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Location: BACLIFF, Texas, United States

My mother was murdered by what I call corporate and political homicide i.e. FOR PROFIT! she died from a rare phenotype of CJD i.e. the Heidenhain Variant of Creutzfeldt Jakob Disease i.e. sporadic, simply meaning from unknown route and source. I have simply been trying to validate her death DOD 12/14/97 with the truth. There is a route, and there is a source. There are many here in the USA. WE must make CJD and all human TSE, of all age groups 'reportable' Nationally and Internationally, with a written CJD questionnaire asking real questions pertaining to route and source of this agent. Friendly fire has the potential to play a huge role in the continued transmission of this agent via the medical, dental, and surgical arena. We must not flounder any longer. ...TSS

Saturday, December 09, 2006

SCRAPIE USA

***UPDATE JANUARY 20, 2007

Subject: REPORT OF THE COMMITTEE ON SCRAPIE ANNUAL REPORT 2006

Date: January 15, 2007 at 7:53 pm PST

REPORT OF THE COMMITTEE ON SCRAPIE

snip...

Infected and Source Flocks

As of September 30, 2006, there were 85 scrapie-infected and source flocks (48 infected and 37
source). There were a total of 116 new infected and source flocks reported for FY 2006. Figure 1
shows the number of new infected and source flocks by year. The total infected and source flock statuses that were released in FY 2006 was 100. A total of 343 positive scrapie cases were confirmed and reported by the National Veterinary Services Laboratories (NVSL). Of these, 70 were RSSS cases, (collected in FY 2006 and confirmed in FY 2006 or FY 2007), and 222 positive field necropsy cases (most of these cases were found during depopulations of scrapie exposed animals in infected/source flocks), 14 necropsies of field cases retained long term for test evaluation, and 37 third eyelid regulatory tests confirmed in FY 2006. Three of the field cases were goats. One goat case, in Colorado, could not be linked to exposure in sheep as a result Colorado goats no longer meet the requirements to be classified as low-risk goats or low-risk commercial goats for interstate movement.

Approximately 3,822 animals were indemnified comprised of 62% non-registered sheep, 30%
registered sheep, 5% non-registered goats and 3% registered goats. This represents a 26% decrease over FY 2005 with a significant shift from registered to grade animals.

Regulatory Scrapie Slaughter Surveillance (RSSS)

RSSS was designed based on 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 2006, collections increased by 9% overall and by 16% for black and mottled faced sheep compared to FY 2005. Improvement in the overall program effectiveness and efficiency is demonstrated by the 33% decrease in percent positive black faced sheep compared to FY 2005 (0.67 to 0.45%, based on test results posted before November 6, 2006). During FY 2006, 37,167 samples were collected. The distribution of these samples is shown in figure 2. There have been 70 NVSL confirmed positive cases that were collected in FY 2006. Face colors of these positives were 62 black and eight mottled. The percent positive by face color is shown in the figure 3 below.

Scrapie Testing

In FY 2006, 42,823 animals were sampled for scrapie testing: 37,167 RSSS; 3,649 regulatory
field cases, 1,934 regulatory third eyelid biopsies, and 73 necropsy validations.

Animal ID

As of October 02, 2006, 118,668 sheep and goat premises have been assigned identification
numbers in the Scrapie National Generic Database. Official eartags have been issued to 96,755 of these premises.

Note: report based on data available as of November 6, 2006

snip...

Descriptive Analysis and Scrapie Infected/Source Flocks and Investigations in FY 2006.

Dianne Norden and Charles Gaiser

Regional Epidemiologists

Veterinary Services

Infected and Source Flocks

On average, Scrapie Infected/Source flocks identified in FY 2006 had an inventory of 98 animals (1,044), 23 animals indemnified on average (1-279), 3.45 positive animals found per flock upon flock cleanup plans. Of all these Infected/Source flocks for which data are available, 4,441 animals were involved in trace forward investigations. The primary breed of these flocks was predominantly blackfaced breeds, however there were 12 white-faced flocks identified (one Shetland, four Polypay Cross, four Southdown, three Dorset) and one flock whose primary breed was Dorper. Most of these flocks (89.7%) underwent a standard genetics based flock plan (flock genotyped and QQ animals removed).

Other flock plans included variations on the standard genetics based flock plan (e.g. some high risk animals retained separately from the genetically less susceptible or resistant animals after lid testing “negative”, other flocks removed QRAV animals in addition to all QQs, and four flocks underwent a whole flock depopulation. These flocks were primarily identified because of a positive found at slaughter (43%). Other detection methods included trace forward of exposed animals (30%), trace back to birth flock of positive animals (19%), investigation of clinical suspects (7%) and voluntary surveillance (1%).

Investigations

Attempts were made to trace 4,889 high risk sheep out of these Infected and Source flocks.
While some of these investigations are still ongoing (9%), 16% were untraceable and 75% were
traceable to a flock. Almost 30 (27) clinically suspicious sheep were investigated in FY 06. Seven of these animals were ultimately diagnosed with scrapie resulting in five newly discovered Infected or Source flocks. Nearly 37,000 (36,891) samples were collected at slaughter. Of these, 55 positive animals were detected, and 31 were successfully traced back to their flock of origin, resulting in 27 newly discovered Infected or Source Flocks. Over 20 (22) traces are still ongoing, and two of these positives were untraceable.

