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From: TSS ()
Subject: 'Atypical' strain of BSE found in U.S. cattle
Date: August 19, 2006 at 7:28 am PST


RapidCityJournal.com
Friday, August 18, 2006

'Atypical' strain of BSE found in U.S. cattle

By Chris Clayton, DTN Staff Reporter, and Journal staff
The two cases of bovine spongiform encephalopathy found in U.S. cattle over the past year came from a rare strain of BSE found largely in Europe that scientists are only beginning to identify, according to research by a French scientist.

Researchers in France and Italy who presented their work at an international conference in London reported two rare strains of bovine spongiform encephalopathy that are harder to detect and affect mainly older cattle.

Thierry Baron of the French Food Safety Agency presented research indicating that a 12-year-old Texas cow testing positive for BSE last June, and the 10-year-old Alabama cow that tested positive in March, showed identical testing patterns to a small number of BSE cases in France, Sweden and Poland.

Animal scientists are calling such strains "atypical" BSE, which is different from the "typical" BSE caused by cattle eating feed with ruminant offal contaminated with a BSE protein.

They don't know whether the atypical strains are caused by something else or simply appear spontaneously in older, susceptible cattle.

Art Davis, a U.S. Department of Agriculture scientist for the Animal and Plant Health Inspection Service at the National Veterinary Services Laboratory in Ames, Iowa, said in his presentation Sunday at the London conference that the Texas and Alabama test results showed completely different prion patterns than the Washington state case discovered in December 2003.

"The classical lesions were not there," Davis said of the cases. The Washington state cow originated in Alberta, Canada, near where several other BSE cases have been found.

The "typical" BSE strain caused a mad cow disease epidemic in Great Britain beginning in the mid-1980s that killed 184,000 cattle and more than 100 people who contracted a human form of the disease caused by eating contaminated beef products.

The scientific evidence shows that in almost all cattle cases, the fatal neurological disorder was contracted through contaminated meat and bone meal fed to the cow, typically at a young age.

However, scientists finding atypical cases of BSE are beginning to question if there has been a change in the abnormal protein that causes BSE or if cattle might be susceptible to a sporadic BSE affecting older cattle.

Danny Matthews, head of transmissible spongiform encephalopathies at England's Veterinary Laboratories Agency, said recent research on atypical cases of BSE raises questions over whether older cattle can sporadically get the disease or if there are more strains of BSE than previously understood. Scientists might also be facing something new, such as "son of BSE," he said.

"We don't fully understand what atypical BSE means," Matthews said. "Is it spontaneous or another source causing it? Time will tell."

Although the test patterns in the U.S. cases and atypical cases in Europe closely matched, Baron said there were no known links among any of the positive animals. The French Food Safety Agency sent a researcher to the United States to study the positive Texas case and compare its results to known cases in France that did not match the typical BSE positive tests.

"You could place them side-by-side and not tell the difference," Baron said.

Baron also raised the prospect that the disease could be sporadic in at least a small number of older cattle. He said, however, such a conclusion would be hard to determine because of the small number of cattle with this atypical strain globally.

Dr. Sam Holland, South Dakota's state veterinarian, said there are many strains of BSE and varying degrees of infectiousness of the agent.

"What if the scenario is there is an atypical prion out there that is much less infective, has a longer incubation period and has not been recognized as part of the Great Britain BSE experience identified in 1985 and '86?" Holland said. "There could be others out there that we haven't recognized yet."

He said it is possible the atypical strains are not caused by contaminated feed.

He said it still makes sense to continue the ban on ruminant offal in cattle feed to prevent the spread of typical BSE and eventually to eliminate that disease.

"Based on what we know about BSE, it makes good sense to, number one, keep some surveillance in place; number two, watch what we import and restrict shipments and movements from places that have had those syndromes; and, number three, with what we know about BSE, it seems to be very prudent to keep our ruminant offal ban in place," Holland said. "At least for typical BSE's, it seems to be very effective. It's probably reasonable to continue the ruminant offal ban even after the last typical BSE case has been eliminated."

Editor's note: DTN, a private company based in Omaha, Neb., provides information to agriculture, energy trading markets and other weather-sensitive industries. The Rapid City Journal received a copy of DTN's story and expanded on it.

http://www.rapidcityjournal.com/articles/2006/05/31/news/local/news05.txt


'Atypical' strain of BSE found in U.S. cattle
By Chris Clayton, DTN Staff Reporter, and Journal staff

snip...

"Is it spontaneous .......

snip...


He said it is possible the atypical strains are not caused by contaminated feed.


snip...

end..........///


Greetings,


FIRST and foremost, there has never been a spontaneous TSE produced that the pathology looks like any natural TSE. spontaneous TSE is a myth.


SECOND, with the amount of potential TSE tainted feed that IS STILL IN COMMERCE IN THE USA, why would this not be a plausible route for any atypical TSE in USA bovine. IF you go back to the old studies of Marsh et al, and the TSE in MINK, these mink were fed 95% deadstock downer cattle. plus the studies at Mission Texas, where USA scrapie to USA bovine in the lab, produced a TSE that was not BSE, it was an atypical TSE.

amplification and transmission is a proven science through feed. ...tss




Subject: MAD COW FEED RECALLS ENFORCEMENT REPORT FOR AUGUST 9, 2006 KY, LA, MS, AL, GA, AND TN 11,000+ TONS
Date: August 16, 2006 at 9:19 am PST

RECALLS AND FIELD CORRECTIONS: VETERINARY MEDICINE - CLASS II
______________________________
PRODUCT
Bulk custom made dairy feed, Recall # V-115-6
CODE
None
RECALLING FIRM/MANUFACTURER
Hiseville Feed & Seed Co., Hiseville, KY, by telephone and letter on or about July 14, 2006. FDA initiated recall is ongoing.
REASON
Custom made feeds contain ingredient called Pro-Lak which may contain ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
Approximately 2,223 tons
DISTRIBUTION
KY

______________________________
PRODUCT
Bulk custom made dairy feed, Recall # V-116-6
CODE
None
RECALLING FIRM/MANUFACTURER
Rips Farm Center, Tollesboro, KY, by telephone and letter on July 14, 2006. FDA initiated recall is ongoing.
REASON
Custom made feeds contain ingredient called Pro-Lak which may contain ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
1,220 tons
DISTRIBUTION
KY

______________________________
PRODUCT
Bulk custom made dairy feed, Recall # V-117-6
CODE
None
RECALLING FIRM/MANUFACTURER
Kentwood Co-op, Kentwood, LA, by telephone on June 27, 2006. FDA initiated recall is completed.
REASON
Possible contamination of animal feed ingredients, including ingredients that are used in feed for dairy animals, with ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
40 tons
DISTRIBUTION
LA and MS

______________________________
PRODUCT
Bulk Dairy Feed, Recall V-118-6
CODE
None
RECALLING FIRM/MANUFACTURER
Cal Maine Foods, Inc., Edwards, MS, by telephone on June 26, 2006. FDA initiated recall is complete.
REASON
Possible contamination of animal feed ingredients, including ingredients that are used in feed for dairy animals, with ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
7,150 tons
DISTRIBUTION
MS

______________________________
PRODUCT
Bulk custom dairy pre-mixes, Recall # V-119-6
CODE
None
RECALLING FIRM/MANUFACTURER
Walthall County Co-op, Tylertown, MS, by telephone on June 26, 2006. Firm initiated recall is complete.
REASON
Possible contamination of dairy animal feeds with ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
87 tons
DISTRIBUTION
MS

______________________________
PRODUCT
Bulk custom dairy pre-mixes, Recall # V-120-6
CODE
None
RECALLING FIRM/MANUFACTURER
Ware Milling Inc., Houston, MS, by telephone on June 23, 2006. Firm initiated recall is complete.
REASON
Possible contamination of dairy animal feeds with ruminant derived meat and bone meal.
VOLUME OF PRODUCT IN COMMERCE
350 tons
DISTRIBUTION
AL and MS

______________________________
PRODUCT
a) Tucker Milling, LLC Tm 32% Sinking Fish Grower, #2680-Pellet,
50 lb. bags, Recall # V-121-6;
b) Tucker Milling, LLC #31120, Game Bird Breeder Pellet,
50 lb. bags, Recall # V-122-6;
c) Tucker Milling, LLC #31232 Game Bird Grower,
50 lb. bags, Recall # V-123-6;
d) Tucker Milling, LLC 31227-Crumble, Game Bird Starter, BMD
Medicated, 50 lb bags, Recall # V-124-6;
e) Tucker Milling, LLC #31120, Game Bird Breeder, 50 lb bags,
Recall # V-125-6;
f) Tucker Milling, LLC #30230, 30 % Turkey Starter, 50 lb bags,
Recall # V-126-6;
g) Tucker Milling, LLC #30116, TM Broiler Finisher,
50 lb bags, Recall # V-127-6
CODE
All products manufactured from 02/01/2005 until 06/20/2006
RECALLING FIRM/MANUFACTURER
Recalling Firm: Tucker Milling LLC, Guntersville, AL, by telephone and visit on June 20, 2006, and by letter on June 23, 2006.
Manufacturer: H. J. Baker and Brothers Inc., Stamford, CT. Firm initiated recall is ongoing.
REASON
Poultry and fish feeds which were possibly contaminated with ruminant based protein were not labeled as "Do not feed to ruminants".
VOLUME OF PRODUCT IN COMMERCE
7,541-50 lb bags
DISTRIBUTION
AL, GA, MS, and TN

END OF ENFORCEMENT REPORT FOR AUGUST 9, 2006

###


http://www.fda.gov/bbs/topics/ENFORCE/2006/ENF00964.html


GOTTA LOVE THOSE FDA/USDA TRIPLE BSE FIREWALLS. ...TSS


WE know now, and we knew decades ago, that 5.5 grams of suspect feed in TEXAS was enough to kill 100 cows ;


http://www.fda.gov/bbs/topics/NEWS/2001/NEW00752.html


NOT TO FORGET THE TEXAS MAD COW THAT DID GET AWAY ;


http://www.fda.gov/bbs/topics/news/2004/NEW01061.html

look at the table and you'll see that as little as 1 mg (or 0.001 gm) caused 7% (1 of 14) of the cows to come down with BSE;


Risk of oral infection with bovine spongiform encephalopathy agent in primates

Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys
Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.


snip...


