplies. We
previously reported preliminary data demonstrating the transmission of BSE and
natural scrapie by blood transfusion in sheep. The final results of this
experiment, reported here, give unexpectedly high transmission rates by
transfusion of 36% for BSE and 43% for scrapie. A proportion of BSE-infected
tranfusion recipients (3/8) survived for up to 7 years without showing clinical
signs of disease. The majority of transmissions resulted from blood collected
from donors at >50% of the estimated incubation period. The high transmission
rates and relatively short and consistent incubation periods in clinically
positive recipients suggest that infectivity titres in blood were substantial
and/or that blood transfusion is an efficient method of transmission. This
experiment has established the value of using sheep as a model for studying
transmission of vCJD by blood products in humans.
Greetings again Dr. Freas et al at FDA,
THIS was like closing the barn door after the mad cows got loose. not only
the red cross, but the FDA has failed the public in protecting them from the TSE
aka mad cow agent. TSE agent i.e. bse, base, cwd, scrapie, tme, and any sub
strains thereof. we do not know if these strains will or have transmitted to
humans as subclinical TSE or clinical disease, and we do not know if they have
or will transmit second, third, forth passage via friendly fire i.e. multiple
potential routes via medical, surgical, pharmaceutical etc.
Saturday, December 08, 2007
Transfusion Transmission of Human Prion Diseases
PRODUCT Recovered Plasma, Recall # B-1660-08 CODE Unit: 5336249 RECALLING
FIRM/MANUFACTURER Florida's Blood Centers, Inc., Orlando, FL, by electronic mail
and facsimile on June 4, 2007. Firm initiated recall is complete. REASON Blood
product, collected from a donor considered to be at increased risk for variant
Creutzfeldt-Jakob Disease (vCJD), was distributed. VOLUME OF PRODUCT IN COMMERCE
1 unit DISTRIBUTION Austria and FL
END OF ENFORCEMENT REPORT FOR JULY 23, 2008
###
see many more blood recalls below ;
Tuesday, October 09, 2007 nvCJD TSE BLOOD UPDATE
Saturday, December 08, 2007 Transfusion Transmission of Human Prion
Diseases
Saturday, January 20, 2007 Fourth case of transfusion-associated vCJD
infection in the United Kingdom
Sunday, May 1, 2011
W.H.O. T.S.E. PRION Blood products and related biologicals May 2011 http://transmissiblespongiformencephalopathy.blogspot.com/2011/05/who-tse-prion-blood-products-and.html
Wednesday, February 1, 2012
CJD and PLASMA / URINE PRODUCTS EMA Position Statements Alberto Ganan
Jimenez, European Medicines Agency PDA TSE Safety Forum, 30 June 2011
TSS
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