Sunday, September 23, 2012

EU-Approved Rapid Tests for Bovine Spongiform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities

EU-Approved Rapid Tests for Bovine Spongiform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities

Daniela Meloni1, Aart Davidse2, Jan P. M. Langeveld2, Katia Varello1, Cristina Casalone1, Cristiano Corona1, Anne Balkema-Buschmann3, Martin H. Groschup3, Francesco Ingravalle1, Elena Bozzetta1*

1 Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy, 2 Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands, 3 Friedrich-Loeffler Institut, Federal Research Institute for Animal Health, Insel Riems, Germany

Abstract Top Since 2004 it become clear that atypical bovine spongiform encephalopthies (BSEs) exist in cattle. Whenever their detection has relied on active surveillance plans implemented in Europe since 2001 by rapid tests, the overall and inter-laboratory performance of these diagnostic systems in the detection of the atypical strains has not been studied thoroughly to date. To fill this gap, the present study reports on the analytical sensitivity of the EU-approved rapid tests for atypical L- and H-type and classical BSE in parallel. Each test was challenged with two dilution series, one created from a positive pool of the three BSE forms according to the EURL standard method of homogenate preparation (50% w/v) and the other as per the test kit manufacturer's instructions. Multilevel logistic models and simple logistic models with the rapid test as the only covariate were fitted for each BSE form analyzed as directed by the test manufacturer's dilution protocol. The same schemes, but excluding the BSE type, were then applied to compare test performance under the manufacturer's versus the water protocol. The IDEXX HerdChek ® BSE-scrapie short protocol test showed the highest sensitivity for all BSE forms. The IDEXX® HerdChek BSE-scrapie ultra short protocol, the Prionics® - Check WESTERN and the AJ Roboscreen® BetaPrion tests showed similar sensitivities, followed by the Roche® PrionScreen, the Bio-Rad® TeSeE™ SAP and the Prionics® - Check PrioSTRIP in descending order of analytical sensitivity. Despite these differences, the limit of detection of all seven rapid tests against the different classes of material set within a 2 log10 range of the best-performing test, thus meeting the European Food Safety Authority requirement for BSE surveillance purposes. These findings indicate that not many atypical cases would have been missed surveillance since 2001 which is important for further epidemiological interpretations of the sporadic character of atypical forms.

Citation: Meloni D, Davidse A, Langeveld JPM, Varello K, Casalone C, et al. (2012) EU-Approved Rapid Tests for Bovine Spongiform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities. PLoS ONE 7(9): e43133. doi:10.1371/journal.pone.0043133

Editor: Corinne Ida Lasmezas, The Scripps Research Institute Scripps Florida, United States of America

Received: March 19, 2012; Accepted: July 16, 2012; Published: September 11, 2012

Copyright: © 2012 Meloni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors have no funding or support to report.

Competing interests: The authors have declared that no competing interests exist.

* E-mail:



In this study we have evaluated the analytical sensitivity of approved rapid tests for the current known atypical BSEs detection. It is to be noted that Seuberlich et al. [38] raised the possibility that a new prion disease not previously encountered and distinct from the known types of BSEs exists. Nevertheless, the information is really limited and the puzzle of the different observations has still to be assembled, considering that the results described remind the features of poorly digested normal PrP (known as the physiologically C2 fragment of PrP [39], [40]).

Referring to the tissues origin, is to be remarked that the investigated H-BSE tissues originated from intracranially challenged cattle, whereas the three other forms derived from field cases. Nevertheless, recent studies showed that biochemical and histopathological features of experimental H-type BSE animals were identical to that found with field H-type [12], [24], [41].

According to our results, all tests were able to detect both H- and L-BSE types at a 1:16 dilution prepared as directed by the manufacturer's instructions, with the same performance as for classical BSE.