BSE bovine brain inoculum

100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg

Primate (oral route)* 1/2 (50%)

Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)

RIII mice (ic ip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)

PrPres biochemical detection

The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was

inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of

bioassays is generally judged to be about plus or minus 1 log. ic ip=intracerebral and intraperitoneal.

Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula


Published online January 27, 2005

http://www.thelancet.com/journal/journal.isa


It is clear that the designing scientists must

also have shared Mr Bradley’s surprise at the results because all the dose

levels right down to 1 gram triggered infection.


http://www.bseinquiry.gov.uk/files/ws/s145d.pdf


2

6. It also appears to me that Mr Bradley’s answer (that it would take less than say 100

grams) was probably given with the benefit of hindsight; particularly if one

considers that later in the same answer Mr Bradley expresses his surprise that it

could take as little of 1 gram of brain to cause BSE by the oral route within the

same species. This information did not become available until the "attack rate"

experiment had been completed in 1995/96. This was a titration experiment

designed to ascertain the infective dose. A range of dosages was used to ensure

that the actual result was within both a lower and an upper limit within the study

and the designing scientists would not have expected all the dose levels to trigger

infection. The dose ranges chosen by the most informed scientists at that time

ranged from 1 gram to three times one hundred grams. It is clear that the designing

scientists must have also shared Mr Bradley’s surprise at the results because all the

dose levels right down to 1 gram triggered infection.


http://www.bseinquiry.gov.uk/files/ws/s147f.pdf


Re: BSE .1 GRAM LETHAL NEW STUDY SAYS via W.H.O. Dr Maura Ricketts

[BBC radio 4 FARM news]


http://www.maddeer.org/audio/BBC4farmingtoday2_1_03.ram


http://www.fda.gov/ohrms/dockets/ac/03/slides/3923s1_OPH.htm


2) Infectious dose:

To cattle: 1 gram of infected brain material (by oral ingestion)


http://www.inspection.gc.ca/english/sci/bio/bseesbe.shtml



USDA Testing Protocols and Quality Assurance Procedures

In November 2004, USDA announced that its rapid screening test produced an inconclusive BSE test result. A contract laboratory ran its rapid screening test on a brain sample collected for testing and produced three high positive reactive results. As required, the contract laboratory forwarded the inconclusive sample to APHIS’ National Veterinary Services Laboratories (NVSL) for confirmation. NVSL repeated the rapid screening test, which again produced three high positive reactive results. Following established protocol, NVSL ran its confirmatory test, an immunohistochemistry (IHC) test, which was interpreted as negative for BSE.

Faced with conflicting results between the rapid screening and IHC tests, NVSL scientists recommended additional testing to resolve the discrepancy but APHIS headquarters officials concluded that no further testing was necessary since testing protocols were followed and the confirmatory test was negative. In our discussions with APHIS officials, they justified their decision to not do additional testing because the IHC test is internationally recognized as the "gold standard" of testing. Also, they believed that

USDA/OIG-A/50601-10-KC/ Page iv

conducting additional tests would undermine confidence in USDA’s testing protocols.

OIG obtained evidence that indicated additional testing was prudent. We came to this conclusion because the rapid screening tests produced six high positive reactive results, the IHC tests conflicted, and various standard operating procedures were not followed. Also, our review of the relevant scientific literature, other countries’ protocols, and discussions with experts led us to conclude that additional confirmatory testing should be considered in the event of conflicting test results.

To maintain objectivity and independence, we requested that USDA’s Agricultural Research Service (ARS) perform the Office International des Epizooties (OIE) Scrapie-Associated Fibrils (SAF) immunoblot test. The additional testing produced positive results. To confirm, the Secretary of Agriculture requested that an internationally recognized BSE laboratory in Weybridge, England (Weybridge) perform additional testing. Weybridge conducted various tests, including their own IHC tests and three Western blot tests. The tests confirmed that the cow was infected with BSE. The Secretary immediately directed USDA scientists to work with international experts to develop new protocols that include performing dual confirmatory tests in the event of an inconclusive BSE screening test.

We attribute the failure to identify the BSE positive sample to rigid protocols, as well as the lack of adequate quality assurance controls over its testing program. Details of our concerns are discussed in Findings 3 and 4.

snip...

Section 2. Testing Protocols and Quality Assurance Controls In November 2004, USDA announced that its rapid screening test, Bio-Rad Enzyme Linked Immunosorbent Assay (ELISA), produced an inconclusive BSE test result as part of its enhanced BSE surveillance program. The ELISA rapid screening test performed at a BSE contract laboratory produced three high positive reactive results.40 As required,41 the contract laboratory forwarded the inconclusive sample to the APHIS National Veterinary Services Laboratories (NVSL) for confirmatory testing. NVSL repeated the ELISA testing and again produced three high positive reactive results.42 In accordance with its established protocol, NVSL ran its confirmatory test, an immunohistochemistry (IHC) test, which was interpreted as negative for BSE. In addition, NVSL performed a histological43 examination of the tissue and did not detect lesions44 consistent with BSE. Faced with conflicting results, NVSL scientists recommended additional testing to resolve the discrepancy but APHIS headquarters officials concluded no further testing was necessary because testing protocols were followed. In our discussions with APHIS officials, they justified their decision not to do additional testing because the IHC is internationally recognized as the “gold standard.” Also, they believed that conducting additional tests would undermine confidence in USDA’s established testing protocols. However, OIG obtained evidence that indicated additional testing was prudent to ensure that USDA’s testing protocols were effective in detecting BSE and that confidence in USDA’s testing procedures was maintained. OIG came to this conclusion because the rapid tests produced six high positive reactive results, confirmatory testing conflicted with the rapid test results, and various standard operating procedures were not followed. Also, our review of scientific literature, other country protocols, as well as discussions with internationally recognized experts led us to conclude that confirmatory testing should not be limited when conflicting test results are obtained. To maintain objectivity and independence in our assessment, we requested the USDA Agricultural Research Service (ARS) perform the Office International des Epizooties (OIE) Scrapie-Associated Fibrils (SAF) 40 ELISA test procedures require two additional (duplicate) tests if the initial test is reactive, before final interpretation. If either of the duplicate tests is reactive, the test is deemed inconclusive. 41 Protocol for BSE Contract Laboratories to Receive and Test Bovine Brain Samples and Report Results for BSE Surveillance Standard Operating Procedure (SOP), dated October 26, 2004. 42 The NVSL conducted an ELISA test on the original material tested at the contract laboratory and on two new cuts from the sample tissue. 43 A visual examination of brain tissue by a microscope. 44 A localized pathological change in a bodily organ or tissue.

immunoblot.45 ARS performed the test at the National Animal Disease Center because NVSL did not have the necessary equipment46 (ultracentrifuge) to do the test. APHIS scientists observed and participated, as appropriate, in this effort. The additional tests conducted by ARS produced positive results. To confirm this finding, the Secretary requested the internationally recognized BSE reference laboratory in Weybridge, England, (Weybridge) to perform additional confirmatory testing. Weybridge conducted various tests, including their own IHC methods, as well as three Western blot methods. The tests confirmed that the suspect cow was infected with BSE. Also, Weybridge confirmed this case as an unequivocal positive case of BSE on the basis of IHC. As a result of this finding, the Secretary immediately directed USDA scientists to work with international experts to develop a new protocol that includes performing dual confirmatory tests in the event of another inconclusive BSE screening test. Finding 3 Rigid Protocols Reduced the Likelihood BSE Could be Detected APHIS relied on a single test method, as well as a histological examination of tissue for lesions consistent with BSE, to confirm the presence of BSE even though discrepant test results indicated further testing may be prudent. When IHC test results were interpreted as negative, APHIS concluded the sample tested negative for BSE. Subsequent independent tests initiated by OIG using a different testing method, as well as confirmatory testing by Weybridge, determined that the suspect sample was a positive case of BSE. APHIS Declares BSE Sample Negative Despite Conflicting Results When the tissue sample originally arrived at NVSL in November 2004 from the contract lab, NVSL scientists repeated the ELISA screening test and again produced three high positive reactive results. NVSL scientists cut out two sections of the brain sample for IHC testing. One section was used for an experimental procedure that was not part of the confirmatory testing protocol, and the other cut was for normal IHC testing using scrapie for a positive control.47 According to NVSL scientists, the experimental test results were inconclusive but the IHC test was interpreted as negative. The NVSL scientists were concerned with the inconsistencies and conducted 45 The OIE SAF immunoblot is an internationally recognized confirmatory test, often referred to as a Western blot test. There are different types of Western blots; the OIE SAF immunoblot includes enrichment steps taken with the sample prior to the standard Western blot steps. 46 APHIS has now ordered the necessary equipment for NVSL. USDA/OIG-A/50601-10-KC Page 32

