Prion detected in blood test
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TSE diagnosed in blood

Thu, 15 Jul 1999 Listserve, seminar notes, conference abstract
Below are some very exciting developments -- TSEs can now be detected in blood, including human, elk, deer, and sheep blood. But how can all this information can be several months old but only be available at obscure seminars? -- the director of the National Institutes of Health is calling for a biomed preprint server yet another branch of government offers no preprint on its own 1 paragraph National Animal Disease Center site.

Why are draft papers not available? The FDA and other agencies need these to make informed decisions on blood donors with CJD. Having already gone public with their data in at least two places, this federal laboratory in Iowa would seemingly have to release all documents under America's Freedom of Information Act. This raises the intriguing question of whether the government can sue itself, ie, can FDA force NADC to divulge their information? [FOIA arises from 5 USC 552(a)(3) which requires that requested records be made available "to any person." If this person is broadly construed, it might apply to another government agency.]

If sporadic CJD human blood is testing positive (and controls are not), preliminary study or not preliminary, in the webmaster's opiunion , this blood should be recalled and destroyed under the precautionary principle, best available science, or just plain common sense. There will be the devil to pay if FDA continues to allow blood sales they know (or should have known) had Prp-Sc.

Are public policy makers really supposed to wait for some journal to go through a tedious process of review and for print publications to be mailed out to libraries? This makes no sense in a situation where policy makers have staff that is perfectly capable of making critical review for themselves and who could care less whether 2 unknown peer reviewers liked a submission.

To quote from the USDA's own FOIA page,

"Nothing could be more irrational than to give the people power and to withhold from them information, without which power is abused." James Madison [early US president]

This important TSE diagnostic news surfaced at a meeting announcement in DC scheduled for Oct 27-28, 1999. A blood test is working in preclinical elk as well as sheep. These may have been from the South Dakota herd that was donated for research purposes rather than random elk surveys. The abstract does not say whether the elk were experimentally infected nor if they went on to develop clinical symptoms and if so, how much lead time was gained by the blood test.

It could take a few years for such a diagnostic test to complete validation procedures. In addition to not requiring a dead animal, animals can be recognized about halfway to clinical onset -- a big improvement but not the end of the story if infected animals have already spread the agent around by then.

1.  Detection of Abnormal Prion Protein in the Blood of Animals Naturally Infected with a Transmissible Spongiform Encephalopathy 
Dr. Mary Jo Schmerr, Research Chemist, Respiratory & Neurologic Disease Research,  
National Animal Disease Center.  tel (515) 239-8287 X287 fax (515) 239-8458 mschmerr@nadc.ars.usda.gov
"The abnormal prion protein was detected using capillary immunoelectrophoresis (CIE) in the blood of sheep infected with scrapie and in the blood of elk infected with chronic wasting disease. These animals were not clinically ill when the blood was taken. There is a good correlation with Western blot of the tonsils of sheep and with CIE of the blood. The assay has potential for development as a preclinical diagnostic test for TSEs."
2. Use of Tonsillar Biopsy and PrPSc Detection in the Preclinical Diagnosis of Scrapie
Dr. Bram E.C. Schreuder, BSE/Scrapie Specialist, 
Department of Immunology, Pathology and Epidemiology, Institute for Animal Science and  Health, ID-DLO
"A possible approach to an early preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) using tonsillar biopsies and scrapie in sheep as a model is reported. In naturally infected live sheep, fully or partially susceptible to scrapie (VRQ/VRQ and VRQ/ARQ), PrPSc could be detected in the tonsils more than one year before the expected onset of clinical disease, at about one-third to less than half of the incubation period. At three-and-a-half months of age no PrPSc could be detected in three homozygous susceptible sheep, whereas the earliest appearance of PrPSc was at four months in one similar sheep. No immunostaining could be detected in animals with a (semi-) resistant genotype. Technical details of taking tonsillar biopsies in sheep will be presented and possible applications of this technique will be discussed.'
3. A Cell Surface Receptor for the Prion Protein
Dr. Neil R. Cashman, Professor, Department of Medicine (Neurology), 
University of Toronto; and  Caprion  Pharmaceuticals, Inc.
" It is likely that the evolutionarily conserved cellular prion protein PrPC would have a similarly conserved cell-surface receptor. We have identified a high-affinity surface binding site for PrPC using PrP-alkaline phosphatase fusion proteins and have cloned a candidate transmembrane protein that confers PrP-AP binding on frog oocytes. The identification of an authentic high-affinity cell surface receptor for prion proteins enables the development of diagnostics and therapeutics based upon this specific interaction."
27-28 Oct 99 Washington DC.
Overall Conference Chairperson
Dr. Paul W. Brown

Registration $395
Hotel $180 Single/$200 Double
Posters: submit by 17 Sept 99

Speakers
Dr. David Asher, Center for Biologics Evaluation and Research/FDA
Dr. Michael Beekes, Robert Koch Institute (Germany)
Dr. Michael Blaese, Kimeragen, Inc.
Dr. No╬lle Bons, University Montpellier II (France)
Dr. Jeri Ann Boose, BioReliance Corporation
Dr. Alex Bossers, ID-DLO (The Netherlands)
Dr. Neil R. Cashman, University of Toronto and Caprion Pharmaceuticals, Inc. (Canada)
Dr. Byron Caughey, National Institute of Health Laboratory for Persistent Viral Disease/NIH
Dr. Linda A. Detwiler, U.S. Department of Agriculture
Dr. Marshall Dinowitz, Genentech, Inc.
Dr. Heino Diringer (Germany)
Dr. David Harris, Washington University School of Medicine
Dr. Kiki Hellman, Center for Devices and Radiological Health/FDA
Dr. Mary Jo Schmerr, National Animal Disease Center
Dr. Lawrence Schonberger, Centers for Disease Control and Prevention
Dr. Bram E.C. Schreuder, ID-DLO (The Netherlands)
Ms. Ailsa Shepherd, Inveresk Research (UK)
Dr. Reed B. Wickner, National Institute of Diabetes and Digestive and Kidney Diseases/NIH

Comment (webmaster):
There also seems to be a new issue of Dev Biol Stand devoted to the topic of detecting prions in blood. In reading the FDA abstract, one is struck by the complete lack of coordination of FDA with the National Animal Disease Center in Iowa, even though Asher and Schmerr surely know each other from conferences (eg, both are speakers on the Oct 27 conference in DC). This was Scmerr's 6th publication on prion capillary electrophoresis (from 1994).

The Iowa group wisely steers clear of detecting TSE agent in human or cow blood (though according to a lab insider they have been working on human blood for the last 18 months; animal tests only correlate with other methods at about 65% at this time). They stick to sheep and elk where results are welcomed by industry, unlike human or cow where detection would be most unwelcome. Despite its long-evident superior sensitivity, there has been no great rush at FDA to adopt this method -- its ramifications are a major political headache for the agency.

If prion capillary electrophoresis was used in the deer and elk hunter case (McEwen's blood was reported sampled), the results have not been released and stockpiled blood products were apparently given out without waiting for CIE results. That makes it a little awkward to release a positive result now (if there was one).

Or for that matter, to release a negative result -- one obtained only after decision was made to use stockpiled material in hundreds of thousands of people.

If CIE was not used in the McEwen case, then the question is why not? FDA should have based recall policy on the best available science -- that surely includes using the best available technology. How long does it take to run 1 sample?

In short, we won't see the results from McEwen's blood test any time soon.

Bovine sera used in the manufacture of biologicals: current concerns and policies of the U.S. Food and Drug Administration regarding the TSEs

Dev Biol Stand 1999;99:41-4
Asher DM
Since 1993, consistent with its statutory responsibility to ensure that regulated products are safe, pure, and free of extraneous organisms, the United States Food and Drug Administration (FDA) has requested that, with certain exceptions, bovine-derived materials from animals born in or residing in countries where bovine spongiform encephalopathy has occurred, should not be used to manufacture products intended for humans.

FDA's Center for Biologics Evaluation and Research (CBER) has specifically recommended that serum used to produce biologicals be obtained from sources certified to be free from contaminants and adventitious agents, such as the agent responsible for the production of Bovine Spongiform Encephalopathy. The United States Department of Agriculture (USDA) has prohibited importation of such serum for use in products [long after the BSE epidemic began -- webmaster].

FDA staff are aware that bovine blood, including foetal blood, and placental tissues and fluids that might contaminate foetal serum have not been found to contain the infectious agent of BSE [sic], and that those tissues are considered by most authorities to have little risk for transmitting disease to humans or animals.