The LOD varied across the tests. The IDEXX® HerdCheck BSE-scrapie short protocol showed the highest analytical sensitivity, as previously reported in a EURL study on classical BSE [21]. The performance of the AJ Roboscreen® BetaPrion, IDEXX® HerdCheck BSE-scrapie ultra short protocol, and Prionics® - Check WESTERN compared favourably with one another at our statistical analysis. The Prionics® - Check PrioSTRIP, Bio-Rad® TeSeE™ SAP and Roche® PrionScreen tests showed the lowest sensitivities for all the BSE types analyzed. These results were confirmed also using other explorative statistical approaches (e.g., Poisson models for number of positive replicates, receiver operating characteristic [ROC] curves) which we had initially applied (results not reported).

The analytical sensitivity of the tests was investigated in accordance with the requirements set by the relevant evaluation protocols established by the European Commission, the SSC and EFSA, using serial dilutions of sample replicates.

Test differences between the last positive dilutions of weak and strong C-BSE samples varies among the different systems from two to four factors (2 base logarithm) for buffer dilutions and from two to five factors for water dilutions. In this context, the different tests showed parallel results between the dilutions prepared following the two protocols. The dynamic range of each rapid test or rather the concentration range of PrPres that results in a change in response is a specific peculiarity of each diagnostic system.

The rate of conversion of substrate to coloured product should be proportional to the amount of PrPres within the well, but there are many limits to this depending on the analyte itself, that tends to aggregate rapidly in solution, and on the combination of methods and materials used within the test kits other than on the equipments.

A gradual stratification of the signal represents a surplus value for TSE rapid assays.

In our study, the Bio-Rad® TeSeE™ SAP test could surprisingly detect only the 1:2 dilution when challenged with positive C BSE weak samples. A loss of analytical sensitivity for this test was observed also during the active surveillance activity carried out from 2004 to 2008 by the Italian Reference Center for TSEs applying Bio-Rad® TeSeE™ test. In that context, a National batch testing was performed on every new batch prior to commercialization to provide reassurance that BSE rapid test kits were fit for the survey purpose. As a consequence, distribution of some kit batches was precluded because of the lack of signal showed on positive reference samples. Further to the unexpected poor performance of Bio-Rad® TeSeE™ within this study even after test repetition, the same Bio-Rad® homogenate sample set, according to previous studies in which its suitability for the IDEXX test was shown, was challenged with the last test revealing signals miming the ones reported for IDEXX test (data not shown). The question of whether the specific kit batch affected the test performance is of concern, but it is noteworthy that all producers were asked to provide a kit for this evaluation. Thereby, our results represent a picture of the kits available on the market.

The seven simple logistic models showed a meaningful difference between the dilution protocols only for the AJ Roboscreen® BetaPrion and Bio-Rad® TeSeE™ SAP. The lower bounds of the 95% confidence intervals for the Roche® PrionScreen and Prionics® - Check WESTERN tests approached 1 (0.9528 and 0.9755, respectively); for the remaining tests, there was no statistical evidence of a higher test sensitivity between the manufacturer's dilution protocol and the 50% w/v protocol (Figure 2).

Whenever in order to evaluate the field performances of BSE rapid post mortem tests the manufacturers' protocol represents the term of reference, the relevance of water dilution-based results relies on the specific Annex X of Regulation (EC) 999/2001 requirements. NRLs for TSE periodically have to verify national diagnostic standards and methods by means of comparative trials. The objectives are to monitor national rapid test activity and to demonstrate to the EC that the rapid surveillance system is effective. EURL itself annually verifies the interlaboratory agreement of the rapid systems used by the NRLs.

As previously reported in the EURL study [20], the analytical sensitivity values obtained under the 50% w/v protocol were from one to three dilutions inferior to those obtained under the specific homogenization protocol. For all the tests except one, the discrepancies between the two modes of dilutions were similar whatever the sample tested. Particularly with the Bio-Rad test the strong positive C -BSE sample was four factors lower when the water protocol was applied. Anyway, this is congruent with the EFSA 2009 results [21], where the discrepancy set at three logarithms. This difference needs to be taken into account when organizing ring trials, during which a less sensitive test could be penalized.