47 A positive control is a sample that is known to contain a given disease or react in the test. The sample then can be used to make sure that the test for that disease works properly. In the case of BSE, tissue infected with either scrapie or BSE can serve as a positive control for an IHC test for BSE since both are different forms of the same disease (transmissible spongiform encephalopathy or TSE).

another IHC test using BSE as a positive control.48 The test result was also interpreted as negative. Also, according to the NVSL scientists, the histological examination of the tissue did not detect lesions consistent with BSE. After the second negative IHC test, NVSL scientists supported doing additional testing. They prepared a plan for additional tests; if those tests had been conducted, BSE may have been detected in the sample. The additional tests recommended by NVSL scientists, but not approved by APHIS Headquarters officials, were the IHC using other antibodies (IHC testing using different antibodies ultimately produced positive results); IHC testing of additional regions of the brain (the cerebellum tested positive); regular and enriched (OIE-like) Western blots (the obex and cerebellum tested positive); and variable rapid tests (the obex and cerebellum tested positive with two different rapid tests). NVSL officials also recommended that the sample be sent to Weybridge for confirmatory testing (to conduct IHC and OIE Western blot tests). In June 2005, Weybridge conducted IHC testing with three different antibodies, including the antibody used in the United States (tested positive), the OIE Western blot (tested positive), a modified commercial kit Western blot (negative) and the NaPTA49 Western blot (tested positive). We obtained information as to the differing protocols used by other countries. We found that while APHIS determined that additional testing was unnecessary after the IHC test, other countries have used multiple tests to confirm positives. In Japan, for example, all reactive screening test samples are examined by both IHC and a Western blot (different from the OIE SAF immunoblot). In the United Kingdom (U.K.), IHC and Western blot (different from the OIE SAF immunoblot) tests are used for all animals that test positive with a screening test. When IHC and the Western blot fail to confirm a positive rapid test, the U.K. resorts to a third test, the OIE SAF immunoblot. With these procedures in place, both Japan and the U.K. have found BSE cases that were rapid test reactive, IHC negative, and finally confirmed positive with a Western blot. Evidence Indicated Additional Testing Would Be Prudent We also spoke with an internationally recognized BSE expert regarding the advisability of limiting confirmatory testing when conflicting results are obtained. This official expressed concern about limiting confirmatory tests to the IHC despite its status as one of the “gold standard” tests. He advised that the IHC is not one test; it is a test method that can vary significantly in sensitivity from laboratory to laboratory. New antibodies can improve or

USDA/OIG-A/50601-10-KC Page 33

48 The NVSL uses scrapie as the positive control as part of its normal IHC testing procedures. Due to the conflicting results between the ELISA and IHC tests, the NVSL conducted another IHC test with BSE as the positive control. Subsequently, the NVSL modified the Confirming Inconclusive Results from BSE Testing Laboratories at the NVSL SOP to show that all IHC tested BSE inconclusive samples from contract laboratories will use BSE as the positive control. 49 Sodium phosphotungstic acid.

USDA/OIG-A/50601-10-KC Page 34

reduce sensitivity, as can variations in many of the reagents50 used. He explained that his laboratory had experienced cases where an initial confirmatory IHC test was challenged by either a more extensive IHC test or “…applying a more sensitive immunoblot.” He emphasized the importance of having additional confirmatory testing to resolve discrepant results since there are many variables, and most of the variability appears to be due to test performance of the laboratory. OIG became concerned that APHIS relied on its confirmatory test methods when rapid screening tests produced high positive reactive results six times.51 Also, we found that APHIS did not pursue and/or investigate why the ELISA produced high reactive positives. An official from the manufacturer of the ELISA test kit told us that they requested, but did not receive, information on the inconclusive reported by USDA in November 2004. These officials requested this information in order to understand the reasons for the discrepant results. The Bio-Rad ELISA rapid screening test is internationally recognized as a highly reliable test and is the rapid screening test used for USDA’s surveillance effort. According to APHIS officials, they felt it would be inappropriate to collaborate on the one sample because Bio-Rad is a USDA-APHIS regulated biologics company and only one of several competing manufacturers. To maintain confidence in USDA’s test protocols, it would have been a prudent course of action for USDA to determine why such significant differing results were obtained. The fact that they did not pursue this matter caused concerns relating to testing quality assurance procedures. In this regard, we found lack of compliance with SOPs relating to laboratory proficiency and quality assurance (see Finding 4), and, in this case, the storage of sampled material and reporting of test results. We found that the NVSL did not prepare a report to document its confirmatory testing of the November 2004 sample. The SOP52 states that the BSE network laboratory initiating the inconclusive will receive a report of the case. NVSL officials could not explain why a final report had not been prepared. We also found that the inconclusive sample was frozen prior to IHC confirmatory testing. APHIS protocols state that samples are not to be frozen prior to laboratory submission. The OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals states that the tissues for histological or IHC examination are not to be frozen as this will provide artefactual53 lesions that may compromise the identification of vacuolation,54 and/or target site location. Although the sample was frozen, APHIS did not conduct a Western 50 A substance used in a chemical reaction to detect, measure, examine, or produce other substances. 51 The six high positive reactive results were from three tests of the submitted sample (multiple runs of the same test). 52 Confirming Inconclusive Results from Bovine Spongiform Encephalopathy Testing Laboratories at the NVSL SOP, dated August 13, 2004. 53 A structure or feature not normally present but visible as a result of an external agent or action, such as one seen in a microscopic specimen after fixation. 54 A small space or cavity in a tissue.

USDA/OIG-A/50601-10-KC Page 35

blot test on the sample. An NVSL official said freezing the sample does not make it unsuitable for IHC. APHIS determined that the sample was suitable for IHC and therefore, in accordance with its SOP, did not conduct a Western blot test. APHIS also handled the December 2003 BSE positive differently than the November 2004 sample. For the December 2003 BSE positive sample, APHIS conducted several confirmatory tests in addition to the IHC testing and histological examination (unlike the November 2004 sample tests, both of these were interpreted as positive). ARS performed two Western blots (Prionics Check Western blot and an ARS developed Western blot). When we questioned why the samples were handled differently, APHIS officials stated that the Western blots were done because the IHC in December 2003 was positive. The additional testing was done to further characterize the case, because it was the first U.S. case; the additional testing was not done to decide whether the case was positive or negative. We discussed our concerns with limiting confirmatory testing, particularly given conflicting results, with the APHIS Administrator and staff in May 2005. He explained that international standards recognized more than one “gold standard” test. In setting up its testing protocols, USDA had chosen one as the confirming test, the IHC test, and stayed with it. APHIS protocols only allow a Western blot in cases where the sample has become unsuitable for IHC tests (e.g., in cases where the brainstem architecture is not evident). International standards, he continued, accept a tissue sample as negative for BSE if its IHC test is negative. Once the test is run in accordance with protocols, additional tests undermine the USDA testing protocol and the surveillance program. He concluded that since APHIS’ protocols accepted the IHC test as confirming the presence or absence of BSE, no further testing was necessary. According to protocol, the tissue sample was determined to have tested negative for BSE. On June 24, 2005, USDA announced that the additional testing by the BSE reference laboratory in England confirmed the presence of BSE in the tissue sample. To obviate the possibility that a future sample would be declared negative and then found positive, the Secretary of Agriculture announced a change to APHIS’ testing protocols that same day. He called for “dual confirmatory tests in the event of another ‘inconclusive’ [reactive] BSE screening test.” He also indicated that he would reinforce proper procedures so that samples will not be frozen, and to spot-check the laboratories to see that they complete reports as required. APHIS issued a SOP on the new confirmatory testing protocols on November 30, 2005.

http://www.usda.gov/oig/webdocs/50601-10-KC.pdf

TSS


[Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirement for the Disposition of Non-Ambulatory Disabled Cattle

03-025IFA
03-025IFA-2
Terry S. Singeltary


Page 1 of 17

From: Terry S. Singeltary Sr. [flounder9@verizon.net]

