Characterization of the Prion Protein in Human Urine*
Ayuna Dagdanova‡,1, Serguei Ilchenko§,1, Silvio Notari‡,1, Qiwei Yang‡, Mark E. Obrenovich‡, Kristen Hatcher‡, Peter McAnulty¶, Lequn Huang?, Wenquan Zou‡, Qingzhong Kong‡, Pierluigi Gambetti‡,2 and Shu G. Chen‡,3 + Author Affiliations
From the ‡Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, the §Center for Proteomics and Mass Spectrometry, Case Western Reserve University, Cleveland, Ohio 44106, the ¶Non-Clinical Consultancy, DK-4000 Roskilde, Denmark, and the ?Nanjing University Medical School, Nanjing 210093, China 2 To whom correspondence may be addressed: Dept. of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-0586; Fax: 216-368-4090; E-mail: pierluigi.gambetti@case.edu. 3 To whom correspondence may be addressed: Dept. of Pathology, Case Western Reserve University, 2103 Cornell Rd., WRB 5533, Cleveland, OH 44106. Tel.: 216-368-8925; Fax: 216-368-0494; E-mail: shu.chen@case.edu. ?1 These authors contributed equally to this work.
Abstract
The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.
Glycosylphosphatidylinositol Anchors Mass Spectrometry (MS) Post-translational Modification Prions Urine Prion Protein
Footnotes * This work was supported, in whole or in part, by National Institutes of Health Grant AG14359. This work was also supported by Centers for Disease Control and Prevention Grant UR8/CCU515004, Department of Agriculture National Research Initiative Grant 2002-35201-2608, Department of Defense National Prion Research Program Grant DAMD17-03-1-0283, and by the Charles S. Britton Fund. Received July 6, 2010. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Received July 6, 2010. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
http://www.jbc.org/content/285/40/30489.abstract?sid=a1dfa5a0-3f41-44d8-ae63-ec27f39d8b4b
ahhh, the fruits of Harash Narang's work finally going to pay off for somebody. ...TSS
1996 NARANG URINE TEST
http://collections.europarchive.org/tna/20081106082948/http://www.bseinquiry.gov.uk/files/yb/1996/02/09003001.pdf
http://collections.europarchive.org/tna/20080102200511/http://www.bseinquiry.gov.uk/files/yb/1996/02/21004001.pdf
http://collections.europarchive.org/tna/20081106042311/http://www.bseinquiry.gov.uk/files/yb/1996/02/21005001.pdf
http://web.archive.org/web/20030527133551/http://www.bseinquiry.gov.uk/files/ws/s113.pdf
http://web.archive.org/web/20010305030338/http://www.bseinquiry.gov.uk/report/volume11/chaptec3.htm
5.289 We have concluded, for the reasons given above, that Dr Narang's work received fair consideration by MAFF scientists. While we would pay tribute to Dr Narang's dedication to research into TSEs, we feel that he had a fair opportunity to demonstrate the validity of his work but did not succeed in doing so.
http://web.archive.org/web/20010301194458/http://www.bseinquiry.gov.uk/report/volume11/chaptec4.htm
No way to treat a pioneer
Apr 20 2003 By Phil Doherty
A leading charity has called for a public inquiry into the way a top mad cow disease expert has been treated by the establishment. Harash Narang was the first scientist to make the link between the illness and its human equivalent- variant Creutzfeldt-Jakob disease - in 1990. Dr Narang says he was made redundant from his job at Newcastle's Public Health Laboratory Services after making his findings known. He claims that he lost his post after the then Health Minister Stephen Dorrell ordered all the lab's work on the killer disease to cease. Since he became a whistle-blower, he says, he has not been able to get lab time in the UK to continue his work.
Dr Narang has since moved to the United States. He is now working at the CJD surveillance unit based at Case Western Reserve University in Cleveland, Ohio.
CJD Foundation head Noel Baldwin, who lost his son Patrick to the killer disease, said: "There is more and more information coming out that proves Dr Narang was right all along.
Medical
"He said years ago that CJD could be found in blood and passed on by transfusions and medical instruments. This has now been accepted.
"He argued that BSE could cause both sporadic as well as vCJD, and recent research has shown this to be correct. He also invented a urine test which shows if someone is harbouring the disease."
Mr Baldwin also rued the Government's decision to pull Medical Research Council funding from Dr Narang.
He said: "Now it looks as if the US will benefit financially from this ground-breaking research. "The UK establishment has ignored him for more than 10 years. We believe those responsible should be made accountable for this because if they had listened to Dr Narang maybe some of those poor people wouldn't have died from this terrible disease.
Scandal
"This is a national scandal that needs to be fully addressed by a public inquiry. "We are planning to launch this campaign in the next few months and will be involving sympathetic MPs to get this issue aired in the House of Commons."
http://icnewcastle.icnetwork.co.uk/0100news/0100local/page.cfm?objectid=12871105&method=full&siteid=50081&headline=No%20way%20to%20treat%20a%20pioneer
2005 NARANG URINE TEST USA CWRU
CJD doc jets off
Mar 9 2003
By Phil Doherty, Sunday Sun
The North scientist who first established a link between mad cow disease and its human form is quitting Britain.
Harash Narang has been head-hunted by a top US university to continue his research into variant Creutzfeldt-Jakob disease.