To rule out a possible decrement of the signal related to the storage of the water aliquots, and because of the scarcity of atypical BSE material, the laboratory test exercise was completed within a 15-day period. This precautionary approach was taken as no data exist on the stability of atypical BSE homogenates, whereas differences in stability have been observed for atypical versus classical scrapie [21], [42], [43], [44]. Further, as it is indeed known that the results of some tests can lapse while approaching the expiring date of kit batches, the kits provided for the evaluation were expected to expire from three to six months after the date of testing. Table S1 lists the kit batches used, the expiring dates and the days of testing.

With regard to the homogeneity of serial dilutions, as PrPres is amyloidogenic, the fibrils tend to aggregate in solution [45], thus potentially hindering a real homogeneity of dilution series. In our study, the ICC of the replicates was higher than 0.99. This ensured that, whenever the amounts of BSE tissues available were extremely limited, the material tested was homogeneous.

When considering the working principle of rapid tests, summarized in the Text S1, all approved tests include a PK digestion step to unmask cryptic epitopes, except for the IDEXX HerdChek® BSE-scrapie EIA, which relies on conformational detection technology using a specific aggregate specific capture ligand on a dextran polymer (Seprion ligand technology, Microsens Biotechnologies, London, UK) [46]. The severe effects of proteinase K (PK) in digesting atypical PrPres are well known. Depending on the PK concentration, signal loss after atypical BSE-related PrPres PK digestion varies from less than 20% for the C-type isolates to more than 50% for both L- and H-type BSE tissues [4]. This could be the reason for the higher sensitivity of the IDEXX test in detecting atypical BSEs compared to the others. However, the type of detergent used in homogenates and the type of TSE strain used do affect the extent of PrPres degradation, and this remains a matter of further study [47].

With regard to the interpretation of results, five of the rapid tests in this study are based on semi-quantitative ELISA methods that produce a qualitative result relative to a cut-off value. To minimize subjectivity, the study's Prionics® - Check PrioSTRIP results were interpreted with the use of the computerized PrioSCAN® software, although visual interpretation by two independent readers was also validated. The Prionics® - Check Western is both a qualitative and quantitative test, as it distinguishes PrPres in non-, mono-, and diglycoforms while expressing their respective quantitative ratio and migration positions. The diagnostic criteria for positive results are based on the exhibition of a three-band signal, the top one corresponding to a protein with an approximate molecular weight of 30 kD. Signal intensity decreases from top to bottom, but the higher band should be clearly visible immediately under the PK band. Significant blot images of atypical BSE dilution series obtained on in the frame of this study are presented in the Figure S1. In addition, extremely weak samples, notably for atypical BSE strains, can vary in their conventional blot pattern that fit positive criteria. Glycoform separation on the Sodium Dodecyl Sulphate PolyAcrylamide gel by electrophoresis causes the PrPres signal to thin out along the migration line rather than concentrate in a narrow area, as occurs with ELISA and immunochromatographic methods. This means that if the relative non-, mono-, and diglycoform immunoreactivity ratios of L-BSE are taken as corresponding roughly to 39%, 35%, and 26% [48], the blot signal characterizing the last tissue ratio meeting the non-negative criteria generates from only 39% of the total prion protein on the migration line. Despite this, the Prionics® - Check Western was found to be among the more sensitive systems, indicating that the interpretation of a specific PrPres marker by an expert reader can increase the test's sensitivity.

In conclusion, despite the evidence of clear differences in relative analytical sensitivity, the LOD of all seven rapid tests included in this study, against all the classes of material used, was within a 2 log10 range of the best-performing test, thus meeting EFSA criteria for rapid tests for BSE monitoring.

No certain conclusions on the field of diagnostic performance of these rapid-test kits can be drawn from our results on their analytical sensitivity, as the two parameters are not directly linked, anyway samples from animals exhibiting subclinical signs [24], could be expected to behave similarly to extremely diluted CNS tissues used in analytical sensitivity studies.

