Wednesday, March 26, 2008

Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

Volume 14, Number 4–April 2008


Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

Stéphanie Simon,* Jérôme Nugier,* Nathalie Morel,* Hervé Boutal,* Christophe Créminon,* Sylvie L. Benestad,† Olivier Andréoletti,‡ Frédéric Lantier,§ Jean-Marc Bilheude,¶ Muriel Feyssaguet,¶ Anne-Gaëlle Biacabe,# Thierry Baron,# and Jacques Grassi* *Commissariat à l'Energie Atomique, Gif-sur-Yvette, France; †National Veterinary Institute, Oslo, Norway; ‡Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Toulouse, France; §Infectiologie Animale et Santé Publique, Tours, France; ¶Bio-Rad, Marnes-la-Coquette, France; and #Agence Française de Sécurité Sanitaire des Aliments, Lyon, France


The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)–infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc.


Analysis of Nor98 Isolates

The typing test was used to analyze 18 sheep isolates from Norway (Table 3). Ratios were almost impossible to calculate because of the large decrease in signal in A´ conditions, as shown in Figure 5, panel A for 3 isolates. Only 1 sample (Lavik) showed characteristics of a conventional scrapie isolate, providing an A/A´ ratio of 0.84 (Figure 5, panel A), a normalized ratio of 0.11, and a Western blot profile close to that of a French scrapie isolate (Figure 4, panel B, lanes 3 and 9; Figure 5, panel B, lanes 2 and 4). Other samples had a pattern that included a 12-kDa band (Figure 5, panel B) (19,22,34), characteristic of the Nor-98 strain.

After adapting the conditions of the PK treatment in the second set of measurements (A´ conditions), we observed (see legend, Figure 6) a much lower A/A´ ratio for those Nor-98, which enables discrimination of highly sensitive PK samples (nos. 24 and 26, Appendix Figure 2 and Table 3) to mildly sensitive PK samples (nos. 8, 11, 16, and 22).


When this study was initiated, no case of natural BSE in small ruminants was recorded, and only a few experimental ovine BSE samples were available, all belonging to the same PrP genotype (ARQ/ARQ), and mainly from a first passage. The possible impact of the genotype, the route of infection, and the number of passages on the biochemical properties of PrPres associated with the BSE strain are poorly understood. Now, further data suggest that, at least during the second passage in sheep, the biochemical properties (glycoform pattern in brain) of the BSE agent are unchanged (35,36). In this study using our ELISA, small ruminant BSE samples clearly behaved differently from conventional scrapie samples. However, slight differences may exist (see ARQ/ARQ vs. ARR/ARR genotype in Appendix Figure 1). We do not know whether these findings reflect differences in the PK sensitivity of PrPres associated with these genotypes or the influence of different tissues.

The main difficulty encountered for the development of a typing test is evaluation of its specificity and sensitivity. In the current study, we unambiguously identified all 37 experimental ovine BSE samples from 25 sheep, including 10 from a second passage. There are few data describing the molecular features of PrPres associated with experimental BSE in goats (37,38). In the framework of the French scrapie strain-typing network, 18 goats were analyzed by this ELISA, and 2 appeared compatible with experimental ovine and caprine BSE. One of them (Ch636), when analyzed with other molecular typing tests, appeared indistinguishable from experimental BSE and was later confirmed as the first natural case of BSE in a goat (1), after experimental transmission in wild-type and transgenic mice. The second BSE compatible sample (TR041528) was later clearly identified as a case of atypical scrapie as defined by its migration pattern (34). All these data suggest a good sensitivity for our test, which unambiguously identified all cases of experimental BSE in the sheep and goats tested, as well as the only natural case identified to date in a goat.

Another key point during the development of this test was to ensure good reproducibility because this parameter clearly influences both sensitivity and specificity. Ratios obtained for the classic scrapie control were highly reproducible, whereas ratios measured for the experimental BSE in sheep and the intermediate scrapie control varied much more, leading to an overlap of the 95% confidence interval (Table 1). To minimize interassay variations, the ratio obtained for each unknown sample was thus normalized by taking as reference the ratio measured for the ovine BSE sample (Figure 2, panel C, and Figure 3) in the same experiment. This enabled us to define the range of normalized ratio compatible with BSE as the mean of experimental ovine BSE ± 2s on the basis of reproducibility experiments recorded in Table 1. This range was experimentally determined between 0.7 and 1.3, leading to 3 categories for field samples: conventional scrapie (ratio <0.7),>1.3).