However, studies of BSE have been limited in size and sensitivity, and several experimental studies of scrapie and CJD in rodents found their blood to be infectious. In addition, a recent unpublished study of BSE (requiring confirmation) reported finding infectivity in the bone marrow of cattle. Possible transmission of BSE from cows to calves, although unlikely to constitute a major mode for maintaining the BSE outbreak, has also not been rigorously ruled out [in fact, it is an accepted fact -- webmaster]. Considering the special nature of biological products, especially of vaccines intended for widespread use in children, it seems prudent for U.S. regulatory authorities to continue current conservative policies that discourage or prohibit the use of bovine serum from countries with BSE. [England continued to use BSE cow serum in vaccines in children until all vaccine stocks on had sold (1993), as with French policy on AIDS and hepatitus. -- webmaster]

Alex Bossers

15 July 99 notes taken at a March 1999 seminar given by Schmerr in Europe
Institute for Animal Science and Health, Lelystad, NL Tel: +31-(0)320-238273

"We had Mary Jo Schmerr visiting a few months ago and she showed some impressive data. I think it is a promissing test which after optimalization can be performed in very short time spans (electrophoresis only takes less than 2 minutes!!) on various tissue types of different species.Some snippets are listed below.

- She seems to use competition between Ab and PrP or Ab and a peptide. As soon as PrP binds to the Ab, free peptide is released and readily detected by capillary electrophoresis.

- Detection level seems to be 25-50 attomoles (0.3 pg) of PrP.

- She found PrP-Sc in blood!

She does a PrP isolation from about 10 ml of blood using this protocol: 10ml EDTA blood, buffy coat isolated, red blood cells lysed in NH4Cl, buffy coat suspended in PBS, homgenized in detergent, DNAse digestion,50-100 ug protease K digestion, organic solv. (n-ethyl-glucoside!?), dry down , finally the whole product is tested in competition assay with peptide and prion antibody.

- Sheep susceptible to scrapie having general survival times of 2 years of age, were preclinically found positive after 4-6 months of age! (about the same as tonsillar biopsy and IHC!!)

- There seems to be some sort of viremia period in blood. Dying animals of TSE, became almost negative in blood!!

- Animals are considered positive at levels around or above 10pg PrP-Sc.

- The test seems to work on CWD (mule deer experimentally challenged orally) as well."

Comment (webmaster):

- "There seems to be some sort of viremea period in blood. Dying animals of TSE, became almost negative in blood!!"

In other words, infectivity in blood may decline from high to low levels by the time of death. Screening blood from blood donors late in their clinical course could thus be highly misleading. Viremia (or prionemia?) inversely correlated with clinical status is counter-intuitive and unfortunate, yet very similar to infectivity time course in some species, as reviewed at length in Dealler's TSE book. It is bad news if applicable to human blood donors: even if blood products are recalled, most will have been used by time of CJD diagnosis. People like McEwen might test negative at time of diagnosis yet have been highly infectious a year or two earlier when donations were being made.

- "Sheep susceptible to scrapie having general survival times of 2 years of age, were preclinically found positive after 4-6 months of age! (about the same as tonsillar biopsy and IHC!!)"

This says capillary immunoelectrophoresis (CIE) is not finding preclinical animals any earlier but at least it is using blood, which is a whole lot more amenable to whole-flock testing or screening of donated blood. It seems possible that Prp-Sc might increase exponentially initially, making it harder and harder to push detection back much farther.

- "The test which after optimalization can be performed in very short timespans (electrophoresis only costs less than 2 minutes!!) on various tissuetypes of different species."

There is reportedly a 2 day turn-around expected for the method. Schmerr's group has been coordinating with an unknown CJD researcher on human blood, presumbably Gambetti and/or Collinge. It would make good sense to try this test on a random sample of people in England. So far, England has resisted any screening of cattle, sheep, or humans with high-sensitivity tests. What would show up in sporadic CJD in the US with the bank of 300 recent cases in the Gambetti lab?

To what extent is method automated now, how much operator attention is needed, how many samples can be run in a day, can the outcome be machine-read? Could machines realistically be set up at slaughterhouses, game processing facilities, and blood centers to determine whether lamb or deer are fit for human consumption and donations safe? These questions have no answers right now.

- "Detection level seems to be 25-50 attomoles (0.3 pg) of PrP. Animals are considered positive at levels around or above 10pg PrP-Sc."

In other words, 0.3 pg of protein fragment with molecular wt 12,000 is 25 x 10 exp -18 or 25 attomoles or 15 million protein molecules, which might translate to 150,000 prion seed crystals. That seems like a remarkable sensitivity. How much lower it must get before we could say with confidence whether or not animal would go on to develop disease. Do animals below 10pg PrP-Sc/10ml blood did not get worse over time in their assay or that they have waited for years and the animals did not become clinical? Or is pg/10ml just irreducible background?

A pharmaceutical company or diagnostic lab would not lose a lot of sleep over automating the protocol you describe..

- "The test seems to work on CWD (mule deer experimentally challenged orally) as well."

Curious. Where are the animals kept? Oral challenge has uncertain relevence to natural routes of CWD transmission but makes more sense than intra-cerebral if aim is to look at blood.

New papers by MJ Schmerr et al

Tue, 20 Jul 1999 
Abstract P325 "Proceedings of the 12th International Symposium on High Performance Capillary Electrophoresis and related microscale techniques, page 134:

The use of capillary electrophoresis and fluroescent labeled peptides to detect the abnormal prion protein in the blood of animals infected with a TSE

MJ Schmerr, AL Jenny, Marie S Bulgin* , JM MIller, AN Hamir, RC Cutlip, K Goodwin
* sheep specialist at Caine Veterinary Teaching Center, U. Idaho, Moscow
"... In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary elcetrophoresis makes it possible to detect the abnormal protein in blood.

A peptide from the carboxy terminal region, amino acid positions 218-232 [in humans, in SwissProt numbering, this would be YERESQAYYQRGS.SM, which includes a bit of GPI cleavage, suggesting sheep TQYQRESQAYYQRG. was used], was labeled with fluorescein during the sythesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immuynoassay. The amount of fluorescein labled peptide in the capillary was 30" attomoles.

Blood was obtained from normnal sheep and elk and from sheep infected with scrapie and elk infected with CWD. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the CIE for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals.

This assay makes a preclinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for the manufacture of pharmaceuticals and products for human consumption."

Note: a journal article is to appear in August, 1999 in the J. of Chromatography A vol 853 (issue 1-2), pg numbers not available. That article will have further information on blood tests for CWD.

Dr. Schmerr kindly answered several questions of the webmaster by email on 20 Jul 99:

(1)Much in the blood test could be simply automated (they're using a Beckmann a P/ACE System 5500 now , order #477172 at tel 800-233-4685 (price not available online), a small instrument about the size of a computer monitor)

(2) The elk tested were not experimentally infected but from herds that had experienced one or more clinical cases of CWD.

(3) Some apparently normal elk tested positive for abnormal prion on the first blood assay conducted (buffy coat) and subsequently succumbed to CWD 6-9 months later. Thus a time series was conducted on these elk; apparently they are still waiting on other elk positive on first assay to see if they will later develop CWD.

(4) They are not involved in random surveys of elk for CWD at this time.

(5) The lower limit of detectability on 20 July 99 was 10 attomoles (about 6.2 million prion molecules)

A brief biography of Mary Jo Schmerr

Iowa State University web site

MARY JO SCHMERR
Research Chemist National Animal Disease Center, USDA, ARS Ames, IA
B.A. in Chemistry, 1968, Clarke College; Ph.D. in Biochemistry, 1975, Iowa State University
Dr. Schmerr has spent over 20 years doing research on animal diseases. The first part of her career was spent characterizing the immune response to the lipolysaccharides of Pasteurella multocida. The next twelve years were spent working on retroviruses. She purified the major internal antigen and the surface glycoprotein from bovine leukemia virus and developed a radioimmunoassay for these proteins. She then moved on to a lentivirus, ovine progressive pneumonia virus, and made monoclonal antibodies to the surface glycoprotein and characterized the carbohydrate moiety of this protein. This research is ongoing.

The most recent research has involved work on the scrapie prion protein. More sensitive diagnostic tests are needed for these transmissible spongiform encephalopathies of which scrapie in sheep is the prototype. New methods of extraction for the prion protein are being explored as well as the use of immunoassay using capillary electrophoresis.