The outcome of this study endorses the current epidemiological follow up and interpretation of all three BSE forms prevalence [49], [50] and means that for epidemiological studies the data obtained in the different countries and regions of EU can be considered equally, as plausibly, most stronger atypical cases have been detected by the different rapid tests.

see full text ;

Performance analysis of rapid diagnostic tests on atypical bovine spongiform encephalopathy

John G. Gray Sandor Dudas Catherine Graham Stefanie Czub1 Canadian Food Inspection Agency, Lethbridge Laboratory, Lethbridge, Alberta, Canada ↵1 Stefanie Czub, Canadian Food Inspection Agency, Lethbridge Laboratory. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.


The preferred method to determine the prevalence of bovine spongiform encephalopathy (BSE) in a country is to use immunology-based rapid-tests. Though these tests are validated to detect C-type BSE disease–associated prion (PrPsc), test-specific properties may influence their ability to detect H- and/or L-type BSE PrPsc, where both are atypical from C-type PrPsc. Molecular characterization shows atypical BSE PrPsc to have a different sensitivity to proteinase activity and different affinities for certain prion-specific antibodies. It is important to understand how atypical BSE PrPsc may affect the performance of rapid-tests, which are typically dependant on the use of specific proteases and antibodies. The current study used experimentally generated C-, H-, and L-type BSE PrPsc to evaluate 3 tests used in various national BSE surveillance programs: an immunochromatographic assay, a standard sandwich enzyme-linked immunosorbent assay (stndELISA), and a PrPsc-conformation–specific ELISA (confELISA). Although BSE PrPsc type had some effects on rapid-test performance, analytical sensitivity for atypical BSE PrPsc on all 3 platforms was not significantly compromised. When testing for atypical BSE PrPsc, the 3 tests were able to meet the same requirements that the European Food Safety Authority set when evaluating the tests for C-type BSE PrPsc.

Tuesday, September 11, 2012

Japan Moves Closer To Raising 20-Month Age Limit For Beef Imports, and further risk consumer to CJD

Monday, September 3, 2012


Sunday, August 26, 2012

Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with BSE

Sunday, August 19, 2012

Susceptibility of cattle to the agent of chronic wasting disease from elk after intracranial inoculation 2012


Friday, February 24, 2012




Subject: BSE--U.S. 50 STATE CONFERENCE CALL Jan. 9, 2001

Date: Tue, 9 Jan 2001 16:49:00 -0800

From: "Terry S. Singeltary Sr."

Reply-To: Bovine Spongiform Encephalopathy


######### Bovine Spongiform Encephalopathy #########

Greetings List Members,

I was lucky enough to sit in on this BSE conference call today and even managed to ask a question. that is when the trouble started.

I submitted a version of my notes to Sandra Blakeslee of the New York Times, whom seemed very upset, and rightly so.

"They tell me it is a closed meeting and they will release whatever information they deem fit. Rather infuriating."

and i would have been doing just fine, until i asked my question. i was surprised my time to ask a question so quick.

(understand, these are taken from my notes for now. the spelling of names and such could be off.)

[host Richard Barns] and now a question from Terry S. Singeltary of CJD Watch.

[TSS] yes, thank you, U.S. cattle, what kind of guarantee can you give for serum or tissue donor herds?

[no answer, you could hear in the back ground, mumbling and 'we can't. have him ask the question again.]

[host Richard] could you repeat the question?

[TSS] U.S. cattle, what kind of guarantee can you give for serum or tissue donor herds?

[not sure whom ask this] what group are you with?

[TSS] CJD Watch, my Mom died from hvCJD and we are tracking CJD world-wide.

[not sure who is speaking] could you please disconnect Mr. Singeltary

[TSS] you are not going to answer my question?

[not sure whom speaking] NO

from this point, i was still connected, got to listen and tape the whole conference. at one point someone came on, a woman, and ask again;

[unknown woman] what group are you with?

[TSS] CJD Watch and my Mom died from hvCJD we are trying to tract down CJD and other human TSE's world wide. i was invited to sit in on this from someone inside the USDA/APHIS and that is why i am here. do you intend on banning me from this conference now?

at this point the conference was turned back up, and i got to finish listening. They never answered or even addressed my one question, or even addressed the issue. BUT, i will try and give you a run-down for now, of the conference.