Sent: Thursday, September 08, 2005 6:17 PM

To: fsis.regulationscomments@fsis.usda.gov

Subject: [Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirements

for the Disposition of Non-Ambulatory Disabled Cattle

Greetings FSIS,

I would kindly like to submit the following to [Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and

Requirements for the Disposition of Non-Ambulatory Disabled Cattle

THE BSE/TSE SUB CLINICAL Non-Ambulatory Disabled Cattle

Broken bones and such may be the first signs of a sub clinical BSE/TSE Non-Ambulatory Disabled Cattle ;

SUB CLINICAL PRION INFECTION

MRC-43-00

Issued: Monday, 28 August 2000

NEW EVIDENCE OF SUB-CLINICAL PRION INFECTION: IMPORTANT RESEARCH

FINDINGS RELEVANT TO CJD AND BSE


Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518

9/13/2005

http://www.fsis.usda.gov/OPPDE/Comments/03-025IFA/03-025IFA-2.pdf


suppressed peer review of Harvard study October 31, 2002

http://www.fsis.usda.gov/oa/topics/BSE_Peer_Review.pdf Mad Cow Cover-Up: NIH Plans to Destroy Brain Samples of Humans Afflicted with Mad Cow-Like CJDSteve Mitchell of UPI has been providing excellent, ongoing coverage of mad cow and its threats in the US. Here is his latest piece. One more strong piece of evidence of the cover-up ‹ it now deserves that label ‹ of mad cow risks here in the US under an administration beholden to big beef.John Stauber-NIH may destroy human brain collection Published online before print February 17, 2004, 10.1073/pnas.0305777101

Medical Sciences
Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob disease

Cristina Casalone *, Gianluigi Zanusso , Pierluigi Acutis *, Sergio Ferrari , Lorenzo Capucci , Fabrizio Tagliavini ¶, Salvatore Monaco ||, and Maria Caramelli *
*Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148, 10195 Turin, Italy; Department of Neurological and Visual Science, Section of Clinical Neurology, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134 Verona, Italy; Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Via Bianchi, 9, 25124 Brescia, Italy; and ¶Istituto Nazionale Neurologico "Carlo Besta," Via Celoria 11, 20133 Milan, Italy

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved December 23, 2003 (received for review September 9, 2003)

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are mammalian neurodegenerative disorders characterized by a posttranslational conversion and brain accumulation of an insoluble, protease-resistant isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Human and animal TSE agents exist as different phenotypes that can be biochemically differentiated on the basis of the molecular mass of the protease-resistant PrPSc fragments and the degree of glycosylation. Epidemiological, molecular, and transmission studies strongly suggest that the single strain of agent responsible for bovine spongiform encephalopathy (BSE) has infected humans, causing variant Creutzfeldt-Jakob disease. The unprecedented biological properties of the BSE agent, which circumvents the so-called "species barrier" between cattle and humans and adapts to different mammalian species, has raised considerable concern for human health. To date, it is unknown whether more than one strain might be responsible for cattle TSE or whether the BSE agent undergoes phenotypic variation after natural transmission. Here we provide evidence of a second cattle TSE. The disorder was pathologically characterized by the presence of PrP-immunopositive amyloid plaques, as opposed to the lack of amyloid deposition in typical BSE cases, and by a different pattern of regional distribution and topology of brain PrPSc accumulation. In addition, Western blot analysis showed a PrPSc type with predominance of the low molecular mass glycoform and a protease-resistant fragment of lower molecular mass than BSE-PrPSc. Strikingly, the molecular signature of this previously undescribed bovine PrPSc was similar to that encountered in a distinct subtype of sporadic Creutzfeldt-Jakob disease.


--------------------------------------------------------------------------------

C.C. and G.Z. contributed equally to this work.

||To whom correspondence should be addressed.

E-mail: salvatore.monaco@mail.univr.it .
www.pnas.org/cgi/doi/10.1073/pnas.0305777101

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

Subject: USDA, SPONTANEOUS MAD COW DISEASE, THE TOOTH FAIRY AND SANTA CLAUS
Date: June 12, 2006 at 5:18 am PST

IF we all believe the BSe that the USDA is trying to put out now about atypical BSE in USA cattle just arising spontaneously,
then we all should believe in the tooth fairy and santa claus as well.

IF USA scrapie transmitted to USA cattle long ago in experiments in a lab in Mission Texas did not produce UK BSE,
but something very different, then why would USA TSE cattle produce the UK human version of mad cow i.e. nvCJD?
IT wouldn't. USA sporadic cjd is increasing, the USA also has atypical human cases of unknown origin as well?

THERE are over 20 strains of scrapie, plus the atypical in sheep, and these strains are increasing in numbers.

SCRAPIE, CWD, AND TSE IN CATTLE i.e. ANIMAL TSE RAMPANT IN USA FOR DECADES, and amplified via rendering and
feeding practices, where USDA triple firewalls against BSE were nothing more than a mere smoke screen.

NO test tube TSE by either Prusiner or Soto, to date, have ever produced a TSE identical to the sporadic CJD. IN fact,
no test tube TSE has ever been produced that resembles _any_ natural field TSE.

IF you feed BSE tainted materials to cattle and primate, you have BSE and nvCJD.
IF you feed USA sheep strain to USA cattle, you get USA TSE.
IF you feed USA tainted cattle to humans, you get USA mad cow disease.
IF you feed sporadic CJD to primate you get a CJD infected primate.
NOTHING spontaneous about it at all.

USA is in a very unique situation. there are more documented TSE in different species than any other country,
all of which have been rendered and fed back to animals for human and animal consumption, for decades. Millions exposed,
and of these Millions, how many surgical and dental procedures have been done on these exposed, to pass on to others,
via the 'friendly fire' mode of transmission?

IF, the spontaneous TSE was true, then this would be Prusiner and everyone else that is trying to cash in on this agent with
there TSE rapid test, this would be there dream come true. IT would require mandatory BSE/TSE testing of all species,
due to the fact you could not ever eradicate it through any intervention. BUT, then again, the spontaneous TSE is like believing
in the tooth fairy or santa claus will be arriving at your house this year.


How long can this sharade continue $

How many more will become exposed and have to die $


Medical Sciences
Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob disease

Cristina Casalone *, Gianluigi Zanusso , Pierluigi Acutis *, Sergio Ferrari , Lorenzo Capucci , Fabrizio Tagliavini ¶, Salvatore Monaco ||, and Maria Caramelli *
*Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148, 10195 Turin, Italy; Department of Neurological and Visual Science, Section of Clinical Neurology, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134 Verona, Italy; Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Via Bianchi, 9, 25124 Brescia, Italy; and ¶Istituto Nazionale Neurologico "Carlo Besta," Via Celoria 11, 20133 Milan, Italy


Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved December 23, 2003 (received for review September 9, 2003)

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are mammalian neurodegenerative disorders characterized by a posttranslational conversion and brain accumulation of an insoluble, protease-resistant isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Human and animal TSE agents exist as different phenotypes that can be biochemically differentiated on the basis of the molecular mass of the protease-resistant PrPSc fragments and the degree of glycosylation. Epidemiological, molecular, and transmission studies strongly suggest that the single strain of agent responsible for bovine spongiform encephalopathy (BSE) has infected humans, causing variant Creutzfeldt-Jakob disease. The unprecedented biological properties of the BSE agent, which circumvents the so-called "species barrier" between cattle and humans and adapts to different mammalian species, has raised considerable concern for human health. To date, it is unknown whether more than one strain might be responsible for cattle TSE or whether the BSE agent undergoes phenotypic variation after natural transmission. Here we provide evidence of a second cattle TSE. The disorder was pathologically characterized by the presence of PrP-immunopositive amyloid plaques, as opposed to the lack of amyloid deposition in typical BSE cases, and by a different pattern of regional distribution and topology of brain PrPSc accumulation. In addition, Western blot analysis showed a PrPSc type with predominance of the low molecular mass glycoform and a protease-resistant fragment of lower molecular mass than BSE-PrPSc. Strikingly, the molecular signature of this previously undescribed bovine PrPSc was similar to that encountered in a distinct subtype of sporadic Creutzfeldt-Jakob disease.


--------------------------------------------------------------------------------

C.C. and G.Z. contributed equally to this work.

||To whom correspondence should be addressed.

E-mail: salvatore.monaco@mail.univr.it.
www.pnas.org/cgi/doi/10.1073/pnas.0305777101


http://www.pnas.org/cgi/content/abstract/0305777101v1

: J Infect Dis 1980 Aug;142(2):205-8


Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.

PMID: 6997404


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract


Atypical cases of TSE in cases of TSE in
cattle and sheep cattle and sheep
H. De H. De Bosschere Bosschere
CODA/CERVA CODA/CERVA
Nat. Ref. Lab. Vet. Nat. Ref. Lab. Vet. TSEs TSEs
Belgium


http://www.var.fgov.be/pdf/1100_TSEDAY.pdf


USDA 2004 ENHANCED BSE SURVEILLANCE PROGRAM AND HOW NOT TO FIND BSE CASES (OFFICIAL DRAFT OIG REPORT)

snip...