Scrapie positive animals

Of the Scrapie positive animals that were found, 75% (116) were female, and most (90%) had
lambed or aborted in their flock of origin. Most (65%) were still in their flock of birth at the time of diagnosis. Nearly all (99.2%) of all positive animals found were QQ, of those that were QQ, most (89.2% were QQAA). One animal has initially tested QRAA; the genotype of this animal is being confirmed. One QRAV positive was detected in FY 2006. Most positive animals were found as part of an Infected or Source flock depopulation (45%). Other methods of detection included RSSS traceback (28%), traceforward investigations (20%), investigation of clinical suspects (5%), and Voluntary Surveillance (2%). The breeds of these positives was predominantly black-faced breeds (99), but there were 63 White-faced breeds identified (40 Southdown, 11 Polypay Cross, two Dorsets, and 10 nonspecified white-faced or white-faced crosses). The average age of scrapie-positive animals was 3.9 years, ranging from six months to 12 years of age.


http://www.usaha.org/committees/reports/2006/report-scr-2006.pdf



=====================================================



Subject: REPORT OF THE COMMITTEE ON SCRAPIE November 9, 2005 USAHADate: February 12, 2006 at 1:03 pm PST

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.

+++++++++++++++++++++++

To reduce/eliminate these problems, the Program has placed additional quality requirements on the testing laboratories: additional review of final reports, additional coding systems for testing operations, strict follow-up and reports to NVSL on corrective actions, dual data entry systems, and more frequent inspections.

The Agricultural Research Services (ARS) Scrapie Research Update was given by Janet Alverson, USDA- ARS. Dr. Alverson reported on the effect of multiple births and fetal position within the uterus on PrP-Sc accumulation in fetal cotyledons. Fetal cotyledons of fetuses with

resistant genotypes can accumulate PrP-Sc when positioned next to a fetus of susceptible genotype with cotyledons positive for PrP-Sc accumulation.

Scrapie Surveillance Evaluation Working Group Update was presented by Tracey Lynn, Epidemiologist with the National Surveillance Unit, Center for Epidemiology and Animal Health (CEAH). The presentation provided a background on evaluation, a quick review of analyses completed to date by the scrapie surveillance evaluation working group, and some of the preliminary findings. The process of surveillance system evaluation is undertaken to assist a disease control program with identifying possible improvements to their surveillance system, and includes an assessment of the overall utility of the system, identification of potential gaps in coverage, and an evaluation of the overall performance of the system. The scrapie surveillance evaluation working group reviewed the structure and processes of the scrapie surveillance program, as well as various quality and effectiveness measures.

Overall, 98-99% of surveillance samples come from the Regulatory Scrapie Surveillance System (RSSS), so the RSSS system has been the primary focus of the evaluation process. The working group developed a flow chart indicating the flow of sheep through RSSS, which identified potential gaps in surveillance coverage, including custom kill plants and sheep being exported to Mexico. Spatial analyses can assist in identifying areas with high density sheep populations with lower levels of RSSS sampling. Identification compliance is being evaluated by reviewing reports from slaughter plants on the proportion of animals with appropriate identification. Additional analyses remain, including defining the most appropriate economic analyses, and comparing the surveillance system with developing surveillance standards. The working group hopes to have a draft written report for review by the end of the year.

Giving the Update on Scrapie Diagnostics and Susceptibility was Katherine O’Roarke, Research Microbiologist, USDA-ARS. "What’s New in Scrapie" -- Biopsy sampling of the third eyelid or tonsillar lymphoid tissue is a useful live animal test for scrapie. The biopsy sample is examined for accumulation of the abnormal prion protein using immunohistochemistry. A joint project conducted by the Veterinary Laboratory Agencies and the Moredun Institute in the United Kingdom has developed an alternative technique in which tissue is collected from the narrow band of lymphoid tissue near the rectal-anal junction. The morphology of the lymphoid follicles is similar in the tonsil, retropharyngeal lymph nodes, third eyelid, and rectal-anal mucosal tissue. A report on more than 300 sheep in the United Kingdom (UK), prepared by Drs. Lorenzo Gonzalez and Jeffrey Martin, will describe the sensitivity, specificity, and optimal collection interval for this technique in a variety of breeds of British sheep. ARS has done a preliminary evaluation of the technique in US sheep. Samples of third eyelid and rectal-mucosal tissue were collected from 56 sheep. Forty-two (42) sheep had negative biopsies at both sites; most of these sheep have been necropsied and no PrP-d was found in retropharyngeal lymph node or tonsil, showing good agreement with the antemortem biopsies. Fourteen (14) sheep had positive rectal biopsy samples; of those, only 12 had positive eyelid biopsies. These sheep will be monitored for disease development. However, the protocol is identical for all samples and it is probable that these sheep represent false negative third eyelid results. Abstracts of reports on the UK studies indicate that sensitivity of the test was 70% in the UK; similar large scale testing on US sheep is necessary. The biopsy tissue is somewhat difficult to handle in the tissue processing laboratory and adaptation to an ELISA format may improve test performance.

Alexia McKnight, Assistant Professor of Radiology, University of Pennsylvania, reviewed magnetic resonance imaging (MRI) diagnostics before the committee. A synopsis containing references is attached at the end of this report. Dr. McKnight asked the question, "could MRI be a cost-effective screening test, estimated at $25-30 each with results immediately available." The committee feels that it is not practical as compared to other alternatives currently available. However, the committee expressed interest in future reference to this technology.

Dr. Diane Sutton lead the Uniform Methods and Rules (UM&R) and Regulatory Issues Discussion. Several modifications to the UM&R were discussed. Eight issues were identified and communicated to the APHIS scrapie program coordinator. The committee acknowledged that APHIS and the industry is adequately addressing the year-to-year industry concerns.

Dr. Kris Petrini representing the North Central United States Animal Health Association District presented five recommendations to the Committee. During the discussions regarding these recommendations it was evident that all five issues had been addressed during the year at this Committee meeting.

The Committee approved a recommendation that USDA-APHIS-VS continue to provide indemnity funds for animals that have been designated for testing in Flocks Under Investigation as an alternative to third eyelid testing after consultation with the designated Scrapie Epidemiologist (DSE) and the Regional Area Epidemiologist (RAE).

The 2004 Resolutions along with their responses were reviewed by the Committee.

A Resolution concerning premises registration and identification was approved by the Committee and forwarded to the Committee on Nominations and Resolutions.