Only 10 (3.8%) of the 260 samples analyzed in the framework of the French epidemiologic surveillance network during 2002–2003 gave a ratio compatible with BSE. Of the 10 BSE suspected samples, only 1 goat sample (Ch636) was later confirmed as a true natural BSE case (1). This result indicates that the specificity of this test is not that good because 9 false-positive results were recorded in 260 samples (specificity 96.5%). However, the test appears useful since it excluded the presence of BSE for most field samples, thus restricting the use of more specific but time-consuming methods, like experimental transmission in mice, to a small number of isolates. Moreover, in a single screening, this test classified all TSE-infected isolates as a function of their PK resistance and thus provided a rapid classification of sheep isolates according to this criterion. The test could also be modified, by adjusting the range of PK sensitivity, to classify Nor-98 isolates.

All these data demonstrate that this ELISA-based typing test is suitable for a routine analysis of field samples, as assessed by the positive evaluation from the European Commission as one of the tests recommended to identify the possible presence of BSE in small ruminant flocks ( These typing tests are mainly designed to identify the BSE strain in small ruminant flocks. They are performed exclusively in national reference laboratories and based on Western blot techniques. In this context, the present ELISA is one of the secondary tests to be used to confirm BSE suspicion. We believe it will help clarify the status of these unusual isolates.



High incidence of subclinical infection of lymphoid tissues in scrapie-affected sheep flocks

Journal Archives of Virology Publisher Springer Wien ISSN 0304-8608 (Print) 1432-8798 (Online) Issue Volume 153, Number 4 / April, 2008 Category Original Article DOI 10.1007/s00705-008-0035-8 Pages 637-644 Subject Collection Biomedical and Life Sciences SpringerLink Date Tuesday, January 29, 2008

Gudmundur Georgsson1 , Jona Adalheidur Adolfsdottir1, Astridur Palsdottir1, Einar Jorundsson1, 3, Sigurdur Sigurdarson2, 4 and Stefania Thorgeirsdottir1

(1) Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavík, Iceland (2) Laboratory of Chief Veterinary Officer, Keldur, Reykjavík, Iceland (3) Present address: Ministry of Education, Science and Culture, Solvholsgata 4, 101 Reykjavík, Iceland (4) Present address: Agricultural Authority of Iceland, Austurvegur 64, 800 Selfoss, Iceland

Received: 12 November 2007 Accepted: 27 December 2007 Published online: 29 January 2008

Abstract Prion diseases are characterized by a long incubation period. In scrapie, sheep may incubate and spread the infection for several years before clinical signs evolve. We have previously studied the occurrence of subclincal infection in the brain. Now, we have studied the occurrence of subclinical infection in the brain and several lymphoid tissues in two scrapie-affected Icelandic sheep flocks by immunohistochemistry for PrPSc, a molecular marker for infectivity, and correlated this with results of PrP genotyping. At culling, one flock had one confirmed scrapie case, while the other flock had two. Analysis of 106 asymptomatic sheep by immunostaining for PrPSc revealed that the incidence of subclinical infection was 58.3% in one flock and 42.5% in the other. PrPSc was only detected in lymphoid tissues. The youngest positive sheep were 4 months old. PrP genotyping showed that over 90% of the sheep were of a genotype which is moderately sensitive to infection and may delay neuroinvasion. Our results show that asymptomatic sheep may spread the infection during the long incubation period of several years, which constitutes an important obstacle in the eradication of scrapie. Our findings indicate that contamination of the environment plays an important part in sustaining the infection.

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Gudmundur Georgsson Email:


prepared February 20, 2008

Infected and Source Flocks

There were 27 scrapie infected and source flocks with open statuses (Figure 3) as of January 31, 2008. Two new source flocks and one new infected flock were reported in January (Figure 4) with a total of 22 reported for FY 2008 (Figure 5). ....


Positive Scrapie Cases

As of January 31, 2008, 58 new scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL) in FY 2008 (Figure 7). Of these, 52 were field cases and 6* were Regulatory Scrapie Slaughter Surveillance (RSSS) cases (collected in FY 2008 and reported by February 20, 2008). There were 8 positive cases for January which are depicted in Figure 8. Seventeen cases of scrapie in goats have been confirmed by NVSL since implementation of the regulatory changes in FY 2002 (Figure 9). The most recent positive goat cases were from the SAME HERD and WERE BOTH CONFIRMED IN JANAURY 2008.