He was working for the Government's Public Health Laboratory Service in Newcastle when he revealed the link and later lost his job.
Dr Narang claimed he was made redundant because he went public with his findings, an allegation which has always been denied. He said: "I now have a job at the United States CJD Surveillance Centre based in Case Western Reserve University, Cleveland, Ohio.
"I'm very excited because it has excellent facilities and is one of the best CJD surveillance centres in the world.
The university is examining a urine test pioneered by Dr Narang which can show whether someone has CJD.
Currently only a post-mortem diagnosis can be made.
Dr Narang said: "Early indications show that my test has performed even better than anticipated. It is expected to be validated very shortly."
And he revealed: "I do not regret telling the truth all those years ago. If I had to do it again then I would."
Ken Bell, a financial backer of Dr Narang's work, claimed he had been forced to go abroad because he cannot get laboratory time in the UK.
He said: "Harash has been blackballed in the UK because he told the public the truth. "The establishment will try anything to stop him working here. It's a disgrace."
Noel Baldwin, of the CJD Foundation charity, said: "He has been proved right about so many things . . . that CJD can be transmitted through blood, that BSE can cause both variant and sporadic CJD and that you can test for the disease through urine samples."
Dr Narang starts work at Case University later this month. Shu Chen, one of his future colleagues, said: "He will be a great asset to our CJD research."
http://icnewcastle.icnetwork.co.uk/0100news/0100local/page.cfm?objectid=12716797&method=full&siteid=50081
Edmonton company boasts cheap BSE test Duncan Thorne , CanWest News Service; Edmonton Journal Published: Friday, July 21, 2006 EDMONTON -- An Edmonton company is confident it has a cheap, ground-breaking test for mad cow disease, but the test's British inventor who claims to have first made the link between BSE and the disease's human form insists he still holds the rights.
Despite their differences, inventor Harash Narang and BSE Prion Solutions Inc. agree the test holds amazing potential to quickly and inexpensively test live cattle for bovine spongiform encephalopathy better known as mad-cow disease. The only approved tests so far for mad cow and its human equivalent depend on removing brain samples after death. A test on live animals would open the way to guaranteeing disease-free herds.
"We have a test that not only works, but works each and every time," said Ron Arnold of BSE Prion Solutions Inc., adding formal validation may take up to two years and regulatory approval. Narang, a former British government scientist who went public about human risks from BSE in 1990, started developing tests for detecting the disease in the late 1980s while at a public health laboratory. He had been studying cases of a fatal but rare human brain illness, Creutzfeldt-Jakob disease (CJD), when he started noticing some cases were different. He has said he was well on the way to establishing a link between BSE and the unusual CJD cases when he was ordered to stop his research. He has also claimed officials rejected his calls for increased testing for BSE and the new form of CJD, now known as variant CJD.
Narang developed three diagnostic tests, including an early version of the urine test that BSE Prion intends to bring to market.
A wide-ranging 1998 inquiry into Britain's response to the mad cow crisis found problems with Narang's claims. It cited evidence that fellow scientists could not get his test to work. Even so, Narang continued development of the urine test. A British company, Biotec Global, sponsored much of his work. He is no longer part of the research, but work on it continues at the United States National Prion Disease Pathology Surveillance Center at Case Western Reserve University in Cleveland. Narang and Case Western researchers reported in 2005 that the urine test could reliably detect a harmless form of the prion protein that is blamed for BSE and variant CJD. It could also detect the bad form if the prion was first added directly to the urine.
They said their findings "may lay the foundation for a future technique," if in fact the bad prion can turn up naturally in urine.
"It needs a lot of work still," Ayuna Dagdanova, one of the test's researchers at the U.S. prion surveillance centre, said from Cleveland.
Without solid data it's not possible to say if they are close to detecting BSE in urine, she said. "No one actually knows, but preliminary experiments show the possibility."
Arnold, a partner in Biotec, said Narang gave Biotec the patent rights in 2003 and it in turn gave BSE Prion the licence for the Americas and Europe.
"We've talked with patent attorneys in London and also in Newcastle. Everyone agrees that the documents and the transfer of ownership of the patents was done judiciously and was extremely well put together by the solicitors," said Arnold.
Edmonton company boasts cheap BSE test Duncan Thorne , CanWest News Service; Edmonton Journal Published: Friday, July 21, 2006 Narang, speaking from Newcastle, acknowledged signing papers, but said it was not clear what he was signing. He said he continues to pay the patent renewal fees.
Biotec has sunk more than $2 million into the research, but BSE Prion has not had to pay a licence fee, Arnold said. That's because the project is humanitarian, with plans to hand over any earnings for research purposes, in the form of grants and scholarships.