CATTLE With CNS Symptoms Were NOT Always Tested


snip...


Between FYs 2002 and 2004, FSIS condemned 680 cattle of all ages due to CNS symptoms. About 357 of these could be classified as adult. We could validate that ONLY 162 were tested for BSE (per APHIS records. ...

snip...

WE interviewed officials at five laboratories that test for rabies. Those officials CONFIRMED THEY ARE NOT REQUIRED TO SUBMIT RABIES-NEGATIVE SAMPLES TO APHIS FOR BSE TESTING. A South Dakota laboratory official said they were not aware they could submit rabies-negative samples to APHIS for BSE testing. A laboratory official in another State said all rabies-negative cases were not submitted to APHIS because BSE was ''NOT ON THEIR RADAR SCREEN." Officials from New York, Wisconsin, TEXAS, and Iowa advised they would NOT submit samples from animals they consider too young. Four of the five States contacted defined this age as 24 months; Wisconsin defined it as 30 months. TEXAS officials also advised that they do not always have sufficient tissue remaining to submit a BSE sample. ...

snip...


FULL TEXT 54 PAGES OF HOW NOT TO FIND BSE IN USA ;


http://www.house.gov/reform/min/pdfs_108_2/pdfs_inves/pdf_food_usda_mad_cow_july_13_ig_rep.pdf

HUMAN TSE USA 2005


Animal Prion Diseases Relevant to Humans (unknown types?)
Thu Oct 27, 2005 12:05
71.248.128.109


About Human Prion Diseases /
Animal Prion Diseases Relevant to Humans

Bovine Spongiform Encephalopathy (BSE) is a prion
disease of cattle. Since 1986, when BSE was recognized,
over 180,000 cattle in the UK have developed the
disease, and approximately one to three million are
likely to have been infected with the BSE agent, most
of which were slaughtered for human consumption before
developing signs of the disease. The origin of the
first case of BSE is unknown, but the epidemic was
caused by the recycling of processed waste parts of
cattle, some of which were infected with the BSE agent
and given to other cattle in feed. Control measures
have resulted in the consistent decline of the epidemic
in the UK since 1992. Infected cattle and feed exported
from the UK have resulted in smaller epidemics in other
European countries, where control measures were applied
later.

Compelling evidence indicates that BSE can be
transmitted to humans through the consumption of prion
contaminated meat. BSE-infected individuals eventually
develop vCJD with an incubation time believed to be on
average 10 years. As of November 2004, three cases of
BSE have been reported in North America. One had been
imported to Canada from the UK, one was grown in
Canada, and one discovered in the USA but of Canadian
origin. There has been only one case of vCJD reported
in the USA, but the patient most likely acquired the
disease in the United Kingdom. If current control
measures intended to protect public and animal health
are well enforced, the cattle epidemic should be
largely under control and any remaining risk to humans
through beef consumption should be very small. (For
more details see Smith et al. British Medical Bulletin,
66: 185. 2003.)

Chronic Wasting Disease (CWD) is a prion disease of elk
and deer, both free range and in captivity. CWD is
endemic in areas of Colorado, Wyoming, and Nebraska,
but new foci of this disease have been detected in
Nebraska, South Dakota, New Mexico, Wisconsin,
Mississippi Kansas, Oklahoma, Minnesota, Montana, and
Canada. Since there are an estimated 22 million elk and
deer in the USA and a large number of hunters who
consume elk and deer meat, there is the possibility
that CWD can be transmitted from elk and deer to
humans. As of November 2004, the NPDPSC has examined 26
hunters with a suspected prion disease. However, all of
them appeared to have either typical sporadic or
familial forms of the disease. The NPDPSC coordinates
with the Centers for Disease Control and state health
departments to monitor cases from CWD-endemic areas.
Furthermore, it is doing experimental research on CWD
transmissibility using animal models. (For details see
Sigurdson et al. British Medical Bulletin. 66: 199.
2003 and Belay et al. Emerging Infectious Diseases.
10(6): 977. 2004.)


http://www.cjdsurveillance.com/abouthpd-animal.html


SEE STEADY INCREASE IN SPORADIC CJD IN THE USA FROM
1997 TO 2004. SPORADIC CJD CASES TRIPLED, and that is
with a human TSE surveillance system that is terrible
flawed. in 1997 cases of the _reported_ cases of cjd
were at 54, to 163 _reported_ cases in 2004. see stats
here;

p.s. please note the 47 PENDING CASES to Sept. 2005

p.s. please note the 2005 Prion D. total 120(8)
8=includes 51 type pending, 1 TYPE UNKNOWN ???

p.s. please note sporadic CJD 2002(1) 1=3 TYPE UNKNOWN???

p.s. please note 2004 prion disease (6) 6=7 TYPE
UNKNOWN???


http://www.cjdsurveillance.com/resources-casereport.html


CWD TO HUMANS = sCJD ???


AS implied in the Inset 25 we must not _ASSUME_ that
transmission of BSE to other species will invariably
present pathology typical of a scrapie-like disease.

snip...

http://www.bseinquiry.gov.uk/files/yb/1991/01/04004001.pdf

snip...end
full text ;

http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf


VERY VERY IMPORTANT THING TO REMEMBER


>> Differences in tissue distribution could require new regulations
>> regarding specific risk material (SRM) removal.


Research Project: Study of Atypical Bse

Location: Virus and Prion Diseases of Livestock

Project Number: 3625-32000-073-07
Project Type: Specific C/A

Start Date: Sep 15, 2004
End Date: Sep 14, 2007

Objective:
The objective of this cooperative research project with Dr. Maria Caramelli
from the Italian BSE Reference Laboratory in Turin, Italy, is to conduct
comparative studies with the U.S. bovine spongiform encephalopathy (BSE)
isolate and the atypical BSE isolates identified in Italy. The studies will
cover the following areas: 1. Evaluation of present diagnostics tools used
in the U.S. for the detection of atypical BSE cases. 2. Molecular comparison
of the U.S. BSE isolate and other typical BSE isolates with atypical BSE
cases. 3. Studies on transmissibility and tissue distribution of atypical
BSE isolates in cattle and other species.

Approach:
This project will be done as a Specific Cooperative Agreement with the
Italian BSE Reference Laboratory, Istituto Zooprofilattico Sperimentale del
Piemonte, in Turin, Italy. It is essential for the U.S. BSE surveillance
program to analyze the effectiveness of the U.S diagnostic tools for
detection of atypical cases of BSE. Molecular comparisons of the U.S. BSE
isolate with atypical BSE isolates will provide further characterization of
the U.S. BSE isolate. Transmission studies are already underway using brain
homogenates from atypical BSE cases into mice, cattle and sheep. It will be
critical to see whether the atypical BSE isolates behave similarly to
typical BSE isolates in terms of transmissibility and disease pathogenesis.
If transmission occurs, tissue distribution comparisons will be made between
cattle infected with the atypical BSE isolate and the U.S. BSE isolate.
Differences in tissue distribution could require new regulations regarding
specific risk material (SRM) removal.


http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408490


3.57 The experiment which might have determined whether BSE and scrapie were
caused by the same agent (ie, the feeding of natural scrapie to cattle) was
never undertaken in the UK. It was, however, performed in the USA in 1979,
when it was shown that cattle inoculated with the scrapie agent endemic in
the flock of Suffolk sheep at the United States Department of Agriculture in
Mission, Texas, developed a TSE quite unlike BSE. 32 The findings of the
initial transmission, though not of the clinical or neurohistological
examination, were communicated in October 1988 to Dr Watson, Director of the
CVL, following a visit by Dr Wrathall, one of the project leaders in the
Pathology Department of the CVL, to the United States Department of
Agriculture. 33 The results were not published at this point, since the
attempted transmission to mice from the experimental cow brain had been
inconclusive. The results of the clinical and histological differences
between scrapie-affected sheep and cattle were published in 1995. Similar
studies in which cattle were inoculated intracerebrally with scrapie inocula
derived from a number of scrapie-affected sheep of different breeds and from
different States, were carried out at the US National Animal Disease Centre.
34 The results, published in 1994, showed that this source of scrapie agent,
though pathogenic for cattle, did not produce the same clinical signs of
brain lesions characteristic of BSE.

http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820543


The findings of the initial transmission, though not of the clinical or
neurohistological examination, were communicated in October 1988 to Dr
Watson, Director of the CVL, following a visit by Dr Wrathall, one of the
project leaders in the Pathology Department of the CVL, to the United States
Department of Agriculture. 33


http://www.bseinquiry.gov.uk/files/yb/1988/10/00001001.pdf

http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820546


The results were not published at this point, since the attempted
transmission to mice from the experimental cow brain had been inconclusive.
The results of the clinical and histological differences between
scrapie-affected sheep and cattle were published in 1995. Similar studies in
which cattle were inoculated intracerebrally with scrapie inocula derived
from a number of scrapie-affected sheep of different breeds and from
different States, were carried out at the US National Animal Disease Centre.
34 The
results, published in 1994, showed that this source of scrapie agent, though
pathogenic for cattle, did not produce the same clinical signs of brain
lesions characteristic of BSE.