Committee on Scrapie

Status Report-Fiscal Year 2005: Cooperative State-Federal Scrapie Eradication Program

Submitted by Diane Sutton, DVM and Gary Ross, DVM

National Center for Animal Health Programs, APHIS, USDA

In Fiscal Year 2005 the Scrapie Eradication Program focused on: (1) utilization of a genetic based approach to flock clean-up plans; (2) cleaning up infected and source flocks; (3) tracing and testing exposed animals and flocks; (4) expansion of regulatory slaughter surveillance (RSSS); (5) conducting considtent state reviews, (6) producer education; (7) upgrading of the Scrapie National Generic Database and (8) publishing the updated Scrapie Eradication Uniform Methods and Rules (UM&R). The current Scrapie Eradication UM&R is posted at


http://www.aphis.usda.gov/vs/nahps/scrapie/umr-scrapie-erad.pdf.



Consistent State Reviews

States must meet the requirements in 9 CFR 79.6 in order to move sheep and goats in interstate commerce with minimal restrictions. Twenty seven states have enacted the required identification rules, the remaining states have submitted a work plan that describes the steps that will be taken to comply and provided a timeline for completing significant milestones. USDA is conducting onsite scrapie program consistent state reviews and has completed reviews in 12 states. States must be in full compliance by the end of their current rule making cycle. States not in full compliance at that time will be removed from the consistent state list. Removal from the list would create a significant impact on the interstate movement of sheep and goats from those States.

Scrapie Flock Certification Program

As of September 30, 2005, there were 1,961 flocks participating in the Scrapie Flock Certification Program (SFCP). Of these flocks 188 were certified flocks, 1,770 were complete monitored flocks, and 3 were selective monitored flocks (figure 2). There were 209 flocks newly enrolled and 53 newly certified (13 with status dates in FY 2005 and 40 with status dates in previous years) in FY 2005 (figure 3).

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





NOW, you can compare with the usda et al choice of the bio-rad at first, this is a long history of questionable decisions ;



-------- Original Message --------Subject: Dr. Ron DeHaven DOING THE MAD COW TEXAS TWO-STEP AGAINDate: Thu, 30 Dec 2004 12:35:24 -0600From: "Terry S. Singeltary Sr." Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE

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

Release No. 0528.04Contact:Office of Communications (202) 720 4623



Statement By Dr. Ron DeHaven Administrator, Animal & Plant HealthInspection Service

December 30, 2004

"USDA is confident that the animal and public health measures thatCanada has in place to prevent BSE, combined with existing U.S. domesticsafeguards and additional safeguards announced yesterday provide theutmost protections to U.S. consumers and livestock.

"Last night Canada announced the finding of a "suspect" animal, which istheir term for inconclusive. If this animal proves to be positive, itwould not alter the implementation of the U.S. rule announced yesterdaythat recognizes Canada as a Minimal-Risk Region. In the extensive riskanalysis conducted as part of the rule making, we considered thepossibility of additional cases of BSE in Canada. Because of themitigation measures that Canada has in place, we continue to believe therisk is minimal.

"When Canadian ruminants and ruminant products are presented forimportation into the United States, they become subject to domesticsafeguards as well. Beef imports that have already undergone Canadianinspection are also subject to re-inspection at ports of entry by theUSDA's Food Safety and Inspection Service (FSIS) to ensure only eligibleproducts are imported

"We are working closely with Canadian officials as they conduct theirinvestigation into this situation."


http://www.usda.gov/wps/portal/!ut/p/_s.7_0_A/7_0_1OB/.cmd/ad/.ar/sa.retrievecontent/.c/6_2_1UH/.ce/7_2_5JM/.p/5_2_4TQ/.d/3/_th/J_2_9D/_s.7_0_A/7_0_1OB?PC_7_2_5JM_contentid=2004%2F12%2F0528.xml&PC_7_2_5JM_navtype=RT&PC_7_2_5JM_parentnav=LATEST_RELEASES&PC_7_2_5JM_navid=NEWS_RELEASE#7_2_5JM



Greetings list members,

IF you remember correctly, i posted this ;

Subject: Re: USDA/APHIS JUNE 2004 'ENHANCED' BSE/TSE COVER UP UPDATEDECEMBER 19, 2004 USADate: Thu, 30 Dec 2004 12:27:06 -0600From: "Terry S. Singeltary Sr.

BSE-L

snip...

>> OH, i did ask Bio-Rad about this with NO reply to date;>>> -------- Original Message --------> Subject: USA BIO-RADs INCONCLUSIVEs> Date: Fri, 17 Dec 2004 15:37:28 -0600> From: "Terry S. Singeltary Sr."> To: susan_berg@bio-rad.com>>>> Hello Susan and Bio-Rad,>> Happy Holidays!>> I wish to ask a question about Bio-Rad and USDA BSE/TSE testing> and there inconclusive. IS the Bio-Rad test for BSE/TSE that complicated,> or is there most likely some human error we are seeing here?>> HOW can Japan have 2 positive cows with> No clinical signs WB+, IHC-, HP- ,> BUT in the USA, these cows are considered 'negative'?>> IS there more politics working here than science in the USA?>> What am I missing?>>>> -------- Original Message --------> Subject: Re: USDA: More mad cow testing will demonstrate beef's safety> Date: Fri, 17 Dec 2004 09:26:19 -0600> From: "Terry S. Singeltary Sr."> snip...end>>> Experts doubt USDA's mad cow results



snip...END

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

Bio-Rad, TSS phone conversation 12/28/04

Finally spoke with ;

Bio-Rad Laboratories2000 Alfred Nobel DriveHercules, CA 94547Ph: 510-741-6720Fax: 510-741-5630Email: XXXXXXXXXXXXXXXXXX

at approx. 14:00 hours 12/28/04, I had a very pleasantphone conversation with XXXX XXXXX about the USDAand the inconclusive BSE testing problems they seemto keep having. X was very very cautious as to speakdirectly about USDA and it's policy of not using WB.X was very concerned as a Bio-Rad official of retaliationof some sort. X would only speak of what other countriesdo, and that i should take that as an answer. I told XI understood that it was a very loaded question and Xagreed several times over and even said a political one.