Narang, who holds shares in Biotec despite the ownership dispute, also said he also wants any profits to go into further research. Meanwhile, he said he's owed back pay and expenses for work he did over the past five years a claim Arnold rejects.
dthorne@thejournal.canwest.com
Edmonton Journal © CanWest News Service 2006
http://www.canada.com/topics/news/national/story.html?id=e0300291-19dd-48a8-8e88-963a06087ce2&k=6995
http://www.canada.com/topics/news/national/story.html?id=e0300291-19dd-48a8-8e88-963a06087ce2&k=6995&p=2
Experimental Biology and Medicine 230:343-349 (2005) © 2005 Society for Experimental Biology and Medicine
----------------------------------------------------------
ORIGINAL RESEARCH ARTICLE
Sensitive Detection of Prion Protein in Human Urine
Harash K. Narang*,2, Ayuna Dagdanova, Zhiliang Xie, Qiwei Yang and Shu G. Chen,1 * BioTech Global, 22-40 Brentwood Avenue, Newcastle Upon Tyne, NE2 3DH, UK; and Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106 1To whom requests for reprints should be addressed at Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106. E-mail: shu.chen@case.edu
ABSTRACT
Transmissible spongiform encephalopathies are a group of infectious diseases typically associated with the accumulation of a protease-resistant and ß-sheet-rich prion protein, PrPSc, in affected brains. PrPSc is an altered isoform derived from the host-encoded glycoprotein, PrPC. The expression of PrPC is the highest in brain tissue, but it can also be detected at low levels in peripheral tissue. However, it is unclear whether a significant amount of PrPC is released into body fluid and excreted into urine. We have developed a simple, rapid method for the reliable detection of PrPC in urine from normal subjects by Western blotting. Our method can easily and reliably detect PrPC in apparently healthy individuals using less than 1 ml of urine in which the amount of urinary PrPC is estimated to be in the range of low micrograms/liter.
http://www.ebmonline.org/cgi/content/full/230/5/343
FULL TEXT ;
http://madcowtesting.blogspot.com/2008/09/identification-of-disease-induced.html
Subject: CJD/BSE/SCRAPIE * Who will get the last laugh...Harash Narang???
Date: Fri, 7 Apr 2000 14:33:50 -0700
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@uni-karlsruhe.de
######### Bovine Spongiform Encephalopathy #########
Greetings List Members, old article, but some interesting data, which 'some' of you, (not all), may find interesting.....TSS
THE AGRICULTURE COMMITTEE
APPENDIX 22 237
Memorandum submitted by Dr H K Narang
SUMMARY 1. SAF are composed of PrP protein which is host derived.
2. Although PrP is incorporated into the SAF it is not the agent of scrapie.
3. There is no such thing as a self replicating protein--a biologically improbable notion.
4. Tubulofilamentous particles, which I described first in 1972, are specific and unique to spongiform encephalopathies and can be demonstrated by simple electron microscopic grid impression technique.
5. Simplified diagnostic procedures would be of great value for epidemiological purposes and surveillance, for BSE and CJD.
6. There are grounds, for postulating that humans might possibly acquire spongiform encephalopathy through ingestion of contaminated tissue. On the current evidence available the hypothesis that CJD in humans is caused by ingestion of infected animal tissue remains open, but it has to be stated that it would be unwise to add potentially contaminated foods into the human food chain.
7. All age groups would be at risk with spongiform encephalopathies.
BACKGROUND Having obtained PhD from Newcastle University in 1969 I joined Professor E J Field. Head of thc Demylinating Unit and developed an interest in spongiform encephalopathies. I worked at the Unit until 1977 on a number of central nervous system diseases. Following the restructuring of MRC Unit, I joined the Public Health Laborators, and have worked in Newcastle in the Virology Department to the present day. A part from my clinical work, I have maintained interest on several clinical diagnostic problems. However. I also have maintained an interest in slow virus infections and have continued to do research in this field in co-operation with Dr D C Gajdusek, National Institute of Health, USA. Dr Gajdusek was awarded The Nobel Prize in medicine for demonstrating that kuru in humans was due to slow virus: Dr R L Chandler, AFRC. Compton and Dr A G Dickinson, AFRC/MRC, Edinburgh. Over the years I have published a number of publications.
TUBULOFILAMENTOUS PARTICLES My first significant paper relating to slow viruses was published in Nature in 1972 in which I described for the first time tubulofilamentous particles in experimental scrapie-infected mice. Subsequently I have found these particles in all other animal species experimentally infected with scrapie, and also in naturally infected sheep and Creutzfeldt-Jakob disease in humans. Tissue from bovine spongiform encephalopathy has not been made available to me for this study. Some workers in early 1981 did not consider these particles to be the infectious agent because they were unable to find these particles in brains of scrapie-infected hamsters However. during a visit to National Institute of Health in 1985 I found these tubulofilamentous particles when I had the opportunity to examine scrapie-infecled hamsters. These findings have since been independently confirmed. It is now well established that the tubulofilamentous particles in tissue can be used as a marker for spongiform encephalopathies.
To simplify the examination of experimentally infected animal tissues by electron microscopy, I developed a simple impression technique, This method was validated by experiments using a variety of known human and animal viruses. I applied this method to experimental scrapie and Creutzfeldt-Jakob diseased brains. Tubulofilamentous particles were identified in all scrapie and Creutzfeldt-Jakob disease inoculated animals.
DEVELOPMENT OF TOUCH IMPRESSION TECHNIQUE The method was also applied to tissue samples taken from hamsters at various stages of the incubational period of the disease and from uninfected tissues without the observer knowing which samples were from normal and which were from infected animals. The status of all tissue under test was correctly identified on the basis of the presence or absence of tubulofilamentmous particles. Furthermore, the samples taken more than 20 day post-inoculation, a quarter of the way through the incubation period were consistently positive, when histologically no spongiform changes were seen in the brains. It would seem reasonable to conclude from this that the technique could be applied for routine sample screening on suspected animals or those late in the incubation phase for diagnostic purposes.