3.58 There are several possible reasons why the experiment was not performed
in the UK. It had been recommended by Sir Richard Southwood (Chairman of the
Working Party on Bovine Spongiform Encephalopathy) in his letter to the
Permanent Secretary of MAFF, Mr (now Sir) Derek Andrews, on 21 June 1988, 35
though it was not specifically recommended in the Working Party Report or
indeed in the Tyrrell Committee Report (details of the Southwood Working
Party and the Tyrell Committee can be found in vol. 4: The Southwood Working
Party, 1988-89 and vol. 11: Scientists after Southwood respectively). The
direct inoculation of scrapie into calves was given low priority, because of
its high cost and because it was known that it had already taken place in
the USA. 36 It was also felt that the results of such an experiment would be
hard to interpret. While a negative result would be informative, a positive
result would need to demonstrate that when scrapie was transmitted to
cattle, the disease which developed in cattle was the same as BSE. 37 Given
the large number of strains of scrapie and the possibility that BSE was one
of them, it would be necessary to transmit every scrapie strain to cattle
separately, to test the hypothesis properly. Such an experiment would be
expensive. Secondly, as measures to control the epidemic took hold, the need
for the experiment from the policy viewpoint was not considered so urgent.
It was felt that the results would be mainly of academic interest. 38


http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820550


http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm


REPORT OF THE COMMITTEE ON SCRAPIE

Chair: Dr. Jim Logan, Cheyenne, WY

Vice Chair: Dr. Joe D. Ross, Sonora, TX

Dr. Deborah L. Brennan, MS; Dr. Beth Carlson, ND; Dr. John R. Clifford, DC; Dr. Thomas F. Conner, OH; Dr. Walter E. Cook, WY; Dr. Wayne E. Cunningham, CO; Dr. Jerry W. Diemer, TX; Dr. Anita J. Edmondson, CA; Dr. Dee Ellis, TX; Dr. Lisa A. Ferguson, MD; Dr. Keith R. Forbes, NY; Dr. R. David Glauer, OH; Dr. James R. Grady, CO; Dr. William L. Hartmann, MN; Dr. Carolyn Inch, CAN; Dr. Susan J. Keller, ND; Dr. Allen M. Knowles, TN; Dr. Thomas F. Linfield, MT; Dr. Michael R. Marshall, UT; Dr. Cheryl A. Miller, In; Dr. Brian V. Noland, CO; Dr. Charles Palmer, CA; Dr. Kristine R. Petrini, MN; Mr. Stan Potratz, IA; Mr. Paul E. Rodgers, CO; Dr. Joan D. Rowe, CA; Dr. Pamela L. Smith, IA; Dr. Diane L. Sutton, MD; Dr. Lynn Anne Tesar, SD; Dr. Delwin D. Wilmot, NE; Dr. Nora E. Wineland, CO; Dr. Cindy B. Wolf, MN.

The Committee met on November 9, 2005, from 8:00am until 11:55am, Hershey Lodge and Convention Center, Hershey, Pennsylvania. The meeting was called to order by Dr. Jim Logan, chair, with vice chairman Dr. Joe D. Ross attending. There were 74 people in attendance.

The Scrapie Program Update was provided by Dr. Diane Sutton, National Scrapie Program Coordinator, United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS), Veterinary Services (VS). The complete text of the Status Report is included in these Proceedings.

Dr. Patricia Meinhardt, USDA-APHIS-VS-National Veterinary Services Laboratory (NVSL) gave the Update on Genotyping Labs and Discrepancies in Results. NVSL conducts investigations into discrepancies on genotype testing results associated with the Scrapie Eradication Program. It is the policy of the Program to conduct a second genotype test at a second laboratory on certain individual animals. Occasionally, there are discrepancies in those results. The NVSL conducts follow-up on these situations through additional testing on additional samples from the field and archive samples from the testing laboratories.

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.

snip...

Infected and Source Flocks

As of September 30, 2005, there were 105 scrapie infected and source flocks. There were a total of 165** new infected and source flocks reported for FY 2005. The total infected and source flocks that have been released in FY 2005 was 128. The ratio of infected and source flocks cleaned up or placed on clean up plans vs. new infected and source flocks discovered in FY 2005 was 1.03 : 1*. In addition 622 scrapie cases were confirmed and reported by the National Veterinary Services Laboratories (NVSL) in FY 2005, of which 130 were RSSS cases. Fifteen cases of scrapie in goats have been reported since 1990. The last goat case was reported in May 2005. Approximately 5,626 animals were indemnified comprised of 49% non-registered sheep, 45% registered sheep, 1.4% non-registered goats and 4.6% registered goats.

Regulatory Scrapie Slaughter Surveillance (RSSS)

RSSS was designed to utilize the findings of the Center for Epidemiology and Animal Health (CEAH) Scrapie: Ovine Slaughter Surveillance (SOSS) study. The results of SOSS can be found at http://www.aphis.usda.gov/vs/ceah/cahm/Sheep/sheep.htm . RSSS started April 1,

2003. It is a targeted slaughter surveillance program which is designed to identify infected flocks for clean-up. During FY 2005 collections increased by 32% overall and by 90% for black and mottled faced sheep improving overall program effectiveness and efficiency as demonstrated by the 26% decrease in percent positive black faced sheep compared to FY 2004. Samples have been collected from 62,864 sheep since April 1, 2003, of which results have been reported for 59,105 of which 209 were confirmed positive. During FY 2005, 33,137 samples were collected from 81 plants. There have been 130 NVSL confirmed positive cases (30 collected in FY 2004 and confirmed in FY 2005 and 100 collected and confirmed in FY 2005) in FY 2005. Face colors of these positives were 114 black, 14 mottled, 1 white and 1 unknown. The percent positive by face color is shown in the chart below.

Scrapie Testing

In FY 2005, 35,845 animals have been tested for scrapie: 30,192 RSSS; 4,742 regulatory field cases; 772 regulatory third eyelid biopsies; 10 third eyelid validations; and 129 necropsy validations (chart 9).

Animal ID

As of October 04, 2005, 103,580 sheep and goat premises have been assigned identification numbers in the Scrapie National Generic Database. Official eartags have been issued to 73,807 of these premises.

*This number based on an adjusted 12 month interval to accommodate the 60 day period for setting up flock plans.


http://www.usaha.org/committees/reports/2005/report-scr-2005.pdf


Date: April 30, 2006 at 4:49 pm PST
SCRAPIE USA UPDATE AS of March 31, 2006


2 NEW CASES IN GOAT, 82 INFECTED SOURCE FLOCKS, WITH 4 NEW INFECTED SOURCE
FLOCKS IN MARCH, WITH 19 SCRAPIE INFECTED RSSS REPORTED BY NVSL


http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html


Published online before print October 20, 2005

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0502296102
Medical Sciences

A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes

( sheep prion | transgenic mice )

Annick Le Dur *, Vincent Béringue *, Olivier Andréoletti , Fabienne Reine *, Thanh Lan Laï *, Thierry Baron , Bjørn Bratberg ¶, Jean-Luc Vilotte ||, Pierre Sarradin **, Sylvie L. Benestad ¶, and Hubert Laude *
*Virologie Immunologie Moléculaires and ||Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogène, 31066 Toulouse, France; Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway


Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)

Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.


--------------------------------------------------------------------------------

Author contributions: H.L. designed research; A.L.D., V.B., O.A., F.R., T.L.L., J.-L.V., and H.L. performed research; T.B., B.B., P.S., and S.L.B. contributed new reagents/analytic tools; V.B., O.A., and H.L. analyzed data; and H.L. wrote the paper.

A.L.D. and V.B. contributed equally to this work.

To whom correspondence should be addressed.

Hubert Laude, E-mail: laude@jouy.inra.fr

www.pnas.org/cgi/doi/10.1073/pnas.0502296102


http://www.pnas.org/cgi/content/abstract/0502296102v1


12/10/76
AGRICULTURAL RESEARCH COUNCIL
REPORT OF THE ADVISORY COMMITTE ON SCRAPIE
Office Note
CHAIRMAN: PROFESSOR PETER WILDY

snip...

A The Present Position with respect to Scrapie
A] The Problem

Scrapie is a natural disease of sheep and goats. It is a slow
and inexorably progressive degenerative disorder of the nervous system
and it ia fatal. It is enzootic in the United Kingdom but not in all
countries.

The field problem has been reviewed by a MAFF working group
(ARC 35/77). It is difficult to assess the incidence in Britain for
a variety of reasons but the disease causes serious financial loss;
it is estimated that it cost Swaledale breeders alone $l.7 M during
the five years 1971-1975. A further inestimable loss arises from the
closure of certain export markets, in particular those of the United
States, to British sheep.

It is clear that scrapie in sheep is important commercially and
for that reason alone effective measures to control it should be
devised as quickly as possible.