Earlier publications on this methodology from Schmerr

Capillary isoelectric focusing of the scrapie prion protein. 
J Chromatogr A 1998 Apr 3;802(1):135-41 
Schmerr MJ, Cutlip RC, Jenny A
Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.
A diagnostic test for scrapie-infected sheep using a capillary electrophoresis immunoassay with fluorescent-labeled peptides.
Electrophoresis 1998 Mar;19(3):409-14 
Schmerr MJ, Jenny A
Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and beta-mercaptoethanol, a monomer form of approximately 27 kDa molecular mass is observed. A competition immunoassay to detect PrPSC from scrapie-infected sheep was developed using free zone capillary electrophoresis with laser-induced fluorescence (LIF) for detection and flourescein-labeled synthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent-labeled peptides bound to the antibody were separated from the unbound peptides using 200 mM Tricine, pH 8.0, containing 0.1% n-octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind approximately 50% of the fluorescent-labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, approximately 2 fmoles of the peptide could be measured. When PrPSC extracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSC could be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142-154 and 155-178, clearly differentiated scrapie-positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopathies in animals and in humans.
Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep. 
J Chromatogr B Biomed Sci Appl 1997 Sep 12;697(1-2):223-9 
Schmerr MJ, Jenny A, Cutlip RC
Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pellet was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCap SDS14-200 Beckman capillary). In infected sheep brain samples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on Western blot (22.4 kDa), a Ferguson plot was made to determine if there were abberations in the molecular mass determination. After correction, the major peak was estimated to be 19.2 kDa. This has a better correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was approximately 50 micrograms. For Western blot, the amount of brain sample was approximately 20 mg. For this assay, this is approximately 100 times less than that needed for Western blot for sheep samples.
Improvements in a competition assay to detect scrapie prion protein by capillary electrophoresis. 
J Chromatogr B Biomed Appl 1996 May 31;681(1):29-35 
Schmerr MJ, Goodwin KR, Cutlip RC, Jenny AL
Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS and beta-mercaptoethanol, a monomer form (prion protein) with a molecular mass of 27 kDa is observed. Free zone capillary electrophoresis and peptides labeled with fluorescein were used to detect the prion protein through competition for a labeled peptide in immune complex formation. The separation of the immune complexes from the unbound peptide using 200 mM Tricine (pH 8.0) was faster and was better resolved than that obtained with phosphate or borate buffer systems. The amount of immune complex formation was dependent on the amount of antibody in the assay. The amount of bound labeled peptide and unbound labeled peptide could be measured directly by calculating the area of each respective peak. As increasing amounts of unlabeled peptide were added to the assay, a concentration dependent reduction in the immune complex peak was observed. The assay could detect less than 10.0 fmol of unlabeled peptide. There was a quantitative difference in the competition of preparations from scrapie infected sheep brain and normal sheep brain.
Capillary electrophoresis of the scrapie prion protein from sheep brain. 
J Chromatogr A 1994 Oct 7;680(2):447-53 
Schmerr MJ, Goodwin KR, Cutlip RC
Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrPsc) that has a molecular mass of ca. 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrPsc. The overall concentration of PrPsc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrPsc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrPsc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrPsc. After treatment with proteinase K and non-ionic detergents, PrPsc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep.

British beef faces long haul back

Wed, Jul 14, 1999 By Eileen Murphy and Geoff Meade, PA News
It has cost the British farming industry more than 1.5 billion pounds since it was imposed in March 1996 and left many farmers bankrupt or living on the breadline but the lifting of the worldwide ban on British beef may not spell the end of the BSE saga.

The British Government and farmers' leaders admit that although the ban will disappear on August 1 it will take much longer for British beef to retake its place on the world's meat markets. For years Britain's troubles with "mad cow disease" were very much a domestic problem. Bovine Spongiform Encephalopathy, first diagnosed in November 1986 [sic] in two cows from different parts of England, turned into an epidemic which peaked in 1993 when more than 1,000 new cases were recorded every week, compared with less than a tenth [sic] of that now.

But when concerns grew about a possible link between BSE in cattle and a new variant of Creutzfeldt Jakob Disease (nvCJD), a similar brain disease in humans, Brussels began to take notice. Agriculture Commissioner Franz Fischler imposed the worldwide ban on Britain's beef exports on March 27, 1996. Germany had already unilaterally banned British beef, contrary to EU rules.

At first few believed the Commission had powers to take such sweeping action, destroying at a stroke much of one member state's beef industry. Mr Fischler was instantly attacked by then Prime Minister John Major for responding to consumer panic rather than considering the Government's anti-BSE safeguards and the scientific evidence of the scale of risk. But later the European Court of Justice would rule against legal claims brought by the Government and the National Farmers' Union. The judges said the worldwide ban was necessary and did not exceed the Commission's authority.

Euro-MPs launched a special committee of inquiry into the handling of the BSE scare as Mr Major ordered all his ministers to vote against any proposed EU decisions, whether the UK was in favour or not. The issue of BSE had by this time become such a Europe-wide crisis that a routine meeting of EU leaders in Florence in June 1996 turned into a "mad cow crisis" summit. The talks resulted in a deal setting out the conditions for a gradual lifting of the ban.

The member states set five pre-conditions which Britain would have to meet first including a selective slaughter scheme and the introduction of animal passports. Since then consecutive agriculture ministers have worked hard to build up a stock of goodwill based on clear evidence that beef standards, from farm gate to dinner plate, are among the highest in the world [sic].

At successive meetings the EU's veterinary and scientific experts began gradually endorsing the British beef safety efforts. In March last year the agriculture ministers agreed the resumption of beef exports from Northern Ireland, impressed by the province's comprehensive computerised cattle monitoring system. But England, Scotland and Wales had to wait until November last year to hear the news that, pending veterinary inspections and compliance with the "Florence Agreement", the worldwide ban on British beef exports would be lifted - bringing to an end the worst agricultural crisis in EU history.

More BSE in France

Tue, 20 Jul 1999
The French ESB-liste has announced last week the 13 th and the 14 th case of BSE in France in 1999, in the "Cotes d'Armor" and "Aube" Departement. The two cows was born in October and September 1994. All the 65 and 83 animals had been slaughtered " as requested ".

Storm over Canada plan for British blood ban

 Sat, Jul 17, 1999 By Alex Richardson, PA News
Canada's national blood agency was coming under pressure today to back down from plans to ban blood donations from people who have visited Britain. The ban is expected to be officially announced shortly, and stems from fears that people who have eaten British beef could have been exposed to new variant CJD, the human form of mad cow disease.

The move has sparked a fierce row in Canada, with a leading health activist resigning from the board of the recently formed Canadian Blood Services - the national blood agency - after accusing it of being over cautious. In the UK the Department of Health and the National Blood Authority said Britain had taken steps to prevent the theoretical risk of CJD transmission through blood products. Any Canadian ban on blood donors who have visited the UK would not affect Britain, they said.

The ban was first proposed in Canada last year, and would cover anyone who had spent a cumulative total of six months or more in the UK since 1980. Earlier this week campaigner Durhane Wong-Rieger, a former president of the Canadian Haemophilia Society, resigned from the board of Canadian Blood Services over the issue. According to the news agency Canadian Press she accused the blood agency of being over-cautious, and predicted the ban would result in a 10-15% drop in donors, triggering a major crisis.

But a spokesman for Canadian Blood Services hit back, telling Canadian Press any ban would be a temporary measure, and was the best way to proceed until more was known about the risks from CJD. The final decision, expected shortly, will be taken by the Bureau of Biologics, an agency of Canada's health department. [Harvey Schipper, a Canadian Blood Services spokesman estimated the agency would lose 4 percent of donors because of a ban -- AP story]

In Britain a spokeswoman for the National Blood Authority said there was a small theoretical risk that new variant CJD could be passed on via blood, but no hard scientific proof that transmission was possible. The spokeswoman said: "What we have done is we no longer use UK plasma for making blood products, and we take out the white blood cells from each donation. "These are Government measures which we have been advised to take to guard against the theoretical risk. "What happens in Canada is a matter for the Canadian authorities."

A spokeswoman for the Department of Health said: "Obviously it is a matter for each country to make its own decisions. "In the UK we have taken every recommended step to make our blood supply as safe as possible. "For someone in need of a blood transfusion the benefits of the transfusion obviously outweigh any small theoretical risk."

The safety of blood transfusions has been a hot political issue in Canada since the 1980s, when thousands of people were infected with the Aids virus and Hepatitis C through tainted supplies. Conservative MP Gerald Howarth today called the proposed ban "rather bizarre".

The Aldershot MP said: "It is not for a member of the British Parliament to tell the Canadians how to run their affairs, but I am somewhat disappointed that this has come at a time when the EU has lifted the beef ban. "The UK has always been totally upfront about all this, although BSE exists in other countries. "Canadian citizens should be far more concerned about countries where they have been less scrupulous about ensuring their public health."