RELATIOINSHIP OF TUBULOFILAMENTOUS PARTICLES TO SAF/PrP In a number of my recent studies I have demonstrated that scrapie associated fibrils/protease resistant protein (SAF/PrP) form the core of these tubulofilamentous particles the coat consists of single stranded DNA (ssDNA) and a protein, that is tubulofilamentous particles consists of an outer protein coat. middle ssDNA and inner most SAF/PrP, and particles are termed nermaridius (Nerma -- filamentous).
238
APPENDICES TO THE MINUTES OF EVIDENCE TAKEN BEFORE
Examination of coded specimens: Subsequently I was invited to AFRC & MRC Neuropathogenesis Unit Edinburgh in September, 1987 to demonstrate the touch technique. Preparation and examination was carried out on six randomised scrapie and normal brains and correct results scored on all six specimens within two and half hours from start to finish. Preparation and examination can be accomplished within half a day.
My December, 1989 application to MAFF for grant to work on the test for BSE in collaboration with the Central Veterinary Laboratory was turned down in January, 1990. However in June 1990, I have been given £20,000 by a private individual to proceed with this work. Naturally necessary co-operation will now be required.
SIGNIFICANCE of SAF/PrP SAF are morphological structures composed of small protein molecules termed "PrP", which is a normal host derived protein. The molecules of this normal protein are more likely to be joined together by a regulatory system from the genome of the scrapie agent, which in turn wraps itself round the SAF. SAF/PrP were considered to be the infectious agent, but now we know through a number of studies that infectivity can be separated from the SAF/PrP.
The host gene for PrP protein is expressed at similar levels both in uninfected and in spongiform encephalopathies infected animals.
The sequence of this PrP gene is known and it is also known that it differs in different species and strains of animals. It is important to state that the primary structure of this gene does not differ between uninfected and infected animal of the same species and strain.
It has been suggested in the Tyrrell Report that by knowing the DNA sequence differences in the PrP gene, one might find a strain of cow which is more resistant to scrapie agent. This phenomenon does not work in other known species of animals where sequence differ in different strains. To find such a difference in cows which will be resistant to scrapie to me it appears wishful thinking and would not in any case provide a short-term solution and even in the tong term would be impractical.
Since scrapie has been established in (all animals inoculated) 23 species and in many different strains of animals by inoculation, it is unlikely that an unchallenged host will be resistant to scrapie agent and therefore humans are not likely to be totally resistant.
Most of the studies on PrP gene will only provide academic answers, and many of the questions asked in Tyrrell Report on molecular studies have already been answered by published studies for example SAF/PrP itself is not the agent.
Since purified PrP from infected animals or PrP synthesised in vitro even in its post-translation modified form has not transmitted disease, it is unlikely that detailed molecular studies on SAF/PrP suggested in Tyrrell Report would add much to the control or eradication of bovine spongiform encephalopathy.
BOVINE SPONGIFORM ENCEPHALOPATHY It is commonly, accepted that the disease in cattle has been transmitted by feed, which contained the infectious agent, It would appear to me that at one stage cows have been fed with high protein supplement, prepared from sheep remains. Due to species cross over and because of long incubation periods there were no apparent clinical cases in cattle. At some stage, into the sheep remains cattle remains were also added (although at the time of killing, clinically cattle appeared healthy) the high protein contained scrapie agent from both sheep and cows. As a result of the passage from cow to cow (cannibalism) the incubation period has been reduced. This phenomenon has also been observed in scrapie experimental animals.
TITRATION INFECTIVITY STUDIES Passage from one species of animal to another can make the incubation period long or short. In mice inoculated from sheep cases the incubation period for primary transmission may extend from 600 days to lifespan while mice inoculated from cases of BSE have consistently shorter incubation periods after primary inoculation, usually extending to about 328 days. Sub-passage from mice inoculated with sheep the incubation period comes down to about 384 days and from mice sub-inoculated from BSE the incubation period is near 184 days. These results suggest that a marked change has occurred in the nature of the scrapie agent, or there is selection of shorter incubation strain of the scrapie agent in cows.
A number of studies have investigated the increased titre of scrapie agent in a variety of organs. infective titre means tissue weight by weight can be diluted with saline water to produce disease. (10 to 1st power = 1:10; 10 to 2ng power = 1: 10(l; l0 to 3 power = 1:1000 and so on) 0.01 gm of diluted material is required to inoculate a mouse or hamster.
THE AGRICULTURE COMMITTEE
In experimental animals earliest titre is found to be associated with spleen, lymph nodes and salivary glands which build up a plateau Concentration of 10 to 3rd power-10 to 7th power (tissue can be diluted, 1:100,000 to 1:10,000,000) titre of infectivity within four weeks of infection which is quarter of the way through the incubation period. Most of the other tissues including muscles (meat) and whole blood from affected sheep and mice also occasionally produce cases of scrapie suggesting brief "viraemic" phase (virus in blood) during incubation period. Although titres may be low to begin with, once replication starts there are relatively minor differences in its maximum rate irrespective of which genetic make up of PrP is present in the host.