Recently the question has again been brought up as to whether
scrapie is transmissible to man. This has followed reports that the
disease has been transmitted to primates. One particularly lurid
speculation (Gajdusek 1977) conjectures that the agents of scrapie,
kuru, Creutzfeldt-Jakob disease and transmissible encephalopathy of
mink are varieties of a single "virus". The U.S. Department of
Agriculture concluded that it could "no longer justify or permit
scrapie-blood line and scrapie-exposed sheep and goats to be processed
for human or animal food at slaughter or rendering plants" (ARC 84/77)"
The problem is emphasised by the finding that some strains of scrapie
produce lesions identical to the once which characterise the human
dementias"

Whether true or not. the hypothesis that these agents might be
transmissible to man raises two considerations. First, the safety
of laboratory personnel requires prompt attention. Second, action
such as the "scorched meat" policy of USDA makes the solution of the
acrapie problem urgent if the sheep industry is not to suffer
grievously.

snip...

76/10.12/4.6

http://www.bseinquiry.gov.uk/files/yb/1976/10/12004001.pdf


Like lambs to the slaughter
31 March 2001
Debora MacKenzie
Magazine issue 2284
What if you can catch old-fashioned CJD by eating meat from a sheep infected
with scrapie?
FOUR years ago, Terry Singeltary watched his mother die horribly from a
degenerative brain disease. Doctors told him it was Alzheimer's, but
Singeltary was suspicious. The diagnosis didn't fit her violent symptoms,
and he demanded an autopsy. It showed she had died of sporadic
Creutzfeldt-Jakob disease.

Most doctors believe that sCJD is caused by a prion protein deforming by
chance into a killer. But Singeltary thinks otherwise. He is one of a number
of campaigners who say that some sCJD, like the variant CJD related to BSE,
is caused by eating meat from infected animals. Their suspicions have
focused on sheep carrying scrapie, a BSE-like disease that is widespread in
flocks across Europe and North America.

Now scientists in France have stumbled across new evidence that adds weight
to the campaigners' fears. To their complete surprise, the researchers found
that one strain of scrapie causes the same brain damage in ...

The complete article is 889 words long.

full text;

http://www.newscientist.com/article.ns?id=mg16922840.300


Neurobiology
Adaptation of the bovine spongiform encephalopathy agent to primates and
comparison with Creutzfeldt- Jakob disease: Implications for human health
Corinne Ida Lasmézas*,, Jean-Guy Fournier*, Virginie Nouvel*, Hermann Boe*,
Domíníque Marcé*, François Lamoury*, Nicolas Kopp, Jean-Jacques Hauw§, James
Ironside¶, Moira Bruce, Dominique Dormont*, and Jean-Philippe Deslys*
* Commissariat à l'Energie Atomique, Service de Neurovirologie, Direction
des Sciences du Vivant/Département de Recherche Medicale, Centre de
Recherches du Service de Santé des Armées 60-68, Avenue du Général Leclerc,
BP 6, 92 265 Fontenay-aux-Roses Cedex, France; Hôpital Neurologique Pierre
Wertheimer, 59, Boulevard Pinel, 69003 Lyon, France; § Laboratoire de
Neuropathologie, Hôpital de la Salpêtrière, 83, Boulevard de l'Hôpital,
75013 Paris, France; ¶ Creutzfeldt-Jakob Disease Surveillance Unit, Western
General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom; and
Institute for Animal Health, Neuropathogenesis Unit, West Mains Road,
Edinburgh EH9 3JF, United Kingdom

Edited by D. Carleton Gajdusek, Centre National de la Recherche
Scientifique, Gif-sur-Yvette, France, and approved December 7, 2000
(received for review October 16, 2000)


Abstract

There is substantial scientific evidence to support the notion that bovine
spongiform encephalopathy (BSE) has contaminated human beings, causing
variant Creutzfeldt-Jakob disease (vCJD). This disease has raised concerns
about the possibility of an iatrogenic secondary transmission to humans,
because the biological properties of the primate-adapted BSE agent are
unknown. We show that (i) BSE can be transmitted from primate to primate by
intravenous route in 25 months, and (ii) an iatrogenic transmission of vCJD
to humans could be readily recognized pathologically, whether it occurs by
the central or peripheral route. Strain typing in mice demonstrates that the
BSE agent adapts to macaques in the same way as it does to humans and
confirms that the BSE agent is responsible for vCJD not only in the United
Kingdom but also in France. The agent responsible for French iatrogenic
growth hormone-linked CJD taken as a control is very different from vCJD but
is similar to that found in one case of sporadic CJD and one sheep scrapie
isolate. These data will be key in identifying the origin of human cases of
prion disease, including accidental vCJD transmission, and could provide
bases for vCJD risk assessment.


http://www.pnas.org/cgi/content/full/041490898v1


USDA CWD PROGRAM

http://www.aphis.usda.gov/vs/nahps/cwd/

USDA CWD MAP (slow to update)

http://www.aphis.usda.gov/vs/nahps/cwd/cwd-distribution.html


DRAFT

WYOMING GAME AND FISH DEPARTMENT

CHRONIC WASTING DISEASE MANAGEMENT PLAN

February 17, 2006


snip...


5. Predicted population effects on free-ranging elk based on captive elk chronically exposed to the CWD prion.
Forty-three female elk calves were trapped at the National Elk Refuge and transported to Sybille in February 2002. Elk were housed in pens, assumed to be environmentally contaminated with the CWD prion. Elk will be held throughout their lifetimes. Elk dying will be examined and cause of death determined. From these data, it will should be possible to model free-ranging elk mortality and population dynamics under extreme circumstances of CWD prion exposure and transmission. As of December 2005 (46 months post capture), 11 of 43 elk have died due to CWD. This compares to 100% mortality in less than 25 months in elk orally inoculated with different dosages of the CWD prion.

REVISED DRAFT


http://gf.state.wy.us/downloads/pdf/CWD2005reviseddraft.pdf


Prions in Skeletal Muscles of Deer with Chronic Wasting Disease


Rachel C. Angers,1* Shawn R. Browning,1*† Tanya S. Seward,2 Christina J.
Sigurdson,4‡ Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3§

1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders
Brown Center on Aging, 3Department of Neurology, University of Kentucky,
Lexington, KY 40536, USA. 4Department of Microbiology, Immunology and
Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5Colorado
Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526, USA.

*These authors contributed equally to this work.

†Present address: Department of Infectology, Scripps Research Institute,
5353 Parkside Drive, RF-2, Jupiter, Florida, 33458, USA.

‡Present address: Institute of Neuropathology, University of Zurich,
Schmelzbergstrasse 12, 8091 Zurich, Switzerland.

§To whom correspondence should be addressed: E-mail: gtell2@uky.edu

Prions are transmissible proteinaceous agents of mammals that cause fatal
neurodegenerative diseases of the central nervous system (CNS). The presence
of infectivity in skeletal muscle of experimentally infected mice raised the
possibility that dietary exposure to prions might occur through meat
consumption (1). Chronic wasting disease (CWD), an enigmatic and contagious
prion disease of North American cervids, is of particular concern. The
emergence of CWD in an increasingly wide geographic area and the
interspecies transmission of bovine spongiform encephalopathy (BSE) to
humans as variant Creutzfeldt Jakob disease (vCJD) have raised concerns
about zoonotic transmission of CWD.

To test whether skeletal muscle of diseased cervids contained prion
infectivity, Tg(CerPrP)1536 mice (2) expressing cervid prion protein
(CerPrP), were inoculated intracerebrally with extracts prepared from the
semitendinosus/semimembranosus muscle group of CWD-affected mule deer or
from CWD-negative deer. The availability of CNS materials also afforded
direct comparisons of prion infectivity in skeletal muscle and brain. All
skeletal muscle extracts from CWD-affected deer induced progressive
neurological dysfunction in Tg(CerPrP)1536 mice with mean incubation times
ranging between 360 and ~490 d, whereas the incubation times of prions from
the CNS ranged from ~230 to 280 d (Table 1). For each inoculation group, the
diagnosis of prion disease was confirmed by the presence of PrPSc in the
brains of multiple infected Tg(CerPrP)1536 mice (see supporting online
material for examples). In contrast, skeletal muscle and brain material from
CWD-negative deer failed to induce disease in Tg(CerPrP)1536 mice (Table 1)
and PrPSc was not detected in the brains of sacrificed asymptomatic mice as
late as 523 d after inoculation (supporting online material).

Our results show that skeletal muscle as well as CNS tissue of deer with CWD
contains infectious prions. Similar analyses of skeletal muscle BSE-affected
cattle did not reveal high levels of prion infectivity (3). It will be
important to assess the cellular location of PrPSc in muscle. Notably, while
PrPSc has been detected in muscles of scrapie-affected sheep (4), previous
studies failed to detect PrPSc by immunohistochemical analysis of skeletal
muscle from deer with natural or experimental CWD (5, 6). Since the time of
disease onset is inversely proportional to prion dose (7), the longer
incubation times of prions from skeletal muscle extracts compared to matched
brain samples indicated that prion titers were lower in muscle than in CNS
where infectivity titers are known to reach high levels. Although possible
effects of CWD strains or strain mixtures on these incubation times cannot
be excluded, the variable 360 to ~490 d incubation times suggested a range
of prion titers in skeletal muscles of CWD-affected deer. Muscle prion
titers at the high end of the range produced the fastest incubation times
that were ~30% longer than the incubation times of prions from the CNS of
the same animal. Since all mice in each inoculation group developed disease,
prion titers in muscle samples producing the longest incubation times were
higher than the end point of the bioassay, defined as the infectious dose at
which half the inoculated mice develop disease. Studies are in progress to
accurately assess prion titers.