my question;

Does Bio-Rad believe USDA's final determination of False positive,without WB, and considering the newatypical TSEs not showing positive with -IHC and -HP ???

ask if i was a reporter. i said no, i was with CJD Watchand that i had lost my mother to hvCJD. X did notwant any of this recorded or repeated.

again, very nervous, will not answer directly about USDA for fear ofretaliation, but again said X tellme what other countries are doing and finding, and thati should take it from there."very difficult to answer"

"very political"

"very loaded question"

outside USA and Canada, they use many different confirmatory tech. inhouse WB, SAF, along withIHC, HP, several times etc. you should see at severaltalks meetings (TSE) of late Paris Dec 2, that IHC- DOES NOT MEAN IT ISNEGATIVE. again, look whatthe rest of the world is doing.said something about Dr. Houston stating;any screening assay, always a chance for humanerror. but with so many errors (i am assumingX meant inconclusive), why are there no investigations, just falsepositives?said something about ''just look at the sheep that tested IHC- but werepositive''. ...

TSS

-------- Original Message --------Subject: Your questionsDate: Mon, 27 Dec 2004 15:58:11 -0800From: To: flounder@wt.net



Hi Terry:

............................................snip Let me know your phonenumber so I can talk to you about the Bio-Rad BSE test.Thank you

Regards



Bio-Rad Laboratories2000 Alfred Nobel DriveHercules, CA 94547Ph: 510-741-6720Fax: 510-741-5630Email: =================================

END...TSS

######### https://listserv.kaliv.uni-karlsruhe.de/warc/bse-l.html ##########



Subject: Re: BSE Update: US Officials Expect BSE Test Results As Early As FridayDate: June 22, 2005 at 9:24 pm PSTIn Reply to: BSE Update: US Officials Expect BSE Test Results As Early As Friday posted by TSS on June 22, 2005 at 8:24 pm:

Greetings list members,

>>>DEFRA's Matthew said that USDA's different results from its BSE tests mayhave more to do with the quality of the samples being studied than theeffectiveness of the tests.<<< >>>That situation, he said, isn't uncommon. "There can be variations in theamount of abnormal prions in the brain stem and you could take one samplethat's got enough in it and another sample that hasn't got enough.... If youcan't confirm your initial result by applying a second test then it's goodpractice to try a third." <<<> > -------- Original Message --------> > Subject: USA BIO-RADs INCONCLUSIVEs> > Date: Fri, 17 Dec 2004 15:37:28 -0600> > From: "Terry S. Singeltary Sr."> > To: susan_berg@bio-rad.com> >> >> >> > Hello Susan and Bio-Rad,> >> > Happy Holidays!> >> > I wish to ask a question about Bio-Rad and USDA BSE/TSE testing> > and there inconclusive. IS the Bio-Rad test for BSE/TSE thatcomplicated,> > or is there most likely some human error we are seeing here?> >> > HOW can Japan have 2 positive cows with> > No clinical signs WB+, IHC-, HP- ,> > BUT in the USA, these cows are considered 'negative'?> >> > IS there more politics working here than science in the USA?> >> > What am I missing?> >> >> >> > -------- Original Message --------> > Subject: Re: USDA: More mad cow testing will demonstrate beef's safety> > Date: Fri, 17 Dec 2004 09:26:19 -0600> > From: "Terry S. Singeltary Sr."> > snip...end> >> >> > Experts doubt USDA's mad cow results>>>> snip...END>> WELL, someone did call me from Bio-Rad about this,> however it was not Susan Berg.> but i had to just about take a blood oath not to reveal> there name. IN fact they did not want me to even mention> this, but i feel it is much much to important. I have omitted> any I.D. of this person, but thought I must document this ;>> Bio-Rad, TSS phone conversation 12/28/04>> Finally spoke with ;>>> Bio-Rad Laboratories> 2000 Alfred Nobel Drive> Hercules, CA 94547> Ph: 510-741-6720> Fax: 510-741-5630> Email: XXXXXXXXXXXXXXXXXX>> at approx. 14:00 hours 12/28/04, I had a very pleasant> phone conversation with XXXX XXXXX about the USDA> and the inconclusive BSE testing problems they seem> to keep having. X was very very cautious as to speak> directly about USDA and it's policy of not using WB.> X was very concerned as a Bio-Rad official of retaliation> of some sort. X would only speak of what other countries> do, and that i should take that as an answer. I told X> I understood that it was a very loaded question and X> agreed several times over and even said a political one.>> my question;>> Does Bio-Rad believe USDA's final determination of False positive,> without WB, and considering the new> atypical TSEs not showing positive with -IHC and -HP ???>> ask if i was a reporter. i said no, i was with CJD Watch> and that i had lost my mother to hvCJD. X did not> want any of this recorded or repeated.>> again, very nervous, will not answer directly about USDA for fear of> retaliation, but again said X tell> me what other countries are doing and finding, and that> i should take it from there.> "very difficult to answer">> "very political">> "very loaded question">> outside USA and Canada, they use many different confirmatory tech. in> house WB, SAF, along with> IHC, HP, several times etc. you should see at several> talks meetings (TSE) of late Paris Dec 2, that IHC- DOES NOT MEAN IT IS> NEGATIVE. again, look what> the rest of the world is doing.> said something about Dr. Houston stating;> any screening assay, always a chance for human> error. but with so many errors (i am assuming> X meant inconclusive), why are there no investigations, just false> positives?> said something about ''just look at the sheep that tested IHC- but were> positive''. ...>>> TSS>> -------- Original Message --------> Subject: Your questions> Date: Mon, 27 Dec 2004 15:58:11 -0800> From: To: flounder@wt.net>>>> Hi Terry:>> ............................................snip Let me know your phone> number so I can talk to you about the Bio-Rad BSE test.> Thank you>> Regards>>>> Bio-Rad Laboratories> 2000 Alfred Nobel Drive> Hercules, CA 94547> Ph: 510-741-6720> Fax: 510-741-5630> Email: =================================>>> END...TSS>>> ######### https://listserv.kaliv.uni-karlsruhe.de/warc/bse-l.html##########>> =====================================================> =====================================================>> END....TSS>

FULL TEXT;

http://www.vegsource.com/talk/madcow/messages/94627.html





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.