Caprine Scrapie Prevalence Study (CSPS)

CSPS was initiated in May 2007 to estimate the national prevalance of scrapie in adult goats at slaughter. If no scrapie is found we will be able to conclude that the prevalence in goats is greater than zero and less than 0.1 percent. AS of January 31, 2008, 2,942 goats have been sampled for scrapie testing (1,515 in FY 2007 and 1,427 in FY 2008). Collection numbers by quarter in FY 2008 is shown in Chart 8. To date, no goats have tested positive for scrapie as part of this surveillance program. HOWEVER, THREE POSITIVE GOATS have been identified this fiscal year through field investigations. One was a clinical suspect submitted for testing and THE OTHER TWO WERE MEMBERS OF THE OF THE BIRTH HERD OF THE CLINICAL CASE.


please see full text ;


The flocks of origin are WY, CO, CA, IN, and MN.

personal communication USDA et al. ...TSS


INFECTED AND SOURCE FLOCKS AS of August 31, 2007, there were 33 scrapie infected and source flocks with open statuses (Figure 3). Five new source flocks and one new infected flock were reported n August (Figure 4) with a total of 64 reported for FY 2007(Figure 5).




If atypical scrapie is found in the United States, an additional control program may be necessary

but it is likely that no changes in the current control program will be needed.

For the period of time from January 1, 2005, until October 15, 2005, there were 23 instances of discrepancies in results from 35 flocks. Of those 23 instances, 14 were caused by laboratory error (paperwork or sample mix-up), 3 results from field error, 5 were not completely resolved, and 1 originated from the use of a non-approved laboratory for the first test. As a result of inconsistencies, one laboratory’s certification was revoked by APHIS-VS.



-------- Original Message --------

Date: Fri, 17 Dec 2004 15:37:28 -0600
From: "Terry S. Singeltary Sr."

Hello Susan and Bio-Rad, Happy Holidays! I wish to ask a question about Bio-Rad and USDA BSE/TSE testing and there inconclusive. IS the Bio-Rad test for BSE/TSE that complicated, or is there most likely some human error we are seeing here? HOW can Japan have 2 positive cows with No clinical signs WB+, IHC-, HP- , BUT in the USA, these cows are considered 'negative'? IS there more politics working here than science in the USA? What am I missing?

-------- Original Message --------

Subject: Re: USDA: More mad cow testing will demonstrate beef's safety
Date: Fri, 17 Dec 2004 09:26:19 -0600
From: "Terry S. Singeltary Sr."


Experts doubt USDA's mad cow results


WELL, someone did call me from Bio-Rad about this, however it was not Susan Berg. but i had to just about take a blood oath not to reveal there name. IN fact they did not want me to even mention this, but i feel it is much much to important. I have omitted any I.D. of this person, but thought I must document this ;

Bio-Rad, TSS phone conversation 12/28/04 Finally spoke with ; Bio-Rad Laboratories 2000 Alfred Nobel Drive Hercules, CA 94547 Ph: 510-741-6720 Fax: 510-741-5630 Email: XXXXXXXXXXXXXXXXXX at approx. 14:00 hours 12/28/04, I had a very pleasant phone conversation with XXXX XXXXX about the USDA and the inconclusive BSE testing problems they seem to keep having. X was very very cautious as to speak directly about USDA and it's policy of not using WB. X was very concerned as a Bio-Rad official of retaliation of some sort. X would only speak of what other countries do, and that i should take that as an answer. I told X I understood that it was a very loaded question and X agreed several times over and even said a political one. my question; Does Bio-Rad believe USDA's final determination of False positive, without WB, and considering the new atypical TSEs not showing positive with -IHC and -HP ??? ask if i was a reporter. i said no, i was with CJD Watch and that i had lost my mother to hvCJD. X did not want any of this recorded or repeated. again, very nervous, will not answer directly about USDA for fear of retaliation, but again said X tell me what other countries are doing and finding, and that i should take it from there. "very difficult to answer" "very political" "very loaded question" outside USA and Canada, they use many different confirmatory tech. in house WB, SAF, along with IHC, HP, several times etc. you should see at several talks meetings (TSE) of late Paris Dec 2, that IHC- DOES NOT MEAN IT IS NEGATIVE. again, look what the rest of the world is doing. said something about Dr. Houston stating; any screening assay, always a chance for human error. but with so many errors (i am assuming X meant inconclusive), why are there no investigations, just false positives? said something about ''just look at the sheep that tested IHC- but were positive''. ... TSS

-------- Original Message --------

Subject: Your questions Date: Mon, 27 Dec 2004 15:58:11 -0800 From: To: Hi Terry: ............................................


Let me know your phone number so I can talk to you about the Bio-Rad BSE test.

Thank you Regards

Bio-Rad Laboratories 2000 Alfred Nobel Drive Hercules, CA 94547 Ph: 510-741-6720 Fax: 510-741-5630 Email: ================================= END...TSS

######### ##########

Executive Summary In June 2005, an inconclusive bovine spongiform encephalopathy (BSE) sample from November 2004, that had originally been classified as negative on the immunohistochemistry test, was confirmed positive on SAF immunoblot (Western blot). The U.S. Department of Agriculture (USDA) identified the herd of origin for the index cow in Texas; that identification was confirmed by DNA analysis. USDA, in close cooperation with the Texas Animal Health Commission (TAHC), established an incident command post (ICP) and began response activities according to USDA’s BSE Response Plan of September 2004. Response personnel removed at-risk cattle and cattle of interest (COI) from the index herd, euthanized them, and tested them for BSE; all were negative. USDA and the State extensively traced all at-risk cattle and COI that left the index herd. The majority of these animals entered rendering and/or slaughter channels well before the investigation began. USDA’s response to the Texas finding was thorough and effective.

