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Detection of infectious prions in urine (Soto et al 2008)

flounder

Well-known member
----- Original Message -----
From: "TERRY SINGELTARY" <[email protected]>
To: <[email protected]> Sent: Tuesday, September 02, 2008 10:35 AM
Subject: [BSE-L] Detection of infectious prions in urine (Soto et al Available online 13 August 2008.)

-------------------- [email protected] --------------------

doi:10.1016/j.febslet.2008.08.003 Copyright © 2008 Published by Elsevier B.V.

Detection of infectious prions in urine

Dennisse Gonzalez-Romeroa, Marcelo A. Barriaa, Patricia Leona, Rodrigo Moralesa and Claudio Soto, a,

aGeorge and Cynthia Mitchell Center for Neurodegenerative diseases, Departments of Neurology, Neuroscience and Cell Biology and Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0646, USA

Received 26 July 2008; accepted 4 August 2008. Available online 13 August 2008.

References and further reading may be available for this article. To view references and further reading you must purchase this article.

Abstract Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrPSc). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrPSc was detected in 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrPSc concentration in urine is around 10-fold lower than in blood. Interestingly, PrPSc present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-4T6KD96-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=10&md5=0e87d8b5ff0021ac6f620d73268f0e38

Detection of infectious prions in urine

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

By Gonzalez-Romero D, Barria MA, Leon P, Morales R, Soto C. At The George and Cynthia Mitchell Center for Neurodegenerative diseases, Departments of Neurology, Neuroscience and Cell Biology and Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0646, USA.

Abstract -------- Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively of the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals [hamster]. PrP(Sc) was detected in approximately 80 per cent of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.

The following paragraphs are extracts from the Discussion section of this paper:

"PrPSc in urine retains infectious properties, since injection of the agent amplified from this fluid produced a disease indistinguishable from the one induced by in vivo isolated material. Interestingly, animals [hamsters] inoculated with PrPSc amplified from the HY strain (both from brain and urine) showed a similar incubation time as those injected with the same quantity of PrPSc from sick brain. Our findings suggest that urine is a possible source of prion transmission. Since urine produced by animals potentially infected with prions is permanently released and likely concentrated in environmental samples, such as soil and grass, this route may prove very relevant for spreading of TSEs [Transmissible Spongiform Encephalopathies] in wild and captive animals such as cervids, sheep and cattle. It is known that PrPSc is highly resistant to degradation, and infectivity can survive in the environment for a long time. Recent studies have shown that PrPSc adsorbs efficiently into soil, where it remains infectious, and that both infectivity and PrPSc can stay intact in soil for long periods. Contamination of soil with urinary prions may contribute to spreading prion disease among animals, which are known to ingest large amounts of soil, including cattle, sheep and cervids. Worrisomely, the continuous excretion of urine and the extremely high resistance of prions may lead to a progressive accumulation of infectious material in the environment, with potentially catastrophic consequences in the future.

"One of the top priorities in the prion field is to minimize further spreading of TSEs to humans or animals by limiting the exposure to contaminated material. This is a difficult problem, because prion diseases have a long clinically-silent incubation period in which infected individuals may unknowingly transmit the disease. In addition, it is possible that many individuals may remain as sub-clinical carriers during their entire life, constituting a permanent source of prions. Therefore, the development and validation of procedures to detect even the tiniest quantities of infectious material is of paramount importance. Implementation of a large scale program to screen animals at risk of infection and diagnosis of the human population requires detection of prions in easily accessible samples, such as blood or urine. Our results showing that PrPSc can be detected in urine of a large proportion of infected animals provide a promising avenue for a sensitive and non-invasive biochemical diagnosis of prion diseases. Adaptation of PMCA [protein misfolding cyclic amplification] for detection of prions in urine of naturally infected animals and humans may offer a great possibility for routine testing of prion infections."

http://apex.oracle.com/pls/otn/f?p=2400:1001:1720436792652856::NO::F2400_P1001_BACK_PAGE,F2400_P1001_PUB_MAIL_ID:1000,73794

The Journal of Infectious Diseases 2008;198:81-89 © 2008 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2008/19801-0015$15.00 DOI: 10.1086/588193 MAJOR ARTICLE Transmission and Detection of Prions in Feces Jiri G. Safar,1,2 Pierre Lessard,1 Gültekin Tamgüney,1,2 Yevgeniy Freyman,1 Camille Deering,1 Frederic Letessier,1 Stephen J. DeArmond,1,3 and Stanley B. Prusiner1,2,4

1Institute for Neurodegenerative Diseases, Departments of 2Neurology, 3Pathology, and 4Biochemistry and Biophysics, University of California, San Francisco, San Francisco

In chronic wasting disease (CWD) in cervids and in scrapie in sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as the mode of transmission of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of 140 days and a rate of prion infection of 80%-100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrPSc) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored 60 ng/g PrPSc and prion titers of 106.6 ID50/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Received 9 October 2007; accepted 15 November 2007; electronically published 27 May 2008.

