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
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.
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 . 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 ,  as well as in species naturally susceptible to prion diseases like sheep and deer , . Moreover, some cases of secondary variant Creutzfeldt Jakob disease (svCJD) have been reported that were caused by blood transfusion from presymptomatic vCJD patients , . 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 . 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, ), quaking induced conversion (QuIC, ), and amyloid seeding assay (ASA, ). QuIC was successfully applied to cerebrospinal fluid samples from sporadic CJD patients ,  and rodent blood . Using PMCA, it has been possible to detect PrPSc in blood from prion-infected hamsters, sheep and deer –. 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 , 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 .
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 ,  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.
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 . According to the literature, infectivity in blood - even in symptomatic experimental hamsters - is as low as 10 infectious units per ml , . In BSE-afflicted cattle infectivity is absent from the lymphatic system and has never been reported in blood , .
However, seeding activity was demonstrated in a small number of BSE serum samples .
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: email@example.com.
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