The effect of developmental maturity on susceptibility: Contrary to normal expectation, newborn mice are less, rather than more susceptible to scrapie infections by an intraperitoneal routes. 50-500-fold less dose is required for adult inoculation by the intraperitoneal route compared to intracerebral route of inoculation. Suggesting that peritoneal cavity of adult is a much more favourable environment for the scrapie agent compared to neonate, therefore both young and adults are at risk of developing Creutzfeldt-Jakob disease.
Mice born of pregnant females inoculated during the gestation period but separated from their own dams at delivery do become infected prenatally or perinatally, all mice except the foster mother (not inoculated) develop the clinically recognisable disease and die following an incubation period not significantly different from that observed as occurring in their inoculated mothers. Affected ewes mated with affected rams produce offspring which nearly always become affected. If this were to be true of cattle, by keeping calves from sick animals, UK would never be free of spongiform encephalopathy.
NATURE OF THE SCRAPIE AGENT The precise nature of the infectious agent(s) responsible for these diseases has not yet been identified and the definitive diagnosis is based on the touch impression technique, histological examination or transmission of the disease to selected animal hosts.
Fears have been expressed that transmission of the disease to man might occur through the consumption of contaminated food. Although it is known that a considerable drop of titre occurs following incubation at 80°C-100*C for 30 minutes, but remarkably a subpopulation of about 10 to 3rd power, from original titre of l0 to 8th power/10th power, often survives even after exposure to 121°C for over one hour. This remaining titre would be enough to produce disease, but with increased length of the incubation period in the host.
It is important to point out that average life of humans is four to eight times the suggested incubation period of the scrapie agent and therefore it is my belief, that however small the amount of the scrapie infective agent is left it could establish itself in host. It has generally been accepted that the disease has a long incubation period and because of this very long incubation and that humans may not clinically develop the disease in their life time, it would be wrong to presume at this early stage that this would be true in all individuals and take it for granted that there is little or no risk to humans. Since the knowledge in the field is limited it would be potentially dangerous to think and predict categorically, that there is no danger of infection from handling or eating the cooked food. It should be remembered that we need only, one infective dose.
So far we have known the disease has always been experimentally established in all animal species which have been inoculated or fed with the infective agent. Once the animals are infected there is a period of incubation, at the end the fatal disease has always declared itself.
Once it was considered that to establish experimentally the disease because of the long incubation period the animals have to be inoculated during weaning. However subsequently it has been found that the incubation period is reduced with age of inoculation, that is the older the animals when infected the shorter the incubation period.
The mode of natural transmission of CJD remains unknown and incubation periods are difficult to estimate. Scrapie, BSE, kuru and CJD have been transmitted to mice, hamsters and squirrel monkeys that have been fed contaminated brain, kidney and spleen.
CATS AND SPONGIFORM ENCEPHALOPATHY
It is important to point out that cats are very susceptible to the scrapie agent. While working with Prof E J Field, I considered that cats might act as an intermediate host for Creutzfeldt-Jakob disease and therefore through our contacts with vets we obtained 20 cat brains with possible underlying neurological disease. We did not find a single cat with spongiform encephalopathy.
In animal exposures where infection has been documented after high dozes of infected tissue feed, transmission was so irregular that the question arises as to whether the true portal was either the gastrointestinal tract itself, or through areas of mucosal abrasions. Ulcerations in the lips, gums and intestines may also play a role. Studying effect of gingival scarification on oral route of infection, demonstrated that 70 per cent of nonscarified compared with 100 per- cent of scarficated mice developed
240 APPENDICES TO THE MINUTES OF EVIDENCE TAKEN BEFORE
the disease and with a significantly shorter incubation time. Single episodes of oral ingestion of the agent alone therefore may not transmit spongiform encephalopathies and is likely that other factors play an important role. including the dose of the agent and length of the incubation period.
In a case control study of dietary risk factors in CJD in the United States patients with CJD had an increased consumption of roast pork, ham, hot dogs, roast lamb, pork chops, liver and scrapple with excess consumption of under cooked meat: It has been reported clustering of CJD cases among Israeli Libyan Jews with average annual incidence of 31.3/million, possibly associated with a dietary, profile which included the consumption of sheep brain and eyes. A high incidence of CJD was also found among North African immigrants to France (4.53/million) who are known to eat sheep brain. These sheep brains are usually prepared by soaking in salt water, followed by gentle simmering in water. Since four patients from Tunisia were from a butcher's family, this might indicate that the transmission occurred via a common source rather than a familial pattern. Since all had common exposure to contaminated food, in this case possibly sheep brain infected with scrapie agent. This point is further solidified that disease can be experimentally transmitted from many of these familial CJD cases.
In a patient occupation analysis of 308 cases of CJD it has been observed that there were 18 cases from the health professions including neurosurgeons, dentists, physicians and nurses. Thirty two from agricultural/meat processing, 78 cooks/housewives/domestic servants. The remainder were from non-health, non-food processing occupations.