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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





NOW ms ostfeld lets look at the vs research on atypical testing ;





Research Project: OVINE PRION & VIRAL INFECTIONS: SCRAPIE & OVINE PROGRESSIVE PNEUMONIA, DIAGNOSIS & CONTROLLocation: Animal Diseases Research

2005 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?The economic losses to the sheep industry due to ovine scrapie can be reduced by a coordinated program of live animal testing, replacement with sheep of lower susceptibility, and reducing transmission within the flock. Scrapie is a transmissible spongiform encephalopathy associated with deposition of an abnormal isoform of a mammalian glycoprotein, the prion protein, in tissues throughout the sheep. The highest level of accumulation is in the brain, although detectable levels are found in lymphoid tissues and placenta/fetal tissues. Detection of prions in peripheral lymphoid tissue can be used to identify and cull infected animals early in infection. Further, the susceptibility of sheep to clinical scrapie and to accumulation of prions is under genetic control. Quantitative determination of the level of protection associated with commonly occurring genotypes could enable producers and regulatory programs to integrate protective genetics with elimination of infected stock to reduce the amount of disease in U.S. flocks. Control of all domestic prion diseases is important in reducing trade barriers for U.S. sheep and germplasm and for assuring the present and future global markets for cattle. Ovine progressive pneumonia virus causes persistent viral infection and development of multi-organ inflammatory disease in some animals. The infection rates in US sheep flocks range from 9 to 49%. A variable and unpredictable percentage of infected animals will progress to clinical disease with high viral titers and increased risk of transmission to flockmates. This project has produced a serologic test for the disease. The current project includes development of a vaccine to reduce infected animals and identification of genes associated with decreased transmission in infected sheep. Sheep scrapie is a member of a group of diseases that includes bovine spongiform encephalopathy (BSE). Sheep scrapie is associated with minor direct losses to the industry. However, loss of international markets for sheep and sheep germplasm and loss of the domestic access to rendering facilities are major economic losses to the industry. Control programs based on epidemiology alone have failed to control the spread of scrapie throughout the U.S. since the disease was introduced in 1947. A control program based on a live animal diagnostic test supplemented by introduction of replacement stock of lower genetic susceptibility is urgently needed. Transmission barriers of these diseases are not understood. Therefore, the presence of any of these diseases within the U.S. represents a continuous threat for emergence in animals not yet found to have been infected in the U.S. The occurrence of BSE in U.S. cattle has caused economic losses in the U.S. cattle domestic market and the world markets. Animals with clinical signs of ovine progressive pneumonia virus represent the main risk of virus transmission because disease progression is associated with much higher virus loads than is nonprogressive persistent infection. Therefore, the immunogenetic basis of disease pathogenesis is an important consideration in SRLV research. The most significant problem associated with SRLV infection is the inability to determine which animals will progress to severe clinical disease and death. Our objectives are to reduce the clinical disease rates and viremia levels through a vaccination program targeting the type 1 immune responses and to determine the immunogenetics associated with this response. SRLV research will lead to methods for the induction of immunologic control and genetic predictors of animals with a low potential for transmission if infected. Our research concerning the transmissible encephalopathies and small ruminant lentiviruses address the following elements of our National Program in Animal Production, Product Value and Safety. Our research encompasses the National Program Initiative 103 Animal Health at 100%, and our research components include pathogen detection and diagnostics, animal immunology, mechanism of disease, genetic resistance to disease, and strategies to control infectious and non-infectious diseases.



2.List the milestones (indicators of progress) from your Project Plan.Year 1 (FY2002)

Validate diagnostic and genetic tests; IHC test validation studies and PrP genotype continue from previous CRIS.

Define transmission route. Continue study on effect of maternal disease status and fetal genotype of PrP-Sc in placenta. Initiate studies on effect of pregnancy and placenta on PrP-Sc.

Initiate blood transfusion study. Complete methods for detecting and quantitating PrP-C and PrP-Sc in dissociated lymph nodes.

Induce type 1 response to CAEV and MVV. Immunize goats with plasmid DNA expressing CAEV env and boost with SU-FIA. Isolate and characterize wild type CAEV for challenge and challenge-immunized goats.

Identify MVV resistant MHC class II haplotypes. Complete titration and isotypes of antibodies from Dubois sheep.

Year 2 (FY2003)

Continue validation of IHC tests.

Continue studies on effect of pregnancy and placenta on PrP-Sc.

Continue FACS and IHC analysis of lymph node cells for PrP-C and PrP-Sc.

Continue examination of postchallenge parameters. Construct and characterize a recombinant plasmid expressing MVV8534 env.

Complete T cell proliferation assays and divide sheep.

Year 3 (FY2004)

Complete validation of IHC tests.

Complete studies on effect of pregnancy and placenta on PrP-Sc.

Complete FACS and IHC analysis of lymph node cells for PrP-C and PrP-Sc.

Continue examination of postchallenge parameters. Construct and characterize a recombinant plasmid expressing MVV8534 env.

Year 4 (FY2005)

Initiate studies on rapid, high volume diagnostic testing; complete PrP genotype study.

Complete study on effect of maternal disease status and fetal genotype of PrP-Sc in placenta Initiate studies on regulation of PrP-C by estrous cycle and pregnancy.

Complete PrP-C distribution and quantitation in peripheral blood cells; initiate immunomagnetic bead enrichment studies if indicated.