IF i were another Country, I would take heed to my notes, BUT PLEASE do not depend on them. ask for transcript from;

RBARNS@ORA.FDA.GOV 301-827-6906

he would be glad to give you one ;-)

Rockville Maryland, Richard Barns Host

BSE issues in the U.S., How they were labelling ruminant feed? Revising issues.

The conference opened up with the explaining of the U.K. BSE epidemic winding down with about 30 cases a week.

although new cases in other countries were now appearing.

Look at Germany whom said NO BSE and now have BSE.

BSE increasing across Europe.

Because of Temporary Ban on certain rendered product, heightened interest in U.S.

A recent statement in Washington Post, said the New Administration (old GW) has a list of issues. BSE is one of the issues.

BSE Risk is still low, minimal in U.S. with a greater interest in MBM not to enter U.S.

HOWEVER, if BSE were to enter the U.S. it would be economically disastrous to the render, feed, cattle, industries, and for human health.

(human health-they just threw that in cause i was listening. I will now jot down some figures in which they told you, 'no need to write them down'. just hope i have them correct. hmmm, maybe i hope i don't ???)

80% inspection of rendering

*Problem-Complete coverage of rendering HAS NOT occurred.

sizeable number of 1st time FAILED INITIAL INSPECTION, have not been reinspected (70% to 80%).

Compliance critical, Compliance poor in U.K. and other European Firms.

Gloria Dunason Major Assignment 1998 goal TOTAL compliance. This _did not_ occur. Mixed level of compliance, depending on firm.

Rendering FDA license and NON FDA license

system in place for home rendering & feed 76% in compliance 79% cross contamination 21% DID NOT have system 92% record keeping less than 60% total compliance

279 inspectors 185 handling prohibited materials

Renderer at top of pyramid, significant part of compliance. 84% compliance

failed to have caution statement render 72% compliance & cross contamination caution statement on feed, 'DO NOT FEED TO CATTLE'


1240 FDA license feed mills 846 inspected

"close to 400 feed mills have not been inspected"

80% compliance for feed.

10% don't have system.

NON-FDA licensed mills There is NO inventory on non licensed mills. approximately 6000 to 8000 Firms ??? 4,344 ever inspected. "FDA does not have a lot of experience with"

40% do NOT have caution statement 'DO NOT FEED'.

74% Commingling compliance

"This industry needs a lot of work and only half gotten to"

"700 Firms that were falitive, and need to be re-inspected, in addition to the 8,000 Firms."

Quote to do BSE inspection in 19 states by end of January or 30 days, and other states 60 days. to change feed status??? Contract check and ask questions and pass info.

At this time, we will take questions.

[I was about the third or fourth to ask question. then all B.S.eee broke loose, and i lost my train of thought for a few minutes. picked back up here]

someone asking about nutritional supplements and sourcing, did not get name. something about inspectors not knowing of BSE risk??? the conference person assuring that Steve Follum? and the TSE advisory Committee were handling that.

Some other Dr. Vet, whom were asking questions that did not know what to do???

[Dennis Wilson] California Food Agr. Imports, are they looking at imports?

[Conference person] they are looking at imports, FDA issued imports Bulletin.

[Linda Singeltary ??? this was a another phone in question, not related i don't think] Why do we have non-licensed facilities?

(conference person) other feed mills do not handle as potent drugs???

Dennis Blank, Ken Jackson licensed 400 non FDA 4400 inspected of a total of 6000 to 8000, (they really don't know how many non licensed Firms in U.S. they guess 6000 to 8000??? TSS)

Linda Detwiler asking everyone (me) not to use emergency BSE number, unless last resort. (i thought of calling them today, and reporting the whole damn U.S. cattle herd ;-) 'not'

Warren-Maryland Dept. Agr. Prudent to re-inspect after 3 years. concerned of Firms that have changed owners.



snip...see full text and more here on tissue donor herds and the TSE Prion disease ;


Tuesday, November 02, 2010


The information contained herein should not be disseminated further except on the basis of "NEED TO KNOW".