The diagnosis of CJD often presents problems because of the time taken to process tissue for histological examination and long incubation periods. This is made more difficult by the unusual format of the CJD amyotrophic form characterised by motor system disturbances without any spongiform changes in the brain. It has also been shown that occasionally CJD is associated with other underlying diseases tumors, brain abscess, Alzheimer's disease, stroke as demonstrated by transmission of the disease from these cases into animals. The problem of correct diagnosis, may explain the low incident of the disease and current known cases may represent only the tip of the iceberg and it is conceivable that large segments of the population are infected without any, or only trivial or subclinical signs or have other major underlying disease. This point is well illustrated by iatrogenic transmission, either corneal transplatafion contaminated electrode implantation and contaminated batches of growth hormone prepared from pool normal pituitary glands.
The problems of accurate diagnosis certainly adds all the difficulties of conducting epidemiological surveys. It is therefore important to use all the available diagnostic procedures, including the simple touch impression technique to confirm CJD cases.
SUGGESTED FUTURE WORK Bearing in mind the very long incubation period of spongiform encephalopathies it would be at this stage prudent to investigate case of CJD in order lo obtain an accurate prevalence rate so that significant increases in future, if any, can be observed.
ROUTINE DIAGNOSIS AND CONFIRMATION OF CJD CASES Since the incidence of CJD appears to be very low, I could provide the diagnosis and confirmation of CJD Cases on National basis by touch impression method.
COURSE OF RESEARCH A study should also be organised with a view to obtain pathological specimens from all suspected CJD cases for confirmation using the electron microscopic technique. Tissue from other neuropathies and normal cases should also be examined for control and clinically misdiagnosed cases. Parallel neuro-histopathological studies of these cases would be carried out. This relatively simple impression technique therefore will provide a means of rapid diagnosis of CJD with minimal tissue handling and reduced exposure risks.
Since we have transmitted CJD to hamsters from one of our two cases, those which will be positive for nemavirus/SAF by touch method, but will have atypical spongiform encephalopathy or are neuro-histopathologically normal should be confirmed by transmission study.
We would also like to purify specific single-stranded nucleic acid for molecular cloning and sequencing. Further information obtained would confirm the nature of the scrapie agent and also provide additional markers of the disease in the living animals and humans.
19 June 1990
***He just knew too damn much, that's why they 'banned him', locked his laboratory. we are now in the year 2000, and in my opinion, not much better off today, than we were on June 19, 1990 (10 years)???
Terry S. Singeltary, Bacliff, Texas
############ http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html ############
Wednesday, September 08, 2010
CWD PRION CONGRESS SEPTEMBER 8-11 2010
snip...
PPo3-19:
Detection of CWD Prions in Salivary and Urinary Tissues of Deer: Potential Mechanisms of Pathogenesis and Prion Shedding Nicholas J. Haley,1 Candace K. Mathiason,1 Glenn C. Telling2 and Edward A. Hoover1 1Department of Microbiology, Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; Fort Collins, Colorado USA; 2Department of Molecular Biology and Genetics; University of Kentucky; Lexington, Kentucky USA
Key words: chronic wasting disease, transmission, PMCA, pathogenesis, excretion, urine, saliva, salivary gland, urinary bladder, kidney, blood
Saliva and urine are thought to play an important role in the transmission and pathogenesis of chronic wasting disease (CWD) in captive and free-ranging cervids. We have previously identified PrPCWD in a variety of excreta using serial PMCA (sPMCA) and bioassay; however the source of infectious prions in urine and saliva has yet to be identified. In the present study, we applied sPMCA to tissues associated with saliva and urine production and excretion in an effort to seek proximal sources of prion shedding. Oropharyngeal and urogenital tissues, along with blood and obex from CWD-exposed cervids (comprising over 300 individual samples) were analyzed blindly in duplicate and scored based on apparent CWD burden. PrPCWD was detected by three rounds of sPMCA in tissues associated with saliva and urine production and excretion, notably salivary gland and urinary bladder; whereas blood samples from the same animals and concurrent negative controls (n = 116 of 117) remained negative. Route of inoculation and CNS burden appeared to play an important role in terminal prion distribution, in that IV-inoculated animals and those with increasing CNS levels of PrPCWD had higher and more widely distributed accumulation in excretory tissues. PMCA identification of PrPCWD in oropharyngeal and urogenital tissues—in the absence of detection by conventional methods—may indicate the presence of protease- sensitive infectious prions in excretory tissues not revealed by assays employing PK digestion or other means to remove PrPC reactivity. Thus, evaluation of peripheral tissues via sPMCA may allow additional insights into prion transmission, trafficking and pathogenesis.
PPo3-26:
Identification of Renal Origin for CWD Urinary Prion Excretion in Deer
Davis M. Seelig,1 Nicholas J. Haley,1 Jan P. Langeveld and Edward A. Hoover1 1Colorado State University; Department of Microbiology, Immunology and Pathology; Fort Collins, CO USA; 2Central Institute for Animal Disease Control (CIDC-Lelystad); Lelystad, The Netherlands
Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids. Although bioassays have confirmed the presence of infectious prions in urine and other body fluids of infected deer, origin and mechanisms of prion transfer to and shedding in excreta remains unknown. To address these questions, we have developed enhanced immunohistochemistry (IHC) methods employing tyramide signal amplification (TSA) on formalin-fixed, paraffin-embedded (FFPE) tissues of n = 20 CWD-infected white-tailed deer. Using these methods we have demonstrated PrPCWD present granular to clumped aggregates both within the cytoplasm of renal tubule cells and in the interstitium. Cytoplasmic PrPCWD aggregates were detected most commonly in proximal convoluted tubule epithelial cells. PrPCWD was not identified in the lower urinary tract (ureters or bladder) of any CWD-infected animal. In summary, we present evidence for PrPCWD accumulation within the renal tubule cells, which may identify a proximate tissue source and explain the manner by which infectious prions are excreted in the urine of infected deer, thereby leading to the high degree of direct and indirect horizontal transmission of chronic wasting disease.