Elk game farms - the disgusting reality

High Country News -- November 10, 1997 (Vol. 29, No. 21)  Hal Herring 
A recent visitor to the ranch writes, "Big Velvet Ranch in Darby, Montana is about 4 miles south of Darby, on the east side of Rt.93 and covers about 4 square miles [placing it 17 miles south of the CWD research facility in Hamilton -- webmaster]. The owner is named Len Wallace.

BVR has been in operation since 1991. Young bull calves are shipped back to Wisconsin for 4 years to grow and develop their antlers. Then they're returned to Wallace's enclosures where they are shot by tourists for fees ranging to $25,000, depending on the rack. The meat is almost an afterthought. Wallace arranges for local butchers to cut & wrap it for his clients - but "they don't really care about the meat or the hide. We don't wanna talk about that part of it."

The BVR "hunting" package includes 4 days: (1) arrival, (2) primary hunting day (3) back-up hunting day, or you can go his friend's operation and "hunt" for a buffalo for $3,000. Day 4 is general braggadocio BS around the lodge, and departure. The cost can range to $25,000

The following is a November '97 article from the High Country News about the Big Velvet Ranch. Of certain note is the part about 49 deer being mismanaged to death as Len Wallace developed his elk ranching business. Montana State Game Wardens called that botched mission and 49 dead animals a 'mixed success.' "

On a Montana ranch, big game and big problems

DARBY, Mont. - It's almost September, and dozens of "shooter bulls" have been turned into the shooting enclosure of Big Velvet Elk Ranch, just south of here, in western Montana's Bitterroot Valley. Ranch owner Len Wallace has booked 80 clients for the fall and every one of them is going to shoot a trophy elk, guaranteed, right there inside the fence.

The clients, rifle and archery enthusiasts alike, put down $5,900 per animal. In the 1,800-acre enclosure, some prefer to shoot from vehicles at close range and others prefer a more realistic hunting scenario and chase their game on foot. Everyone leaves with the meat, the head, and the antlers. This is trophy hunting, 1990s-style: Safe, fast and expensive.

"All of our hunters go home very happy boys," says Wallace. What happens on his ranch, he explains, is like plucking a live lobster out of a water tank to cook. "Hunting covers a wide range of things people do."

After four state-approved expansions, Big Velvet Ranch has become Montana's biggest game farm: 2,000 acres and 800 elk set in the middle of some of the nation's most significant wildlife habitat. Wallace owns another 3,000 acres nearby that are not part of the operation. The ranch - one of a dozen game farms in the West which offer "shoot for pay" hunting - has become a flashpoint in a debate over the environmental implications and ethics of game farming, including harvesting the velvet antlers of bull elk, a $3 billion international business that supplies the south Asian traditional medicine market.

A year ago, when Wallace tried to fence off another 1,100 acres he owns adjacent to his ranch, the conflict erupted. Ravalli County Sheriff Jay Printz calls the situation "a big hoorah" of harassment complaints involving Wallace, sporting groups and conservationists campaigning against the ranch. Wallace says people have pulled down fencing around the ranch, snipers have fired on ranch employees and helicopters have buzzed his property.

"It's a very big mess," says Printz. "It's the Hatfields and McCoys, in a manner of speaking." Critics call Wallace the "arrogant" master of a ranch that threatens wildlife and natural habitat. "Big Velvet Ranch is a rogue operation," says Rich Day, of the National Wildlife Federation office in Missoula.

Wallace describes his critics as "anti-game-farm scum," and himself as Montana's chief spokesman for the industry. He says game farming, and his ranch in particular, are targets of government regulation gone awry. "There's somewhat of an anti-business bias in Montana these days," Wallace explains. "They don't want you to cut trees or mine, they don't want you to have cattle, they don't want you to do anything."

A big pink house

To understand Big Velvet Ranch and the controversy, it helps to know what the ranch looks like. Four miles south of Darby, Mont., east of Highway 93, Rye Creek winds its way down from the Sapphire Range through sagebrush country of hard-grazed steeps and gullies, and crosses the ranch on its way to union with the Bitterroot River. From the highway, the ranch is marked by the Wallaces' sprawling, bright-pink log home on the edge of Rye Creek, and by miles of raw roadcuts that crisscross the ranch on hills above and to the north of the creek. When Wallace bought the property in 1991, he clear-cut 640 acres of Douglas fir and pine to make pasture.

Nine-foot-high barbed wire fences framed by heavy wood posts bisect and surround the property. The ground on the hillsides remains bare and rocky, trampled by penned elk along the perimeter, by the maintenance crew's four wheelers, and snowmelt and storm runoff. Outside the fences, the land is less battered, and yellow flowers of balsamroot and antelope bitterbrush dot hillsides of a much deeper green.

On Rye Creek Road, a mile east of Highway 93, the holding pens and feedlots come into view, a maze of tall wire mesh with a heavy-gauge electrified wire running on both sides. Red-lettered signs warn away gawkers: "No Stopping On Roadway." The elk in the pens stand together or lie in groups, and when a car passes on the road, they pay close attention, their wide ears cocked like exclamation points.

Earl Butler, who worked as a foreman at Big Velvet Ranch until he quit his job last year, knows the operation well. "I left the Big Velvet because I am a hunter," he says, "and what they do up there is not a hunt." Butler tells of guiding one ranch client who refused to leave the cab of a ranch pickup until the elk he wanted to shoot was driven on the road at close range. Another client shot a bull elk in the lungs, but didn't kill it. For reasons Butler has never understood, the man refused to kill the animal with a follow-up shot. The elk staggered down a hillside to the ranch fence beside a public road, where it stood before passersby, blood spraying from its nostrils with every breath for 20 minutes before collapsing.

And then there's the "velveting" process, in which Butler worked, and which he found repellent. Velvet antlers, which sell for $60 a pound in south Asia, are ground into powder and used in tonics for common maladies and as an aphrodisiac. During the antler harvest, bull elk are driven one by one down a narrow corridor to the ranch's handling facilities. At the end of the corridor, the bull stands trapped in a chute as a hydraulic vise tightens around his body and lifts him off the floor, while a separate vise closes around his head.

This, says Butler, prepares the elk for a dose of electric current. Ranch hands use electricity to immobilize the animal in place of anesthetic because Asian buyers complain that drugs contaminate the product. The ranch hands attach an electrode to the bull's lip and another to the skin around his anus. Then they flip a switch, directing current through the animal. They tie a tourniquet where the antler joins the skull, and amputate the antlers with a handsaw. Blood and tissue stain the plywood walls around the chute.

The shoot-for-pay activities and the velveting harvests are legal, though ethically questionable to environmentalists and hunting purists. And Wallace admits that no animal shot on his farm would be knowingly recognized as a trophy on the Boone and Crockett point system, the traditional scoring scale for hunters.

Building tension

Wallace, a tall and stocky man from Southern California, wore a baseball cap when he sat for an interview in his cluttered, dusty office to discuss the ranch he calls, "my retirement. That's the reason I get up every morning at three o'clock to work on it." The office is decorated with the enormous heads of elk. One head is wrapped in a feather boa with a piece of rolled paper stuck in its mouth like a cigarette.

"Hunting here on the ranch is no different than anywhere else," Wallace says. "All over the country animals are confined. It may be by an interstate highway, a city, a river, whatever, these boundaries are not any different than a fence." Wallace made a fortune in the California electronics business, building and selling electronic power packs, before coming to the Bitterroot Valley with his wife in 1991 to start Big Velvet Ranch. But as the ranch has grown, so have protests. Last year, when Wallace applied for a fifth permit to expand, things came to a head.

Terry Klampe, a former state senator who lives 45 miles away in Florence, Mont., began citing it in a campaign to get the state Legislature to declare a moratorium on game farming. "The damage to clean water, fish, and wildlife that has been caused by (Big Velvet Ranch) reflects a total lack of concern for public property and public interests," Klampe says.

But state officials aren't likely to act on a moratorium soon. Game farming in Montana has become big business. This year, 30 more game-farm applications have been filed; 90 farms are already operating. But Klampe and his supporters - including the National Wildlife Federation, the Montana Wildlife Federation and Ducks Unlimited - are pushing hard. They've mounted an information campaign and they spoke against the ranch at last spring's state-sponsored public meetings on the Big Velvet Ranch expansion.

In June, the state, citing the need to protect a wildlife migration corridor, rejected the permit. To date, the state has spent $200,000 on the Big Velvet Ranch issue, and it's not over yet. Wallace has appealed the permit rejection and will get a hearing before state wildlife commissioners in December.