Immunize sheep with plasmid DNA expressing MVV8534 env and boost with MVV SU-FIA. Challenge immunized goats with MVV8534.

Complete PCR-RFLP of OvLa DRB-1 alleles.

Year 5 (FY2006)

Complete PrP-Sc rapid test development and transfer technology.

Complete studies on regulation of PrP-C by estrous cycle and pregnancy.

Complete blood transfusion study.

Continue examination of postchallenge parameters.

Identify OvLa DRB-1 alleles that associate with type 1 responses. Identify MMV-free sheep with appropriate DRB-1 haplotypes and challenge with MVV8534.

3a.List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why.

Initiate studies on rapid, high volume diagnostic testing; complete PrP genotype study.Milestone Substantially Met

Complete study on effect of maternal disease status and fetal genotype of PrP-Sc in placenta Initiate studies on regulation of PrP-C by estrous cycle and pregnancy.Milestone Substantially Met

Complete PrP-C distribution and quantitation in peripheral blood cells; initiate immunomagnetic bead enrichment studies if indicated.Milestone Fully Met

Immunize sheep with plasmid DNA expressing MVV8534 env and boost with MVV SU-FIA. Challenge immunized sheep with MVV8534.Milestone Not Met

Complete PCR-RFLP of OvLa DRB-1 alleles.Milestone Fully Met

3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone?Year 5, 6 and 7 milestones are listed below with a description of the anticipated outcomes. The entire project will be completed during FY 2006, and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2007.

Year 5 (FY2006)

Complete rapid scrapie test development and transfer technology.

Complete PrP genotype study in goats.

Complete sequencing of expressed OvLa MHC Class II DRB-1 alleles.

Continue oral scrapie transmission study in goats.

Potential impact: A rapid test for scrapie will facilitate diagnosis. A map of the PrP gene and the mutations for goats will aid in determining if certain genotypes are more susceptible or resistant to scrapie once challenge experiments are initiated. Characterization of the expressed OvLa MHC Class II DRB1 alleles will allow development of a rapid high-throughput system for typing sheep.

Year 6 (FY 2007)

Continue oral scrapie transmission study in goats.

Initiate experimental inoculation of OPPV into sheep with specific OvLa MHC Class II DRB-1 expressed alleles.

Develop rapid MHC Class II DRB1-1 typing tests.

Potential impact: Data on oral transmission of scrapie to goats will aid in determining the impact of goats on scrapie transmission to sheep flocks in the US. Development of a rapid MHC Class II DRB1 typing test will allow rapid screening of sheep for specific MHC Class II types followed by experimental inoculation of OPPV.

Year 7 (FY 2008)

Continue experimental inoculation of OPPV into sheep with specific OvLa MHC Class II DRB-1 expressed alleles.

Initiate TSE strain studies.

Complete oral scrapie transmission study in goats.

Potential impact: Examining strains of scrapie will aid in understanding transmission of sheep scrapie. Data on oral transmission of scrapie to goats will aid in determining the impact of goats on scrapie transmission to sheep flocks in the US. Monitoring of experimental inoculation of OPPV into sheep with specific MHC Class II DRB1 types will allow associations to be made between disease progression and specific MHC Class II DRB1 types.

4a.What was the single most significant accomplishment this past year?We found that multiple fetuses in the uterus of a pregnant ewe have an effect on PrPSc accumulation in the placental tissue. We previously showed that if a sheep fetus has a scrapie-resistant genotype, PrPSc does not accumulate in the placenta tissue of scrapie-infected ewes. We have now found, however, the placenta tissue associated with a fetus of a scrapie-resistant genotype can accumulate PrPSc if it is positioned next to a fetus of a scrapie susceptible genotype in utero in scrapie infected ewes. This has potential impact to the sheep industry in relying on the genetics to breed for resistance to scrapie.

4b.List other significant accomplishments, if any.When applying OPPV diagnostic tests to the field, it is necessary to identify the immunodominant antigen in OPPV-infected sheep. One publication has resulted from identifying the surface envelope glycoprotein (SU) as the B-lymphocyte immunodominant antigen in mature OPPV-infected sheep. Because the CAEV cELISA detects anti-SU antibodies, this result ensures that most mature OPPV-infected sheep will have measurable titers of anti-SU antibodies which can be detected in the CAEV cELISA This result has direct implications for the sheep industry in terms of accurate OPPV diagnostic testing using the CAEV cELISA.

Previous studies have shown that sheep with Prnp diploid genotypes of AA, QR and AA, RR at codons 136, 171, respectively, are more resistant to scrapie. However, it is unknown whether breeding toward these scrapie-resistant Prnp diploid genotypes would affect lamb production traits. ADRU is currently collaborating with University of Wyoming (5348-32000-019-03S) to show that breeding flocks toward the scrapie resistant PrP genotypes does not influence lamb production. Results of this research have yielded one publication and has impact on the sheep industry.

We previously showed that if a sheep fetus has a scrapie-resistant genotype, PrPSc does not accumulate in the placenta tissue of scrapie-infected ewes. Another question we wanted to address, which relates to sheep with scrapie-resistant Prnp diploid genotypes, is whether ewes with scrapie resistant PrP genotypes born from scrapie-infected ewes will accumulate PrPSc in their placental tissue after being bred back to a ram with a scrapie susceptible PrP genotype. In addition, ADRU and North Dakota State University (5348-32000-019-04S) are collaborating to evaluate the natural passage of scrapie through a flock with Prnp diploid genotypes at 136 of AV and VV. Since this flock is the first documented case of valine 136-based scrapie in the U.S., it is extremely important to understand the Prnp genetics of all flock mates and the transmission of valine 136-based scrapie.



4c.List any significant activities that support special target populations.None.