BSE - ATYPICAL LESION DISTRIBUTION (RBSE 92-21367) statutory (obex only) diagnostic criteria CVL 1992

APHIS Factsheet
Animal and Plant Health Inspection Service April 2012
BSE Confirmatory Tests
Immunohistochemistry (IHC)
• Primary confirmatory test for USDA’s BSE surveillance program.
• Recognized by the World Organization for Animal Health, or OIE.
• Allows scientists to determine if a sample is positive for BSE in two distinct ways: visually (spongiform changes), and through a staining technique (presence of abnormal prion protein).
• Involves looking at an intact portion of the brain, the obex, to see if there are lesions (holes or a “spongy” appearance) present that are characteristic for BSE, and using a staining process using antibodies that detect the abnormal protein prion.
• Takes four to seven days to run.
• Freezing samples does not interfere with performing the IHC test as long as the sample is confirmed as obex. Western Blot
• Several types, with the SAF Immunoblot and commercially available Western blot kits recognized by OIE.
• Used under USDA protocol when a sample is “not suitable for IHC”, i.e., if it is autolyzed (or degraded) or brain stem architecture is not evident microscopically.
• Uses a large portion of obex brain tissue; the abnormal prion protein in brain material is enriched, and the sample is exposed to protease, an enzyme, to destroy any normal prion proteins that may be present, leaving only abnormal prion proteins. Remaining sample is then run through a gel to separate the abnormal prion protein components by molecular weight. After the transfer of the proteins to a membrane, proteins are stained using antibodies that can identify a specific banding pattern associated with prion diseases including BSE. A diagnosis is made by recognizing three distinctive bands that are identified as a result of a reaction with the anti-prion protein antibody.
• Freezing samples does not interfere with the performance of western blot tests.
• Both IHC and the Immunblot (Western blot) are internationally recognized as confirmatory tests for BSE.
• The tests use different methods to determine if the abnormal prion protein is present in brain tissue of an animal.
• The IHC test additionally allows for the viewing of brain tissue to determine if lesions characteristic to BSE are present.
• Both tests are equally effective at detecting the classical form of BSE.
United States Department of Agriculture • Animal and Plant Health Inspection Service • Safeguarding American Agriculture
USDA is an equal opportunity provider and employer.


in the url that follows, I have posted

SRM breaches first, as late as 2011.


MAD COW FEED BAN BREACHES AND TONNAGES OF MAD COW FEED IN COMMERCE up until 2007, when they ceased posting them.



Friday, May 18, 2012

Update from APHIS Regarding a Detection of Bovine Spongiform Encephalopathy (BSE) in the United States Friday May 18, 2012

Saturday, May 26, 2012

Are USDA assurances on mad cow case 'gross oversimplification'?


What irks many scientists is the USDA’s April 25 statement that the rare disease is “not generally associated with an animal consuming infected feed.”

The USDA’s conclusion is a “gross oversimplification,” said Dr. Paul Brown, one of the world’s experts on this type of disease who retired recently from the National Institutes of Health.

"(The agency) has no foundation on which to base that statement.”

“We can’t say it’s not feed related,” agreed Dr. Linda Detwiler, an official with the USDA during the Clinton Administration now at Mississippi State.

In the May 1 email to me, USDA’s Cole backed off a bit. “No one knows the origins of atypical cases of BSE,” she said

The argument about feed is critical because if feed is the cause, not a spontaneous mutation, the California cow could be part of a larger outbreak.



Saturday, August 4, 2012

Final Feed Investigation Summary - California BSE Case - July 2012



Summary Report BSE 2012

Executive Summary

Saturday, August 4, 2012

Update from APHIS Regarding Release of the Final Report on the BSE Epidemiological Investigation

Sunday, August 26, 2012

Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with BSE

2011 Monday, September 26, 2011

L-BSE BASE prion and atypical sporadic CJD

Tuesday, June 26, 2012

Creutzfeldt Jakob Disease Human TSE report update North America, Canada, Mexico, and USDA PRION UNIT as of May 18, 2012

type determination pending Creutzfeldt Jakob Disease (tdpCJD), is on the rise in Canada and the USA

Monday, July 23, 2012

The National Prion Disease Pathology Surveillance Center July 2012


Wednesday, September 12, 2012

Blood test closer for mad cow disease, Alzheimers and Parkinson's

Blood test closer for mad cow disease, Alzheimers and Parkinson's

by: Pia Akerman From:The Australian

September 12, 201210:51AM

BLOOD tests to diagnose Alzheimers disease, Parkinson's and the human form of mad cow disease could be closer thanks to new Australian research.