Report on Food & Drug Administration Dallas District Investigation of Bovine Spongiform Encephalopathy Event in Texas 2005 Executive Summary: On June 24, 2005, USDA informed FDA that a cow in Texas tested positive for Bovine Spongiform Encephalopathy (BSE). Information provided by APHIS was that the BSE positive cow was born and raised in a herd in Texas and was approximately 12 years old. The animal was sampled for BSE at a pet food plant in Texas on November 15, 2004, as part of USDA’s enhanced surveillance program.

Texas even had a 'secret' test that showed that mad cow positive; experimental IHC test results, because the test was not a validated procedure, and because the two approved IHC tests came back negative, the results were not considered to be of regulatory significance and therefore were not reported beyond the laboratory. • A Western blot test conducted the week of June 5, 2005, returned positive for BSE.

48 hr BSE confirmation turnaround took 7+ months to confirm this case, so the BSE MRR policy could be put into place. ...


-------- Original Message --------

Subject: re-USDA's surveillance plan for BSE aka mad cow diseaseDate: Mon, 02 May 2005 16:59:07 -0500

From: "Terry S. Singeltary Sr."

To:,, Greetings Honorable Paul Feeney, Keith Arnold, and William Busbyet al at OIG, ...............


There will be several more emails of my research to follow. I respectfully request a full inquiry into the cover-up of TSEs in the United States of America over the past 30 years. I would be happy to testify...

Thank you,
I am sincerely,
Terry S. Singeltary Sr.
P.O. Box 42 Bacliff, Texas USA 77518 xxx xxx xxxx

Date: June 14, 2005 at 1:46 pm PST
In Reply to: Re: Transcript Ag. Secretary Mike Johanns and Dr. John Clifford, Regarding further analysis of BSE Inconclusive Test Results posted by TSS on June 13, 2005 at 7:33 pm:

Secretary of Agriculture Ann M. Veneman resigns Nov 15 2004, three days later inclusive Mad Cow is announced. June 7th 2005 Bill Hawks Under Secretary for Marketing and Regulatory Programs resigns. Three days later same mad cow found in November turns out to be positive. Both resignation are unexpected. just pondering... TSS


-------- Original Message --------

Subject: Re: BSE 'INCONCLUSIVE' COW fromTEXAS ??? Date: Mon, 22 Nov 2004 17:12:15 -0600 From: "Terry S. Singeltary Sr." To: Carla EverettReferences: <[log in to unmask]><[log in to unmask] us>

Greetings Carla, still hear a rumor;

Texas single beef cow not born in Canada no beef entered the food chain?

and i see the TEXAS department of animal health is ramping up for something, but they forgot a url for update?


can you confirm??? terry


-------- Original Message --------

Subject: Re: BSE 'INCONCLUSIVE' COW from TEXAS ???

Date: Fri, 19 Nov 2004 11:38:21 -0600

From: Carla Everett

To: "Terry S. Singeltary Sr."References: <[log in to unmask]>

The USDA has made a statement, and we are referring all callers to the USDA web site. We have no information about the animal being in Texas.


At 09:44 AM 11/19/2004, you wrote:

Greetings Carla,

i am getting unsubstantiated claims of this BSE 'inconclusive' cow is from

TEXAS. can you comment on this either way please?

thank you,

Terry S. Singeltary Sr.>>


-------- Original Message --------

Subject: Re: BSE 'INCONCLUSIVE' COW from TEXAS ???

Date: Mon, 22 Nov 2004 18:33:20 -0600

From: Carla Everett

To: "Terry S. Singeltary Sr."References: <[log in to unmask]><[log in to unmask] us><[log in to unmask]> <[log in to unmask]us> <[log in to unmask]>

our computer department was working on a place holder we could post USDA's announcement of any results. There are no results to be announced tonight by NVSL, so we are back in a waiting mode and will post the USDA announcement when we hear something.

At 06:05 PM 11/22/2004,

you wrote:

why was the announcement on your TAHC site removed?

Bovine Spongiform Encephalopathy:

November 22: Press Release title here

star image More BSE information


Carla Everett wrote:

no confirmation on the U.S.'inconclusive test...

no confirmation on location of animal.>>>>>>