snip...see more ;
http://chronic-wasting-disease.blogspot.com/2010/09/cwd-prion-2010.html
Sunday, December 06, 2009
Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer Long after Oral Exposure to Urine and Feces from CWD+ Deer
http://chronic-wasting-disease.blogspot.com/2009/12/detection-of-sub-clinical-cwd-infection.html
Friday, May 14, 2010
Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure
Published Online May 13, 2010 Science DOI: 10.1126/science.1187107 Science Express Index
http://chronic-wasting-disease.blogspot.com/2010/05/prion-strain-mutation-determined-by.html
Thursday, June 03, 2010
Prion Strain Mutation and Selection John Collinge MEDICINE
http://chronic-wasting-disease.blogspot.com/2010/06/prion-strain-mutation-and-selection.html
Wednesday, March 18, 2009
Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse Bioassay
http://chronic-wasting-disease.blogspot.com/2009/03/detection-of-cwd-prions-in-urine-and.html
Tuesday, February 09, 2010
Chronic Wasting Disease: Surveillance Update North America: February 2010
***
>>> In addition, we documented horizontal transmission of CWD from inoculated mice and to un-inoculated cohabitant cage-mates. <<<
http://ajp.amjpathol.org/cgi/content/abstract/ajpath.2010.090710v1
http://chronic-wasting-disease.blogspot.com/2010/02/chronic-wasting-disease-surveillance.html
NOT only muscle, but now fat of CWD infected deer holds infectivity of the TSE (prion) agent. ...TSS
Monday, July 06, 2009
Prion infectivity in fat of deer with Chronic Wasting Disease
http://chronic-wasting-disease.blogspot.com/2009/07/prion-infectivity-in-fat-of-deer-with.html
2002
Subject: MAD DEER/ELK DISEASE AND POTENTIAL SOURCES Date: Sat, 25 May 2002 18:41:46 -0700 From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@uni-karlsruhe.de
now, what about those 'deer scents' of 100% urine', and the prion that is found in urine, why not just pass the prion with the urine to other deer...
Mrs. Doe Pee Doe in Estrus Model FDE1 Mrs. Doe Pee's Doe in Estrus is made from Estrus urine collected at the peak of the rut, blended with Fresh Doe Urine for an extremely effective buck enticer. Use pre-rut before the does come into heat. Use during full rut when bucks are most active. Use during post-rut when bucks are still actively looking for does. 1 oz. www.gamecalls.net/ ELK SCENT/SPRAY BOTTLE * Works anytime of the year * 100 % Cow Elk-in-Heat urine (2oz.) * Economical - mix with water in spray mist bottle * Use wind to your advantage Product Code WP-ESB $9.95 www.elkinc.com/Scent.asp prions in urine? [PDF] A
URINE TEST FOR THE IN-VIVO DIAGNOSIS OF PRION DISEASES
http://www.sigov.si/vurs/PDF/diagnoastika-bse-urin.pdf
http://p079.ezboard.com/fwolftracksproductionsfrm2.showMessage?topicID=54.topic
http://www.fda.gov/ohrms/dockets/dailys/03/Jan03/012403/8004be07.html
tss
CWD/POTENTIAL SOURCE/URINE/HUNTERS ?
Mrs. Doe Pee Doe in Estrus Model FDE1 Mrs. Doe Pee's Doe in Estrus is made from Estrus urinecollected at the peak of the rut, blended with Fresh Doe Urine for anextremely effective buck enticer. Use pre-rut before the does come intoheat. Use during full rut when bucks are most active. Use duringpost-rut when bucks are still actively looking for does. 1 oz. http://www.gamecalls.net/huntingproducts/deerlures.html
http://www.google.com/search?hl=en&q=Mrs.+Doe+Pee+Doe+in+Estrus&btnG=Search&aq=f&aqi=&aql=&oq=&gs_rfai=
ELK SCENT/SPRAY BOTTLE Works anytime of the year* 100 % Cow Elk-in-Heat urine (2oz.)* Economical - mix with water in spray mist bottle* Use wind to your advantage Product Code WP-ESB $9.95 http://www.elkinc.com/Scent.asp prions in urine? [PDF]
A URINE TEST FOR THE IN-VIVO DIAGNOSIS OF PRION DISEASES
http://www.sigov.si/vurs/PDF/diagnoastika-bse-urin.pdf
TSS ########### http://mailhost.rz.uni-karlsruhe.de/warc/bse-l.html ############
http://www.michiganwalleye.com/forum/printthread.php?t=23313
http://www.wapiti.net/discussion/showPost.cfm?post=10918
http://www.fda.gov/OHRMS/Dockets/dailys/03/Jan03/012403/8004be07.html
Subject: DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability
Date: Fri, 16 May 2003 11:47:37 -0500
From: "Terry S. Singeltary Sr."