In Montana, the state can deny an operating license to a game farm that threatens critical habitat. State wildlife enforcement officer Karen Zackheim says Big Velvet Ranch already consumes half the winter range for elk, mule deer and white-tailed deer, and the expansion would have taken another 2 percent of the habitat. "We're not asking (Wallace) to do anything anyone else is not already doing," she says. "We are concerned about the wildlife resource."

In 1994, Wallace completed the ranch's fourth expansion to include the 1,800-acre upper enclosure and the lodge. Under Montana law, the "public's wildlife" - that is, any free-roaming wild game animal - must not be permanently enclosed by private fences, so Wallace and state game officials undertook an effort over several weeks to displace wild big game from the area.

Wallace hired local teenagers to run through rough country, hazing animals - including 775 mule deer - to the outside of the fencing perimeter. The state and Wallace each hired a helicopter to assist. When the drive was over, state game wardens had to shoot 49 mule deer that had were trapped behind the new fence. The wardens called the project a "mixed success," at a cost to the state of $26,000. The operation so soured relations with conservationists and sporting groups that when Wallace proposed the fifth expansion, they quickly organized.

Wallace blames the state for stirring opposition to his ranch, which he says competes with the state for hunters. "People who hunt on my property don't have to buy a hunting license," he says, "and it's not to the (state's) benefit to have a game farm in existence." Zackheim dismisses Wallace's accusation. The state, she says, sells a set number of hunting licenses at $450 apiece every year and doesn't have enough licenses to meet demand.

Running afoul of the law

1994 is also when Wallace began piling up violations with federal and state authorities. The U.S. Army Corps of Engineers has cited Big Velvet Ranch for Clean Water Act violations in connection with five dams Wallace had built that year on Lowman Creek, which also flows across the ranch and empties into Rye Creek. When the two creeks flooded during a winter thaw, overflow from the dams sent tons of sand and rock debris down Rye Creek and onto lands off the ranch. But Wallace says there was nothing unusual about the debris-flow. "If you look at the earth, things are changing constantly," he says. "Sand is always flowing down that creek."

State officials have also cited the ranch for improper fencing and for not maintaining riparian areas along the two creeks. And they have documented 24 cases of wild animals breaking into the ranch or captive game escaping. As a result, wildlife officials have had to shoot a dozen wild mule deer and three white-tailed deer.

Risk of disease

Conservationists and state officials say game farms, Big Velvet Ranch especially, must be monitored because of the risk they will spread diseases, such as tuberculosis, as well as pass the domestic traits of captive animals to wild game. Wyoming banned game farms for this reason.

"If you bring something that is captive into a (wild) herd, it passes on captive traits," says Rick Sylvester, a Wyoming Wildlife Commission spokesman. This means wild game would develop reduced immunity to disease and the inability to survive in the wild. "That's what we're trying to prevent."

Recent history compounds the worry. In 1994, an epidemic of bovine tuberculosis exploded among elk on game farms in Alberta, Canada. To protect wild herds, government shooters destroyed 2,700 head of captive elk and paid $24 million in compensation to elk ranchers. As a result, Canada's livestock industry lost its TB-free status, though there have been no recorded breakouts since.

Two years earlier, in 1992, near Hardin, Mont., tuberculosis was found in a wild mule deer doe shot outside the fence of a game farm owned by Greg Stires, of Chino, Calif. The farm had been under a TB quarantine. [Stires ranch, the Elk Valley Game Farm, received a 4 shipments 18 elk from an apparently CWD- contaminated Philipsburg facility. -- webmaster] The year before, a herd of infected game farm elk was destroyed in Colorado, and outbreaks occurred in Nebraska and South Dakota.

Last year, Big Velvet Ranch itself suffered an outbreak of cryptosporidium, an intestinal infection that breeds in fecal matter in crowded feedlots and standing water. The disease killed some 30 head of elk. But Wallace accepts no blame. He says the disease is commonly present in standing water anywhere, a claim state officials dispute. "The whole controversy," Wallace says, "is a bogus issue."

Wallace shows no sign of giving up his fight. He's preparing for his December appeal hearing on the ranch expansion, arguing that activists have been tearing down ranch fencing to let captive and wild game mix and make the ranch look bad. They're the same people, he says, that have been shooting at ranch hands and buzzing the place with helicopters. "These people are scum," Wallace says of his opponents. "They are as dishonest as the day is long." Sheriff Jay Printz has found no evidence to support the claims.

Wyoming, federal officials oppose Crow elk entrapment

10 Jul 99 Billings< Montana Gazette
CASPER, Wyo. - A Utah management group is proposing to establish a game farm on the Crow Indian Reservation in Montana stocked with wild elk captured as they winter within reservation boundaries, Wyoming wildlife managers say. [The reservation is located south of Billings, Montana and extends to the Wyoming border NW of Sheridan. Elk come off the Bighorn National Forest to escape winter snow. -- webmaster] Sponsors plan to capture 550 elk this December and a minimum of 300 elk each year for 10 subsequent years under the proposal, and manage the ranch under a Crow tribal ordinance, state officials said. The ranch would use the elk for commercial purposes, including trophy hunting and commercial slaughter. The initial capture would amount to nearly half the North Big Horn elk herd of northeastern Wyoming, which winters on the Crow reservation, Wyoming Game and Fish Department officials said.

Wyoming and federal wildlife managers oppose the concept. Taking that many elk from a herd that has about 1,300 elk will devastate the herd, Wyoming state officials said. "These are migratory elk that basically winter on the reservation ... but they don't pay any attention to political boundaries," said Jay Lawson, chief of the Wyoming department's wildlife division. Kim Floyd, president of the Wyoming Wildlife Federation, said the WWF fears the elk ranch would "completely wipe out" elk in Wyoming's Hunt Area 39, where herd spends its summer and fall. "This would decimate that herd," he said. "It's going to be a tragedy if this ranch is actually located there."

Ron Skates, a supervisor with the U.S. Fish and Wildlife Service in Bozeman, Mont., said the federal agency also is "very much against" the proposal. "If this is approved, we're looking at substantially reducing this herd, absolutely," he said. "... All our survey information going back 20 years indicated that herd is somewhere between 1,200 and 1,400 head. This will involve a lot of adverse impacts biologically on the herd."

Jack Lynch, an attorney for the Montana Department of Fish, Wildlife and Parks, said the agency does not yet have enough information about the proposal to take a position. For now, the matter is in the hands of the federal wildlife agency, he said. "We're just deferring to the feds until we're called in," Lynch said. "No one has come to us with a specific proposal."

James Innis and Wildlife Resource Management LCC of West Jordan, Utah, are sponsoring the proposal. Under its terms, the ranch would be wholly owned by the Crow tribe but operated by WRM under the name of the Castle Rock Elk Ranch. Skates said he attended a meeting last month with WRM and Crow Indian tribal members to engage in "very preliminary" discussions about the feasibility of the project. According to a spokesman at the Crow Tribal Game and Fish office, the tribal council has scheduled a meeting Saturday to further discuss the proposal. No one was immediately available to answer questions about the proposal.

Skates said that because of tribal sovereignty, the Crow tribe can establish a game ranch within its boundaries without regard for most state and federal law. "But once those elk come off that reservation, then some of these laws concerning disease and the movement of animals, hybrids and tests ... would apply," he added.

Lynch said Montana's jurisdiction over the proposed game farm would depend on who owns the land where the farm is located and whether nontribal members operate the facility. Before the proposal can go further, Skates said the tribe would have to approve the project and pass the tribal resolution. And the Utah management group will have to come up with more answers before that time, he said. "They would have to provide (us and) the tribe a lot more information about the economics of the package, how much it will cost, how much it will benefit the tribe, how many people he will employ," he said. "They didn't have those details at the first meeting."

BSE/CJD blood test by Paradigm Genetics / Prionics collaboration

BW HealthWire --July 20, 1999
RESEARCH TRIANGLE PARK, N.C. Paradigm Genetics Inc. of the United States and Prionics AG of Switzerland have signed a contract under which they will collaborate in the joint development of innovative diagnostics for the rapid detection of BSE in cattle and CJD in humans.

"The few tests available today detect prions only in brain and spinal cord tissue as opposed to whole blood and can only be administered after the patient or animal has died or been sacrificed," said Sandy Stewart, Paradigm's vice president of Biochemistry, who will head up the project for Paradigm. These tissue tests are more difficult to perform than blood tests and must be performed in a research laboratory which usually require 1-2 working days for results. In the interest of industry, faster and simpler tests will facilitate their application and thereby improve consumer safety."

Stewart explained that Prionics AG has developed proprietary antibodies that recognize and differentiate the disease-specific from the normal form of the prion protein, while Paradigm Genetics is a functional genomics company with expertise in developing state of the art immunoassay technology. Last week the European Commission confirmed the current Prionics test to be 100 percent specific and sensitive in identifying diseased from healthy animals.