4d.Progress report.None

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.A practical live animal test for scrapie and preclinical postmortem tests for scrapie were developed and transferred to the regulatory agencies for use in the US. Monoclonal antibodies useful in assays on routinely formalin fixed tissue from infected sheep, deer, elk, cattle, humans, mink, domestic cats and a wide variety of captive wildlife potentially exposed to prion diseases were developed. Mechanisms for preventing transmission of scrapie through genetic selection of sires were demonstrated. Along with sire testing, genetic testing in bred ewes will ensure less transmission of scrapie from ewe to lamb.

The specific immune cells in lymph nodes involved in PrPSc accumulation remain unknown. Therefore, we analyzed lymph nodes for the presence of PrPSc and macrophage or follicular dendritic cell (FDC) markers using dual immunohistochemistry. Results indicated that lymph node follicular macrophages acquire PrPSc by phagocytosis of CD21+ FDC processes and process full-length PrPSc to N-terminally truncated PrPSc. These data provide the first knowledge in determining the pathogenesis of sheep scrapie in FDC and macrophages.

A new caprine arthritis-encephalitis virus (CAEV) competitive inhibition enzyme-linked immunosorbent assay (cELISA) had previously been developed. We tested 200 goat sera for the presence of CAEV antibodies using cELISA against the standard of comparison, immunoprecipitations (IP) of [S35] methionine-labeled CAEV lysate. The CAEV cELISA validation resulted in 100% sensitivity and 96.4% specificity against the standard of comparison. By annually testing goats for CAEV using cELISA, a CAEV-free herd could be established.

Because of the high sensitivity and specificity of the CAEV cELISA in goats, validation of the CAEV cELISA in sheep also ensued. By using IP of [S35] methionine-labeled OPPV lysate as the comparable standard, detection of serum antibodies to ovine progressive pneumonia virus (OPPV) in sheep using the CAEV cELISA yielded a sensitivity 98.6% and a specificity of 96.9%. This indicates that one diagnostic test, the CAEV cELISA, can be utilized for detection of small ruminant lentiviruses.

When applying the CAEV cELISA diagnostic test to the field, it is necessary to identify OPPV field strains. We isolated 7 new OPPV field strains from the colostrum cells of 10 OPPV-infected sheep. We characterized and compared the surface envelope glycoprotein (SU) deduced amino acid sequences from these sheep against previously characterized OPPV, CAEV and maedi-visna virus strains. The new OPPV field strains had high sequence identity to OPPV and CAEV strains more than MVV strains. This result is important for future CAEV cELISA testing of U.S. sheep and sheep outside the U.S. infected with OPPV or MVV, respectively.

Standardized, validated diagnostic tests are needed for eradication of scrapie. In FY2001-FY2003, a cooperative ARS-APHIS-state test validation program resulted in submission of samples from more than 2,000 sheep. These samples are being used to validate the third eyelid live animal test, the postmortem immunohistochemistry test, and to develop novel rapid, high throughput tests suitable for slaughter surveillance. A panel of internationally accepted tests suitable for diagnostic and surveillance purposes will result.

Control of scrapie and the small ruminant lentiviruses (SRLV) directly benefits small farms that raise sheep and goats for supplementary income. Direct and indirect losses to these producers because of these diseases are significant. Control programs that include identification of infected flocks and animals should reduce the economic consequences of SRLV and prion diseases.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?Two monoclonal antibodies to the prion protein and their use in combination as detection reagents for prions have been patented. Both antibodies are commercially available. Only non-exclusive licenses have been offered, to insure the widest possible use of these reagents in diagnostics, industry, and research. The antibodies are in use internationally and collaborative programs to train personnel in Canada, Mexico, and China are in progress. The preclinical test for scrapie has been transferred to the National Veterinary Services Laboratory. APHIS has established a national testing network, through which veterinary and state diagnostic laboratories will apply the technology under contract with APHIS. CAEV cELISA will be licensed and commercially available in the U.S. in 6-12 months.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).1. Scrapie: Prions in the Placenta of AAQQ and AAQR sheep. Invited presentation at American Sheep Industry Convention, Reno, NV, January 28, 2005.

2. Prion accumulation in the sheep placenta / Goat scrapie genotyping project. Invited presentation at USAHA Sheep and Goat Committee, United States Animal Health Association Annual Meeting, Greensboro, NC, October 24, 2005.

3. Sheep scrapie: maternal and fetal genetics. Invited presentation at Animal Prion Diseases and the Americas, Ames, IA, October 14-16, 2005.

4. Using host immunogenetics as a prediction tool for ovine progressive pneumonia virus clinical disease. Invited presentation at the American Sheep Industry Convention, Reno, NV, January 2005.

5. Natural Sheep Scrapie: What have we learned? Invited presentation to the Department of Chemistry and Biochemistry at the University of Denver, Denver, CO, November 2004.

6. Predicting ovine progressive pneumonia virus loads using MHC Class II DRB1 immunogenetics. Invited presentation at the United States Animal Health Association Sheep and Goat Committee, Greensboro, NC, October 2004.

7. Lack of Natural Ovine Progressive Pneumonia Virus Transmission from OPPV-Infected Ewes to Their Lambs. Presentation at the Keystone Symposia HIV Pathogenesis, Banff, Canada, April 2005.

Review PublicationsAlverson, J. 2005. Data sheet for scrapie. Animal Health and Production Compendium. Available: http://www.cabicompendium.org/ahpc/home.asp



Alexander, B.M., Stobart, R.H., Russell, W.C., Orourke, K.I., Lewis, G.S., Logan, J.R., Duncan, J.R., Moss, G.E. 2005. The incidence of genotypes at codon 171 of the prion protein gene (prnp) in five breeds of sheep and production traits of ewes associated with those genotypes. Journal of Animal Science. 83(2):455-459.

Herrmann, L.M., Mcguire, T.C., Hotzel, I., Lewis, G.S., Knowles Jr, D.P. 2005. Surface envelope glycoprotein is b-lymphocyte immunodominant in sheep naturally infected with ovine progressive pneumonia virus. Clinical and Diagnostic Laboratory Immunology. 12(6):797-800.