To: fdadockets@oc.fda.gov
http://madcowfeed.blogspot.com/2008/07/docket-03d-0186-fda-issues-draft.html
http://www.buckmasters.com/bm/Community/Forums/tabid/60/forumid/14/postid/10141/view/topic/Default.aspx
Science 14 October 2005:Vol. 310. no. 5746, pp. 324 - 326DOI: 10.1126/science.1118829
Reports Coincident Scrapie Infection and Nephritis Lead to Urinary Prion Excretion
Harald Seeger,1* Mathias Heikenwalder,1* Nicolas Zeller,1 Jan Kranich,1 Petra Schwarz,1 Ariana Gaspert,2 Burkhardt Seifert,3 Gino Miele,1 Adriano Aguzzi1
Prion infectivity is typically restricted to the central nervous and lymphatic systems of infected hosts, but chronic inflammation can expand the distribution of prions. We tested whether chronic inflammatory kidney disorders would trigger excretion of prion infectivity into urine. Urinary proteins from scrapie-infected mice with lymphocytic nephritis induced scrapie upon inoculation into noninfected indicator mice. Prionuria was found in presymptomatic scrapie-infected and in sick mice, whereas neither prionuria nor urinary PrPSc was detectable in prion-infected wild-type or PrPC-overexpressing mice, or in nephritic mice inoculated with noninfectious brain. Thus, urine may provide a vector for horizontal prion transmission, and inflammation of excretory organs may influence prion spread.
snip... SEE FULL TEXT ;
http://chronic-wasting-disease.blogspot.com/2008/04/prion-disease-of-cervids-chronic.html
http://creutzfeldt-jakob-disease.blogspot.com/2008/08/excretion-of-transmissible-spongiform.html
CHRONIC WASTING DISEASE BLOG
http://chronic-wasting-disease.blogspot.com/
Docket APHIS-2010-0056 National Veterinary Services Laboratories; Bovine Spongiform Encephalopathy Surveillance Program Documents COMMENT SUBMISSION Docket No. APHIS-2010-0056
http://madcowtesting.blogspot.com/2010/09/docket-aphis-2010-0056-national.html
Tuesday, September 14, 2010
Transmissible Spongiform Encephalopathies Advisory Committee; Notice of Meeting October 28 and 29, 2010 (COMMENT SUBMISSION)
http://tseac.blogspot.com/2010/09/transmissible-spongiform_14.html
Friday, September 24, 2010
USA Blood products, collected from a donor who was at risk for vCJD, were distributed SEPTEMBER 2010
http://vcjdtransfusion.blogspot.com/2010/09/usa-blood-products-collected-from-donor.html
Wednesday, September 08, 2010
Emerging Infectious Diseases: CJD, BSE, SCRAPIE, CWD, PRION, TSE Evaluation to Implementation for Transfusion and Transplantation September 2010
http://vcjdtransfusion.blogspot.com/2010/09/emerging-infectious-diseases-cjd-bse.html
Sunday, April 12, 2009
CWD UPDATE Infection Studies in Two Species of Non-Human Primates and one Environmental reservoir infectivity study and evidence of two strains
snip...
From: TSS (216-119-163-189.ipset45.wt.net)
Subject: CWD aka MAD DEER/ELK TO HUMANS ???
Date: September 30, 2002 at 7:06 am PST
From: "Belay, Ermias"
To:
Cc: "Race, Richard (NIH)" ; ; "Belay, Ermias"
Sent: Monday, September 30, 2002 9:22 AM
Subject: RE: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Dear Sir/Madam,
In the Archives of Neurology you quoted (the abstract of which was attached to your email), we did not say CWD in humans will present like variant CJD. That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091). Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.
Ermias Belay, M.D. Centers for Disease Control and Prevention
-----Original Message-----
From:
Sent: Sunday, September 29, 2002 10:15 AM
To: [log in to unmask]">[log in to unmask]; [log in to unmask]">[log in to unmask]; [log in to unmask]">[log in to unmask]
Subject: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS
Sunday, November 10, 2002 6:26 PM ......snip........end..............TSS
snip...
full text ;
http://chronic-wasting-disease.blogspot.com/2009/02/exotic-meats-usa-announces-urgent.html
Wednesday, March 18, 2009
Noah's Ark Holding, LLC, Dawson, MN RECALL Elk products contain meat derived from an elk confirmed to have CWD NV, CA, TX, CO, NY, UT, FL, OK RECALLS AND FIELD CORRECTIONS: FOODS CLASS II
http://chronic-wasting-disease.blogspot.com/2009/03/noahs-ark-holding-llc-dawson-mn-recall.html
see full text ;
http://chronic-wasting-disease.blogspot.com/2009/04/cwd-update-infection-studies-in-two.html
Monday, August 9, 2010
National Prion Disease Pathology Surveillance Center Cases Examined (July 31, 2010)
(please watch and listen to the video and the scientist speaking about atypical BSE and sporadic CJD and listen to Professor Aguzzi)
http://prionunitusaupdate2008.blogspot.com/2010/08/national-prion-disease-pathology.html
URINE IS USED IN MANY PHARMACEUTICALS AND COSMETICS. Premarine is made from horse urine for instance. ...
Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518