"We believe that the superior immunological reagents developed by Prionics can be employed in a rapid and high throughput test format to be developed by Paradigm, increasing prion detection sensitivity, reducing incubation time to literally minutes or a few hours and allowing testing of blood instead of tissues for the pre-symptomatic detection of the diseases," he said. "The ability to test for the presence of prions in blood is a major step forward in developing a rapid, reliable and simple detection assay."

The research is expected to be completed during the next year with the first diagnostics becoming available for testing on cattle during that time. Human diagnostics could be available in the next several years depending on the length of the regulatory process.

"The exact prevalence and magnitude of the disease in cattle or humans is completely unknown primarily due to the lack of testing and the long length of incubation of the disease, especially in humans," noted Dr. John Ryals, president and chief executive officer of Paradigm Genetics. "There is clear evidence linking the new variant of CJD to consumption of BSE-contaminated foodstuff. "

Iatrogenic scrapie from sheep dura mater?

Mon, 19 Jul 1999 Listserve and Lancet  January 9, 1993 pp 123-124
An orthopaedic surgeon employed by the Lyodura company [Braun Melsungen] extracted dura mater from sheep and human cadavers and came down with fast CJD 22 years later. The ratio of sheep to human dura mater he collected was150 sheep to 12 humans. Apparently the surgeon and the sheep were German. Scrapie has long been present in Germany but reported levels are very low, about a flock a year has to be destroyed. I am not aware of any high sensitivity tests or random screening every being used in Germany to assess the levels of preclinical animals.

This raises the question, what did the lyodura company do with so much dura mater from sheep? The market for specialty surgical products was overwelmingly in humans in 1968. Are there companies today that sell sheep dura mater for research? The Lancet article only says it was for research -- but in what species? Perhaps dura mater gives an immune response across species after processing, ruling out its use in humans. But blood type or other genetic differences do not matter within humans, ie, there is no tissue matching with dura mater.

How CJD could show up from a handful of human dura mater donations with sporadic CJD supposedly so rare -- this would be extraordinary bad luck that any of 150 sheep + 12 humans could have carried the disease. On the other hand, there would be no surprise at all if a case of subclinical scrapie showed up in 150 sheep. [While Germany only reports one case a year of scrapie and destroys the flock, the disease nonetheless persists, indicating under-reporting.]

This raises the question, have dura mater recipients or the surgeon subsequently been strain-typed? This might give a very different outcome than other forms of iatrogenic CJD or simply co-classify with pituitary growth hormone if route of infection (injected, oral, hereditary, etc.) is more important than strain source.

Otherwise, iatrogenic scrapie (like cwdCJD) is somwhat unpredictable in its gel pattern (though strains of scrapie in other primate species might be re-examined). The original scrapie strain is not be identifiable directly because material was not likely retained. Strain-typing was not available at the time of the article -- but Collinge was one of the authors.

There is little doubt that scrapie could be transfered to humans by intracerebral injection (based on lack of species barrier in primates) and that processed pooled (human?) dura mater can carry sufficient infectivity to cause CJD. Animal experiments have not commonly used sheep dura mater as experimental dose source.

If any humans received sheep dura mater, it is doubtful that this will be disclosed or that specific recipients will be identified to their doctors. It is probably better to trace backwards from affected recipients -- see if they strain-type out to be sheep.

Japan has been particularly hard hit by dura mater CJD (curious in itself) and researchers there might be motivated to find out what happened. I am not aware of agricultural agencies that would impede research over there.

Transmission of Creutzfeldt-Jakob disease by handling of dura mater.

The Lancet Volume 341(8837)  January 9, 1993 pp 123-124
Weber, Thomas; Tumani, Hayrettin; Holdorff, Bernd; Collinge, John; Palmer, Mark; Kretzschmar, Hans A.; Felgenhauer, Klaus
Sir,- Creutzfeldt-Jakob disease (CJD) can be transmitted iatrogenically by human pituitary growth hormone, corneal transplants, and dura mater grafts (1). Possible accidental transmission has been reported in only four people-a neurosurgeon (2), a pathologist (3), and two laboratory technicians (4,5) . We have encountered an unusually rapid case of CJD probably acquired through handling of sheep and human dura mater.

In May, 1992, a 55-year-old orthopaedic surgeon developed paraesthesia of the left arm. A few days later he had spatial disorientation, apraxia, and gait ataxia. In June he was admitted and a neurologist suspected CJD on the basis of the clinical signs, typical electroencephalogram (EEG) pattern, and history. An EEG in June revealed a typical pattern of periodic biphasic and triphasic sharp wave complexes.

We saw the patient in July, 1992. He was awake and oriented, with dyscalculia, dysgraphia, disturbed vision, apraxia mainly of the left side, rigidity of wrists, spasticity of all muscles, myoclonus of the left arm, increased tendon reflexes, ataxia of limbs and trunk, and incoordination of left arm. Within 3 weeks he had impaired consciousness and attention, mildly impaired memory, and threatening visual hallucinations with restless turning. He had periodic states with movements of his head and eye-bulbs resembling tonic adversive seizures. During sleep these motor disturbances stopped. 2 1/2 months later the patient died.

This patient had worked with sheep and human dura mater from 1968 to 1972. He handled about 150 specimens of ovine origin and at least a dozen human preparations for research. Handling involved opening skulls with a band saw, removing dura, and testing them either fresh (usually), preserved, or lyophilised for mechanical qualities.

These specimens were sent to a company that has sold dura mater preparations by which CJD was transmitted in six instances. No information was available from the company about a possible connection with this patient's disease and the earlier cases of transmitted CJD.

Brain biopsy was consistent with diagnosis of CJD. Cerebrospinal fluid obtained in July showed neuron-specific enolase (NSE) at 82.0 ng/mL, compared with 16.7 ng/mL in serum of other cases (6). Proton magnetic resonance spectroscopy of parieto-occipital and temporal grey matter, parietal white matter, and thalamus revealed a 20-30% reduction of N-acetylaspartate, as described (7). DNA was genotyped with allele-specific oligonucleotides (8) and was homozygous for methionine at the polymorphic codon 129. Subsequent direct DNA sequencing for the PrP gene open-reading frame demonstrated normal sequence on both alleles, excluding known or novel pathogenic PrP mutations.

It is tempting to speculate that prions were transmitted to this patient from sheep or human dura mater through small lacerations of his skin, but the patient and his wife did not remember any significant injury during his four years of working with these samples. It cannot be excluded that this was a case of sporadic CJD although this assumption is unlikely in view of the clinical course which was similar to iatrogenic CJD transmitted by peripheral inoculation, such as with human pituitary growth hormone or gonadotropin or to kuru (1). Iatrogenic cases resulting from intracerebral inoculation with the transmissible agent, for instance following dura mater grafts (2-5), present with a dementing picture, as is usual in sporadic CJD, rather than with ataxia as in this case.

 
1. Brown P, Preece MA, Will RG. "Friendly fire" in medicine: hormones,
homografts, and Creutzfeldt-Jakob disease. Lancet 1992; 340: 24-27. 

2. Schoene WC, Masters CL, Gibbs CJ Jr, et al. Transmissible spongiform
encephalopathy (Creutzfeldt-Jakob Disease): atypical clinical and
pathological findings. Arch Neurol 1981; 38: 473-77. 

3. Gorman DG, Benson DF, Vogel DG, Vinters HV. Creutzfeldt-Jakob disease in
a pathologist. Neurology 1992; 42: 463. 

4. Miller DC. Creutzfeldt-Jakob disease in histopathology technicians. N
Engl J Med 1988; 318: 853-54.  

5. Sitwell L, Lach B, Atack E, Atack D, Izukawa D. Creutzfeldt-Jakob disease
in histopathology technicians. N Engl J Med 1988; 318: 854. 
[Context Link]

6. Wakayama Y, Shibuya S, Kawase J, Sagawa F, Hashizume Y. High
neuron-specific enolase level of cerebrospinal fluid in the early stage of
Creutzfeldt-Jakob disease. Klin Wochenschr 1987; 65: 798-801.  

7. Bruhn H, Weber T, Thorwirth V, Frahm J. In-vivo monitoring of neuronal
loss in Creutzfeldt-Jakob disease by proton magnetic resonance spectroscopy.
Lancet 1991; 337: 1610-11. 

8. Collinge J, Palmer MS, Dryden AJ. Genetic predisposition to iatrogenic
Creutzfeldt-Jakob disease. Lancet 1991; 337: 1444-42.  