Herrmann, L.M. 2005. Needles: To Re-use or Not to Re-use?. Sheep Industry News. 9(5):2.

Johnson, M.L., Evoniuk, J.M., Stoltenow, C.L., Orourke, K.I., Redmer, D.A. 2005. Development of an assay to determine single nucleotide polymorphisms (snp) in the prion gene for the diagnosis of genetic susceptibility to scrapie in sheep. American Society of Animal Science. 56:151.

Evoniuk, J.M., Stoltenow, C.L., O'Rourke, K.I., Moore, B.L., Redmer, D.A. 2005. Assessment of the genetic risk and impact of lateral transmission in a valine-associated scrapie outbreak in sheep. American Journal of Veterinary Research. 66(8):1-6.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405202&fy=2005



Research Project: Study of Atypical BseLocation: Virus and Prion Diseases of Livestock

2005 Annual Report

4d.Progress report.This report serves to document research conducted under a specific cooperative agreement between ARS and the Italian Reference Centre for Animal TSE (CEA) at the Istituto Zooprofilattico Sperimentale, Turin, Italy. Additional details of research can be found in then report for the parent project 3625-32000-073-00D, Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies. The aim of the cooperative research project conducted by the CEA and ARS is to compare the U.S. bovine spongiform encephalopathy (BSE) isolate and the bovine amyloidotic spongiform encephalopathy isolates (BASE) identified in Italy. The first objective was to determine whether diagnostic methods routinely used by USDA are able to identify the Italian BASE cases. For this purpose, CEA received the immunohistochemistry (IHC) protocol developed by APHIS-USDA. The IHC protocol was reproduced and standardized in the CEA laboratory and will be applied to the Italian BSE and BASE cases. Furthermore, fixed brainstem sections and frozen brainstem material from Italian BSE and BASE cases will be sent to ARS for analysis using USDA IHC and Western blot (WB) methods. These studies will enable us to determine whether the present diagnostic tools (IHC and WB) employed at the USDA will be able to detect the Italian BASE cases and also enable us to compare Italian BSE and BASE with the U.S. BSE cases.





http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408490&showpars=true&fy=2005



3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone?FY 2006 Mouse bioassay with various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S.



http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408808&showpars=true&fy=2005



Published online before print October 20, 2005

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

12/10/76AGRICULTURAL RESEARCH COUNCILREPORT OF THE ADVISORY COMMITTE ON SCRAPIEOffice NoteCHAIRMAN: PROFESSOR PETER WILDY

snip...

A The Present Position with respect to ScrapieA] The Problem

Scrapie is a natural disease of sheep and goats. It is a slowand inexorably progressive degenerative disorder of the nervous systemand it ia fatal. It is enzootic in the United Kingdom but not in allcountries.

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

It is clear that scrapie in sheep is important commercially andfor that reason alone effective measures to control it should bedevised as quickly as possible.

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

Whether true or not. the hypothesis that these agents might betransmissible to man raises two considerations. First, the safetyof laboratory personnel requires prompt attention. Second, actionsuch as the "scorched meat" policy of USDA makes the solution of theacrapie problem urgent if the sheep industry is not to suffergrievously.

snip...

76/10.12/4.6

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

1: 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/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=6997404&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus



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 infectedwith scrapie?FOUR years ago, Terry Singeltary watched his mother die horribly from adegenerative brain disease. Doctors told him it was Alzheimer's, butSingeltary was suspicious. The diagnosis didn't fit her violent symptoms,and he demanded an autopsy. It showed she had died of sporadicCreutzfeldt-Jakob disease.

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

Now scientists in France have stumbled across new evidence that adds weightto the campaigners' fears. To their complete surprise, the researchers foundthat 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§, JamesIronside¶, Moira Bruce, Dominique Dormont*, and Jean-Philippe Deslys** Commissariat à l'Energie Atomique, Service de Neurovirologie, Directiondes Sciences du Vivant/Département de Recherche Medicale, Centre deRecherches du Service de Santé des Armées 60-68, Avenue du Général Leclerc,BP 6, 92 265 Fontenay-aux-Roses Cedex, France; Hôpital Neurologique PierreWertheimer, 59, Boulevard Pinel, 69003 Lyon, France; § Laboratoire deNeuropathologie, Hôpital de la Salpêtrière, 83, Boulevard de l'Hôpital,75013 Paris, France; ¶ Creutzfeldt-Jakob Disease Surveillance Unit, WesternGeneral Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom; andInstitute for Animal Health, Neuropathogenesis Unit, West Mains Road,Edinburgh EH9 3JF, United Kingdom

Edited by D. Carleton Gajdusek, Centre National de la RechercheScientifique, 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 bovinespongiform encephalopathy (BSE) has contaminated human beings, causingvariant Creutzfeldt-Jakob disease (vCJD). This disease has raised concernsabout the possibility of an iatrogenic secondary transmission to humans,because the biological properties of the primate-adapted BSE agent areunknown. We show that (i) BSE can be transmitted from primate to primate byintravenous route in 25 months, and (ii) an iatrogenic transmission of vCJDto humans could be readily recognized pathologically, whether it occurs bythe central or peripheral route. Strain typing in mice demonstrates that theBSE agent adapts to macaques in the same way as it does to humans andconfirms that the BSE agent is responsible for vCJD not only in the UnitedKingdom but also in France. The agent responsible for French iatrogenicgrowth hormone-linked CJD taken as a control is very different from vCJD butis similar to that found in one case of sporadic CJD and one sheep scrapieisolate. These data will be key in identifying the origin of human cases ofprion disease, including accidental vCJD transmission, and could providebases for vCJD risk assessment.

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




USA NOR-98 ATYPICAL SCRAPIE UPDATE AUGUST 31, 2007 RISES TO 5 DOCUMENTED CASES



http://nor-98.blogspot.com/




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

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