While the risk of exposure to CWD infectivity following consumption of
prions in muscle is mitigated by relatively inefficient prion transmission
via the oral route (8), these


results show that semitendinosus/semimembranosus muscle, which is likely to
be consumed by humans, is a significant source of prion infectivity. Humans
consuming or handling meat from CWD-infected deer are therefore at risk to
prion exposure.

References and Notes

1. P. J. Bosque et al., Proc. Natl. Acad. Sci. U.S.A. 99, 3812 (2002).

2. S. R. Browning et al., J. Virol. 78, 13345 (2004).

3. A. Buschmann, M. H. Groschup, J. Infect. Dis. 192, 934 (2005).

4. O. Andreoletti et al., Nat. Med. 10, 591 (2004).

5. T. R. Spraker et al., Vet. Pathol. 39, 110 (2002).

6. A. N. Hamir, J. M. Miller, R. C. Cutlip, Vet. Pathol. 41, 78 (2004).

7. S. B. Prusiner et al., Biochemistry 21, 4883 (1980).

8. M. Prinz et al., Am. J. Pathol. 162, 1103 (2003).

9. This work was supported by grants from the U.S. Public Health Service
2RO1 NS040334-04 from the National Institute of Neurological Disorders and
Stroke and N01-AI-25491 from the National Institute of Allergy and
Infectious Diseases.

Supporting Online Material

www.sciencemag.org/

Materials and Methods

Fig. S1

21 November 2005; accepted 13 January 2006 Published online 26 January 2006;
10.1126/science.1122864 Include this information when citing this paper.


Table 1. Incubation times following inoculation of Tg(CerPrP)1536 mice with
prions from skeletal muscle and brain samples of CWD-affected deer.

Inocula Incubation time, mean d ± SEM (n/n0)*

Skeletal muscle Brain

CWD-affected deer

H92 360 ± 2 d (6/6) 283 ± 7 d (6/6)

33968 367 ± 9 d (8/8) 278 ± 11 d (6/6)

5941 427 ± 18 d (7/7)

D10 483 ± 8 d (8/8) 231 ± 17 d (7/7)

D08 492 ± 4 d (7/7)

Averages 426 d 264 d

Non-diseased deer

FPS 6.98 >523 d (0/6)

FPS 9.98 >454 d (0/7) >454 d (0/6)

None >490 d (0/6)

PBS >589 d (0/5)

*The number of mice developing prion disease divided by the original number
of inoculated mice is shown in parentheses. Mice dying of intercurrent
illnesses were excluded.


http://www.sciencemag.org/


www.sciencemag.org/


Supporting Online Material for

Prions in Skeletal Muscles of Deer with Chronic Wasting Disease

Rachel C. Angers, Shawn R. Browning, Tanya S. Seward, Christina J.
Sigurdson,

Michael W. Miller, Edward A. Hoover, Glenn C. Telling§

§To whom correspondence should be addressed: E-mail: gtell2@uky.edu

Published 26 January 2006 on Science Express

DOI: 10.1126/science.1122864

This PDF file includes:

Materials and Methods

Fig. S1

Supporting Online Materials

Materials and Methods

Homogenates of semitendinosus/semimembranosus muscle (10% w/v in phosphate

buffered saline) were prepared from five emaciated and somnolent mule deer,
naturally

infected with CWD at the Colorado Division of Wildlife, Wildlife Research
Center.

These deer were identified as D10, D08, 33968, H92, and 5941. CWD infection
was

confirmed in all cases by the presence of histologic lesions in the brain
including

spongiform degeneration of the perikaryon, the immunohistochemical detection
of

disease-associated PrP in brain and tonsil, or by immunoblotting of
protease-resistant,

disease associated PrP (CerPrPSc). Semitendinosus/semimembranosus muscle was
also

obtained from two asymptomatic, mock inoculated deer, referred to as FPS
6.68 and 9.98,

that originated from a CWD non-endemic area and which were held indoors at
Colorado

State University from ten days of age. These control deer were confirmed
negative for

CWD by histopathological and immunohistochemical analysis of brain tissue at
autopsy.

The utmost care was taken to avoid inclusion of obvious nervous tissue when
muscle

biopsies were prepared and to ensure that contamination of skeletal muscle
samples with

CNS tissue did not occur. Fresh, single-use instruments were used to collect
each sample

biopsy and a central piece from each sample was prepared with fresh,
disposable

instruments to further isolate muscle tissue for inoculum preparation. Brain
samples for

transmission were prepared separately from muscle as additional insurance
against cross

contamination.

1

Groups of anesthetized Tg(CerPrP)1536 mice were inoculated intracerebrally
with 30 µl

of 1 % skeletal muscle or brain extracts prepared in phosphate buffered
saline (PBS).

Inoculated Tg(CerPrP) mice were diagnosed with prion disease following the
progressive

development of at least three neurologic symptoms including truncal ataxia,
‘plastic’ tail,

loss of extensor reflex, difficultly righting, and slowed movement. The time
from

inoculation to the onset of clinical signs is referred to as the incubation
time.

For PrP analysis in brain extracts of Tg(CerPrP)1536 mice, 10 % homogenates
prepared

in PBS were either untreated (-) or treated (+) with 40 µg/ml proteinase K
(PK) for one

hour at 37oC in the presence of 2% sarkosyl. Proteins were separated by
sodium dodecyl

sulfate polyacrylamide gel electrophoresis, analyzed by immunoblotting using
anti PrP

monoclonal antibody 6H4 (Prionics AG, Switzerland), incubated with
appropriate

secondary antibody, developed using ECL-plus detection (Amersham), and
analyzed

using a FLA-5000 scanner (Fuji).

2

Fig. S1

PrP in brain extracts from representative Tg(CerPrP)1536 mice receiving
muscle or CNS

tissue inocula from CWD-affected or CWD-negative deer. Extracts were either
treated

(+) or untreated (-) with proteinase K (PK) as indicated. The positions of
protein

molecular weight markers at 21.3, 28.7, 33.5 kDa (from bottom to top) are
shown to the

left of the immunoblot.

3


http://www.sciencemag.org/

Chronic Wasting Disease and Potential Transmission to Humans
Ermias D. Belay,* Ryan A. Maddox,* Elizabeth S. Williams,† Michael W. Miller,‡ Pierluigi Gambetti,§ and Lawrence B. Schonberger*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; †University of Wyoming, Laramie, Wyoming, USA; ‡Colorado Division of Wildlife, Fort Collins, Colorado, USA; and §Case Western Reserve University, Cleveland, Ohio, USA

Suggested citation for this article: Belay ED, Maddox RA, Williams ES, Miller MW, Gambetti P, Schonberger LB. Chronic wasting disease and potential transmission to humans. Emerg Infect Dis [serial on the Internet]. 2004 Jun [date cited]. Available from: http://www.cdc.gov/ncidod/EID/vol10no6/03-1082.htm


http://www.cdc.gov/ncidod/EID/vol10no6/03-1082.htm


Research

Environmental Sources of Prion Transmission in Mule Deer
Michael W. Miller,* Elizabeth S. Williams,† N. Thompson Hobbs,‡ and Lisa L. Wolfe*
*Colorado Division of Wildlife, Fort Collins, Colorado, USA; †University of Wyoming, Laramie, Wyoming, USA; and ‡Colorado State University, Fort Collins, Colorado, USA

Suggested citation for this article: Miller MW, Williams ES, Hobbs NT, Wolfe LL. Environmental sources of prion transmission in mule deer. Emerg Infect Dis [serial on the Internet]. 2004 Jun [date cited]. Available from: http://www.cdc.gov/ncidod/EID/vol10no6/04-0010.htm


http://www.cdc.gov/ncidod/EID/vol10no6/04-0010.htm


ATYPICAL TSEs in USA CATTLE AND SHEEP ?


http://www.bseinquiry.gov.uk/files/sc/seac17/tab03.pdf

UKBSEnvCJD only theory Singeltary et al 2006
(please note, et al in this term means all victims and familes of the sporadic CJD
that are still looking for answers. ...TSS)


http://www.microbes.info/forums/index.php?act=Attach&type=post&id=13


http://www.microbes.info/forums/index.php?showtopic=306


NEW STRAIN OF TSE USA CATTLE OR JUST INCOMPETENCE IN TESTING???


http://www.fsis.usda.gov/OPPDE/Comments/03-025IFA/03-025IFA-2.pdf

Terry S. Singeltary Sr.
P.O. Box 42
Bacliff, Texas USA 77518






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