The US would never think of pooling dura mater in the same container because of the risk of cross-contamination. However, even after the lyodura experience, apparently it was thought safe to use the same rinse water on 26 consecutive dura mater donations: Comment (webmaster): Statistical issues and various red herrings were raised in a subsequent comment letter from two consistent TSE minimalists are not applicable to the case of this surgeon:

Occupational risk of Creutzfeldt-Jakob disease.

The Lancet Volume 341(8845) Mar 6, 1993 pp 641-642[Letters to the Editor]
Ridley, R.M.; Baker,H.F.
Division of Psychiatry, Clinical Research Centre, Harrrow, Middlesex HA1 3UJ, UK.

Sir,- Readers should be cautious about Dr Weber and colleagues' (Jan 9, p 123) suggestion that occupational transmission of Creutzfeldt-Jakob disease (CJD) may have taken place in a neurosurgeon, a pathologist, 2 histology technicians, and an orthopaedic surgeon. Large epidemiological surveys (1,2) have failed to find a link between occupation and CJD. This disease has been reported in several people working in occupations in which exposure to neural tissue could have happened (eg, butchers, farmers, and various health professionals (3) ) but the number of these cases is not in excess of that which would be expected by random association. In the absence of a clear excess of cases, as has occurred in the iatrogenic transmission of spongiform encephalopathy by exposure to human derived growth hormone (4), the occurrence of CJD in people from the medical and paramedical professions is no more remarkable than its occurence in people of any other profession. Brown et al (1) rep! orted six cases among clerics, but this does not necessarily implicate their occupation in their ultimate demise.

The notion that CJD is always acquired (as opposed to idiopathic) and that the existence of any hypothetical risk factor must therefore be the cause of the disease led to the much cited claim that the high incidence of CJD among Libyan Jews was due to their consumption of sheep's eyeballs (5), despite a lack of evidence that their dietary habits differed from their ethnic neighbours in whom no increased incidence of this disease was recorded. The high frequency of CJD in the Libyan Jews is now known to be due to a codon 200 mutation in the PrP gene in affected families in that ethnic group (6).

CJD is a peculiar disease that does not fit into any single pattern of distribution. The great majority of cases cannot be attributed to environmental exposure. Very particular precautions are required to prevent transmission from cases of human and animal spongiform encephalopathy since, when this does occur, a major outbreak of disease can arise. Under these circumstances it is especially important that the occurrence of CJD is viewed from an epidemiological rather than an anecdotal perspective.

CJD in a Recipient of a U.S.-Processed Dura Mater Graft: Cause or Coincidence?

Jifsan meeting web site
Belay E.D., ...Schonberger, L.B 
In 1997, CDC was notified about a 72-year-old man who developed CJD 54 months [ie, in 1992] after he received a dura mater graft during removal of a meningioma. CDC confirmed that CJD diagnosis was based on standard clinical criteria, including typical electroencephalographic changes. Investigation of patients who underwent craniotomy within 4 months before or after the case-patient's surgery revealed no evidence for nosocomial transmission of CJD. The dura donor was a previously healthy 34-year-old man with no known risk factors for CJD who had died in a motor vehicle collision. [Accidents are not at all unusual in CJD -- evidently no tests were done for CJD prior to use as donor -- webmaster]

The dura was processed in the United States with no direct contact with other dura. However, possible indirect contact through water used to rinse dural grafts from about 25 other donors simultaneously could not be ruled out; tracing of recipients of these grafts is in progress. If this case-patient remains the only recipient of a U.S.-processed dural graft with CJD, then this graft-CJD association is more likely to be coincidental than causal. This report underscores the potential importance of recent recommendations to minimize the risk of CJD transmission by such grafts, including neuropathologic screening of all donors and removal of opportunities for cross-contamination among grafts.

Old FDA Safety Alert

IA#84-03 --- 6/27/87
 
A recent reported case of Creutzfeldt-Jakob Disease (CJD) in a 28 year-old patient who had received a human dura mater graft indicates that the graft may have been the source of this always fatal disease. The woman died 22 months after receiving the lyophilized, irradiated human cadaveric dura mater graft.

The dura mater used in the graft was packaged in October 1982 under lot #2105 by B. Braun Melsungen AG of West Germany, shipped to Tri Hawk International, Inc., Montreal, Quebec, Canada and sold to Saint Francis Hospital, Hartford, Connecticut, on April 4, 1985.

This is the first known case of CJD transmission associated with a dura mater graft. Present methods of sterilizing the dura mater do not completely inactivate the CJD agent.

The dura mater is manufactured by the West German firm under the trade name Lyodura. Although the material is primarily used in neurosurgery, it is also used in orthopedic, otologic, dental, urologic, gynecologic, and cardiac surgical procedures.

We have been unable to determine the total number of packages of Lyodura that were imported into the United States because the Canadian distributor failed to maintain adequate records of distribution for all lots which may have been distributed by mail to hospitals in the United States and Canada.

As stated in the FDA Safety Alert which issued April 28, 1987, we strongly recommend that users of dura mater choose only products from known sources which retrieve, process and handle the material according to guidelines such as those of the American Association of Tissue Banks.

Zeidler reviews dura mater history

Dr. Martin Zeidler 8 June 1998 JIFSAN meeting 
Thank you very much and I'd like to start this by thanking JIFSAN for kindly inviting me here today.

I became involved in the whole issue of Creutzfeldt-Jakob disease and dura mater grafts about a year ago when I had the good fortune to be working with the World Health Organization. It was at this time that WHO had a consultation addressing the issue of safety of medicinal and other products in relation to human and animal TSEs. This meeting recommended that dura mater grafts could no longer be used, which caused some controversy and I was involved in the debate which ensued with various dura mater manufacturers....

So, what is dura mater? Well, it's the outer covering of the meninges, this is the fibrous sheath which encircles the central nervous system. It really has two functions, first, to keep the spinal fluid in, and, second, to stop infection from getting into the central nervous system. Surgical procedures or trauma that broach the dura mater may result in a hole, that because of the fibrous, inelastic nature of dura, may not be possible shut by primary closure. If the defect is to be filled, perhaps the obvious tissue to do this is a dural graft.

Since the 1950s, dura mater grafts have been utilised ; some of the first grafts came from the U.S. Navy's Medical School here in the United States. The popularity of dural grafts came to the fore in the 1970s and 1980s when they were commercially produced. It appeared that surgeons were actually very happy with these particular materials - they provided a good barrier to infection and stopped the leakage of CSF.

It was in 1987 that the first case was reported of a person with CJD who had previously been known to have received a dura mater graft. The patient was a 28-year-old woman who had an operation 18 months previously to remove a cholesteatoma and she subsequently developed histologically confirmed CJD. To date I've managed to find a total of 83 case reports of dura mater related CJD cases, and I am grateful Dr Paul Brown for providing me with data. There are 3 additions to the number which Paul mentioned during his talk earlier, although these three cases are slightly questionable. Two of these were reported from France, and both had undergone embolisation procedures with dura mater rather than receiving a conventional graft. The third case is from Thailand and has not yet been pathologically confirmed.

The clinical phenotype of patients with dura mater-associated CJD is similar to that seen in the classical sporadic form of CJD: rapidly progressive dementia, myoclonus, and in the majority of patients a characteristic EEG. However, there are some differences: dura-associated cases tend to have more prominent early cerebellar symptoms and a somewhat more prolonged clinical course. In sporadic CJD the median illness duration is 4 1/2 months and this is doubled on average in dura mater cases. The age at onset is about 10-15 years younger on average than we see with sporadic CJD.

I think the youngest dura mater case documented was 16 or 17. The average incubation period from the time the patient received their graft to onset of their illness is approximately 6 years, but ranges from 16 months to 16 years. Although most of these cases have arisen through the use of dura mater for cranial surgery, there are some cases which have been known to have resulted from ear, nose and throat surgery or from spinal surgery. Two cases from France, as mentioned, followed embolism procedures, in one case the patient had a nasopharyngeal tumor and dura mater was cut up into lots of little pieces and injected into the external carotid artery to embolise this, and in the other case the dura mater was inoculated into an artery in the chest to embolise an area of infection.

I would like to just go back and show you the countries which are known to have had dura mater cases of CJD. Most of the reports come from Japan, and we were rather surprised at the WHO consultation last year in Geneva, to hear reports from Dr Tateishi of a recent study conducted in Japan which had shown the presence of 43 cases of dura mater CJD.