Research by University of Melbourne scientists, published in the Nucleic Acids Research journal this week, has identified specific particles (exosomes) released by cells infected with prions, the pathogen that causes diseases such as human variant Creutzfeldt-Jakob disease.

It raises the possibility of a diagnostic blood test identifying the exosomes in the blood stream.

Thousands of Australians are currently banned from donating blood because they lived in the UK when mad cow disease was present in local beef and there is no easy test to identify the risk.

Since 2004 a small number of British residents have been diagnosed with vCJD transmitted through blood transfusion.

University of Melbourne associate professor Andrew Hill, who worked on the research, said further testing was needed.

He said while Alzheimer's and Parkinson's were not transmissible like prion diseases such as vCJD, they similarly released the exosome particles.

"Our thinking is that they might too have a unique signature on them that we could potentially pick up in blood as well," he said.

"Obviously these brain diseases are hard to diagnose because you need very extensive imaging tests to see what's going on inside the brain."

The Red Cross has previously said its ban on blood donations from people who lived in the UK between 1980 and 1996 for more than six months, or received blood transfusions there, would remain until there was a reliable blood screening test for vCJD.

Nucleic Acids Research Advance Access published September 10, 2012

Nucleic Acids Research, 2012, 1–13 doi:10.1093/nar/gks832

Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells

Shayne A. Bellingham1,2, Bradley M. Coleman1,2 and Andrew F. Hill1,2,3,*

1Department of Biochemistry and Molecular Biology,

2Bio21 Molecular Science and Biotechnology Institute and

3Mental Health Research Institute, The University of Melbourne, Parkville, Victoria 3010, Australia

Received December 8, 2011; Revised and Accepted August 9, 2012


Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrPC, and the abnormal infectious form, PrPSc, are found associated with exosomes, which are small 50–130nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, noncoding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.


While this hypothesis remains to be tested, these observations leave open the possibility that miRNAs are packaged into exosomes as a result of PrPC binding AGO1 and AGO2 promoting formation of miRISCs on the MVB, which functions as checkpoint for scanning mRNAs. Therefore selecting AGO-bound complementary mRNA:miRNAs that are to be repressed, while non-complementary miRNAs are packaged into ILVs along with PrPC and released with exosomes. Whether misfolding of PrPC into PrPSc during prion disease infection alters its ability to bind to Argonaute proteins, modulates the function of miRISC on the MVB and subsequent release of miRNA in exosomes during prion diseases certainly deserves investigation. Given that, we have identified significant changes in particular miRNA species released in association with exosomes from prion-infected cells, its plausible to suggest that miRNAs are selectively packaged as a direct result of PrPC and PrPSc and its influence on the miRNA biogenesis pathway. In summary, our results strongly support the hypothesis that exosomes released from prion-infected neuronal cells have a distinct miRNA signature that may be utilized for the identification of prion infection. This signature comprises significant increases in let-7 b, let-7i, miR-128 a, miR-21, miR-222, miR-29 b, miR-342-3 p and miR-424 with decreased miR-146 a detection and agrees to some extent to previously reported miRNA changes detected in brains of terminally infected mouse and primate models of prion disease, and sporadic CJD samples (17,18). Evaluation of our exosomal miRNA signature in circulating exosomes derived from clinical plasma samples from sporadic and variant forms of human prion disease and in animal models infected with different prion strains will be the subject of our further studies. Importantly, it has been shown the miRNAs deregulated in prion-infected exosomes identified in this study have also been detected in circulating exosomes isolated from human serum samples (14), and that neither have currently been detected in disease-associated exosomes in the current literature and a search of ExoCarta database (58), suggesting that this miRNA signature has significant and specific diagnostic potential. However, it should be noted that our study also identified other ncRNAs and mRNA fragments (Supplementary Figure S1) that may also be deregulated in exosomes released from prioninfected neuronal cells. Furthermore, it has been identified that extracellular miRNA released from cells into plasma can associate in two populations, both dependent and independent of exosomes either bound to AGO2 (59–61) or high-density lipoproteins (62). Therefore, targeted exosomal purification strategies for enrichment of circulating miRNA biomarkers may be required to increase biomarker sensitivity (14,15,23). This research also has potential diagnostic implications for other neurodegenerative diseases in which exosomes have been identified to play a role including Alzheimer’s disease (63– 65), amyotrophic lateral sclerosis (66) and Parkinson’s disease (67).