I think you will agree looking at the slide that the other cases have been reported really quite widely throughout the world. Virtually all of these patients received one particular form of dura mater graft that was commercially manufactured by a single German company. The product was called Lyodura, and most of the patients had received grafts that had been manufactured during a 4 year period between 1983 and 1987. Lyodura was pooled during this time, so there was a potential for cross contamination and the sterilization procedures used involved 10% hydrogen peroxide for 24 hours and ionizing radiation. Subsequent animal experiments have shown that this is not an adequate form of sterilization.

An important question is whether any of the dura mater cases were recipients of grafts that were treated with more thorough and adequate sterilisation. By this, I mean treatment with 1N sodium hydroxide, which is in the standard step which was introduced in the treatment of Lyodura after 1987. There are four cases out of this series of 83 which perhaps I'll talk about in a little bit more detail. Two cases were clearly not Lyodura. These were locally procured grafts, one from Italy and the other from the United Kingdom - these were used between 1969 and 1981.

Furthermore, there was the a recently reported case from America which we heard about yesterday. Perhaps the case of most interest is one from the Japanese series. This was a lady in her mid-60s who received a graft in 1991 and later developed clinically probable CJD, but this was not histologically confirmed. The hospital records did not note whether or not her graft was Lyodura or the other form of dura used in that hospital at that time. It was concluded in the report of the Japanese cases that it was unlikely that this patient had received Lyodura produced before 1987.

So the possibility exists that this patient had received a form of dura which was considered to be adequately sterilised. It is important to note two points, first, as this case did not undergo histological examination the diagnosis of CJD is not 100% certain, and second, we can not be absolutely sure that in this or some of the of other 82 cases that the history of receiving a dural mater graft is coincidental. In none of these cases is there data which tells if the graft donor had CJD.

Following the announcement of the first case here in the United States, doctors in the United Kingdom, Australia and New Zealand decided that they were going to use alternatives to cadaveric dura homografts, and here in the States I believe there was an importation ban on Lyodura.

So what alternatives are there to cadaveric-derived dura? There are several - I'll just run through these. One of the most popular is fascia lata, this is the fibrous covering of the lateral thigh muscle. The removal of this can add about 30 minutes to the length of the operation and of course leaves a wound which, as with all wounds, can potentially become infected or have other complications.

Other alternatives include pericranium (the covering of the skull bone), temporalis fascia (the membrane which surrounds the temporalis muscle at the side of the head) and synthetic materials - a number of such materials have been tried over the years including gold foils, cellophane, and dacron grafts. However, there has been some concern about the safety of synthetic materials and neurosurgeons have felt that these were rather inferior, although I think with the newer materials that's not so clearly the case. I should note that there is no controlled trial that has ever been conducted to answer the question as to whether or not these substitutes are better or worse than cadaveric-derived dura.

I think there are two key questions that need to be addressed, first, are there situations where cadaveric dura is better than available alternatives? If the answer is no then we need to question why we are using cadaveric dural grafts at all. If the answer is yes, then the next question is how can dura be made as safe as possible? I'd like to show you some of the report from the WHO meeting over a year ago. I'll read it to you.

"Because over 50 cases of CJD have resulted from cadaveric dura mater grafts, the group strongly recommended that dura mater no longer be used, especially in the case of neurosurgery, unless no alternative is available. If dura mater is to be used, only material which is from non-pooled sources originating from carefully screened donors subjected to validated inactivated treatment should be considered."

Following this recommendation the Japanese authorities decided that they were no longer going to issue a license for the use of dura mater and the TSE Advisory Committee here in the States met again to discuss the issue of dura mater. I just want to run through their recommendations, there were some differences from WHO's: although they also discouraged use of dura mater, the final decision on its usage was left up to the individual physicians, but certain additional safeguards were put into place.

For instance, it was felt mandatory that for every donor a full brain autopsy should be performed and examined histologically and with immunocytochemistry, which is probably the most sensitive method that we have, other than transmission studies. It was further recommended that a sample of the dura and the brain should be kept for further testing as needed.

Additionally, standard protocols for determining donors eligibility and tissue procurement were recommended, and dura should be collected before the brain at autopsy - which obviously makes sense to avoid contamination of the graft. Furthermore, decontamination with 1N sodium hydroxide for one hour should be used. This had previously been confirmed by Paul Brown and colleagues to be an effective decontamination procedure. There should be no pooling of grafts, to prevent cross-contamination and there should be documentation to allow tracking from the donor to the recipient and from the recipients to the donor. I think there can be little doubt that if these recommendations are adopted, then the safety of dura mater grafts will be dramatically improved.

However, I would like to just play the devil's advocate here and to mention a few cautions. We know from animal experiments that infectivity can predate any pathological changes and this includes immunocytological changes as well. We also know that standard decontamination procedures using sodium hydroxide, as David Taylor mentioned yesterday, may not completely be effective.

I think we have to remember that dura is a potentially high-risk material, and that studies also performed by David Taylor have shown that dura mater can have 106 ID50 per gram. Perhaps through the use of current decontamination procedures we will produce grafts which are much safer than those previously used, with but with low-level residual infectivity which may lead to disease with a potentially long incubation period. (For TSE agents it is known that dose administered is inversely proportional to incubation period)...

Summary statistics provided by Paul Brown

3   corneal
  2  sterotactic
  4  neurosurgery
 80  dura mater
106  growth hormone
...25 US (includes 5 New Zealand + 1 Brazilian case using US-prepared hormone)
...28 UK
...53 France
  4   gonadotrophin

Blood banks turn away tattooed donors

July 19, 1999 By CHARLES WOLFE  AP
FRANKFORT, Ky. Chris Wood queued up for the blood drive at Western Hills High School, ready to do his public service. First, a scan of the list of disqualifiers: anemia, antibiotic usage, abnormal blood pressure. No problem, Wood thought. But, as he plunged farther down the list, he spied another disqualifier:

Tattoos.

Oops. He wasn't expecting that. "It came out of nowhere," said Wood. That was in 1998, his junior year. Wood, who is bound for Western Kentucky University in the fall, had gotten a jester's head tattoo on his hip while on spring break. At the same time, he unwittingly disqualified himself as a blood donor for one year. He wasn't alone. Blood-donation workers say it now happens routinely.

The rage for body art, including tattoos and piercing, is cutting into the pool of prospective blood donors, particularly teens and young adults. At Iroquois High School in Louisville, about 20 students who pre-registered for a Red Cross blood drive this year were turned away when they returned from spring break with freshly inked tattoos.

"Everything has a ripple effect on the blood supply," said Melissa McMillan, spokeswoman for America's Blood Centers, an umbrella organization based in Washington, D.C. Blood centers adhere to Food and Drug Administration rules. Anyone who gets a tattoo or has a piercing of a body part other than the ear cannot donate blood for 12 months.

"That's just to make sure that if any infection developed, it wouldn't pose any potential threat to the U.S. blood supply. It's a layer of safety," McMillan said. The loss of potential donors because of tattoos has been palpable if not drastic, blood-center officials said.

At the Red Cross in Louisville, 6 percent of deferrals this year through June - 273 people in all - were due to tattoos, piercings and accidental needle sticks, spokeswoman Lisa Brosky said. Most were tattooed, she said. Of that total, 185 were under age 30, she said. A fifth of those deferred were 17, the minimum age for blood donors, or 18, the legal age for a tattoo or piercing without parental or guardian consent.

The Red Cross needs 500 pints of blood per day. It collected 65,000 pints in the first six months of the year. "Losing 273 doesn't sound like much, except these people could have donated three times" in six months, Brosky said, adding that the actual toll lost probably is higher.

For blood drives at Louisville-area schools, the Red Cross routinely posts a sign at the door about the deferral rule for people with tattoos. Many turn away on the spot. "They don't even have a chance to be deferred," Brosky said. At Lexington-based Central Kentucky Blood Center, deferrals are highest in the March-April and September-October periods, traditional times for blood drives on campuses, spokeswoman Marsha Berry said.

Many of those turned away are first-time donors who do not know the rules, "particularly on the high school scene, because that's the first time in their life they can donate," Berry said. Tattoos and body piercings are lumped in a category with acupuncture and electrolysis. It has been the No. 4 reason for deferrals for the last two years, trailing anemia, blood pressure and use of antibiotics, according to figures supplied by Berry.

Some question the need. Sean McNally, a new Western Hills graduate, said the tattoo parlor he patronized during spring break this year in Destin, Fla., was "completely sterile and almost hospital-like." A regular blood donor, McNally said he knew the tattoo on his back - a Chinese symbol meaning "to seek" - would disqualify him for a year.

"I was wondering at the time why I couldn't donate. The needle he was using came right out of the package," said McNally. "I guess if there's a surplus of blood, better safe than sorry," he said. But if there were a shortage, "I'd take the risk."

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