Endogenous Viral Etiology of Prion Diseases

Thursday, August 16, 2012

Blood products, collected from a donor who was at risk for variant Creutzfeldt-Jakob disease ( vCJD) USA JUNE, JULY, AUGUST 2012


Wednesday, May 16, 2012

Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?

Proposal ID: 29403


Thursday, September 6, 2012

UK Breaches of BSE controls in consignments of beef 2011 communications missing four reports

UK Breaches of BSE controls in consignments of beef 2011 communications missing four reports

Last updated on 6 September 2012

Breaches of BSE controls in consignments of beef

Due to an oversight, four breaches of BSE controls in British beef identified last year were not publicised immediately on the Agency’s website in the normal manner. .

As all specified risk material (SRM) was removed from the beef carcasses it is highly unlikely that there was any health risk to members of the public. SRM is those parts of the animal most likely to contain BSE infectivity. Under European law, SRM must be removed from the carcass after slaughter, stained and disposed of safely.

In the interests of transparency, the Agency is now issuing reports on these incidents. ..

BSE breaches August 2011

During routine inspections in August 2011 by staff in the Department of Agriculture and Rural Affairs in Northern Ireland (DARD), breaches of BSE controls were discovered in three separate consignments of beef sides received at Omagh Meats, an approved slaughterhouse and cutting plant in Northern Ireland.

On 17 August 2011, a consignment of 76 beef sides was received from West Devon Meat Ltd, an approved slaughterhouse near Okehampton. The documentation accompanying this consignment did not separately identify sides originating from animals aged over 30 months (OTM) and animals under 30 months of age (UTM).

On 23 August 2011, two other consignments of beef sides were received from West Scottish Lamb, an approved slaughterhouse in Carlisle.

The first of these consignments contained 108 sides, 52 originating from OTM animals and 56 from UTM animals. Six of the OTM sides were labelled incorrectly as being from UTM cattle.

The second consignment consisted of 118 sides. Sixteen of the sides listed in the documentation as being from OTM animals were found to be labelled incorrectly as UTM. In addition, two OTM sides were listed incorrectly as UTM on the accompanying documentation.

The vertebral column of OTM cattle is SRM. The European Union transmissible spongiform encephalopathy controls require that UTM and OTM carcasses are identified separately on consignment documentation and are also labelled differently. Although the sides in these consignments were identified correctly by DARD inspectors and Omagh Meats, in accordance with normal practice at Omagh Meats, all the sides were treated as originating from OTM cattle and the vertebral column was removed and treated as SRM.

The receiving business, Omagh Meats, was not responsible for the breaches. ..

BSE breaches September 2011

In a separate incident, on 1 September 2011, during routine inspection at a different approved slaughterhouse and cutting plant in Northern Ireland, spinal cord was found in a health-marked beef quarter received in a consignment of 224 quarters from ABP Sturminster Newton, an approved slaughterhouse in Dorset. All the other quarters were checked and no further SRM was discovered.

The spinal cord in cattle over 12 months of age is SRM and must be removed. The non-compliant quarter was destroyed and no SRM entered the food chain.

The receiving business was not responsible for the breach.

The official vets in all the originating plants have been informed of the breaches and steps have been taken, in collaboration with the plant management, to prevent a recurrence.

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