(See the editorial commentary by Bosque and Tyler, on pages 8-9.)

Potential conflicts of interest: none reported.

Financial support: National Institutes of Health (grants AG02132, AG010770, NS22786, and NS14069); G. Harold and Leila Y. Mathers Foundation; Sherman Fairchild Foundation.

Reprints or correspondence: Dr. Stanley B. Prusiner, 513 Parnassus Ave., HSE-774, San Francisco, CA 94143-0518 ([email protected]).

http://www.journals.uchicago.edu/doi/abs/10.1086/588193

http://stanford.wellsphere.com/healing---recovery-article/transmission-and-detection-of-prions-in-feces/13816

http://www.michigan-sportsman.com/forum/showthread.php?p=2218816

http://creutzfeldt-jakob-disease.blogspot.com/2008/08/excretion-of-transmissible-spongiform.html

Subject: Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease

Date: October 5, 2006 at 1:45 pm PST

Infectious Prions in the Saliva

and Blood of Deer with Chronic

Wasting Disease

Candace K. Mathiason,1 Jenny G. Powers,3 Sallie J. Dahmes,4 David A. Osborn,5 Karl V. Miller,5

Robert J. Warren,5 Gary L. Mason,1 Sheila A. Hays,1 Jeanette Hayes-Klug,1 Davis M. Seelig,1

Margaret A. Wild,3 Lisa L. Wolfe,6 Terry R. Spraker,1,2 Michael W. Miller,6 Christina J. Sigurdson,1

Glenn C. Telling,7 Edward A. Hoover1*

A critical concern in the transmission of prion diseases, including chronic wasting disease (CWD) of cervids, is the potential presence of prions in body fluids. To address this issue directly, we exposed cohorts of CWD-nai¨ve deer to saliva, blood, or urine and feces from CWD-positive deer.

We found infectious prions capable of transmitting CWD in saliva (by the oral route) and in blood (by transfusion). The results help to explain the facile transmission of CWD among cervids and prompt caution concerning contact with body fluids in prion infections.

SNIP...

Deer cohorts 1 (blood), 2 (saliva), and 3 (urine and feces) were electively euthanized at 18 months pi to permit whole-body examination for PrPCWD. The greatest scrutiny was directed toward those tissues previously established to have highest frequency of PrPCWD deposition in infected deer and generally regarded as the most sensitive indicators of infection- medulla oblongata and other brainstem regions, tonsil, and retropharyngeal lymph node. We found unequivocal evidence of PrPCWD in brain and lymphoid tissue of all six tonsil biopsy- positive deer in cohorts 1 (blood) and 2 (saliva), whereas all deer in cohorts 3 and 5 were negative for PrPCWD in all tissues (Table 2 and

Figs. 1 and 2).

The transmission of CWD by a single blood transfusion from two symptomatic and one asymptomatic CWDþ donor is important in at least three contexts: (i) It reinforces that no tissue from CWD-infected cervids can be considered free of prion infectivity; (ii) it poses the possibility of hematogenous spread of CWD, such as through insects; and (iii) it provides a basis for seeking in vitro assays sufficiently sensitive to demonstrate PrPCWD or alternate prion protein conformers in blood-one of the grails of prion biology and epidemiology.

The identification of blood-borne prion transmission has been sought before with mixed results (9-11). Bovine spongiform encephalopathy and scrapie have been transmitted to naBve sheep through the transfer of 500 ml of blood or buffy coat white blood cells from infected sheep (12, 13). In addition, limited but compelling evidence argues for the transmission of variant Creutzfeldt-Jakob disease (vCJD) through blood from asymptomatic donors (14-16). Even in sporadic CJD, PrPres has been found in peripheral organs of some patients (17). The present work helps establish that prion diseases can be transmitted through blood.

The presence of infectious CWD prions in saliva may explain the facile transmission of CWD. Cervid-to-cervid interactions (SOM text), especially in high density and captive situations, would be expected to facilitate salivary crosscontact (11, 18, 19). Salivary dissemination of prions may not be limited to CWD. Proteaseresistant prion protein has been demonstrated in the oral mucosa, taste buds, lingual epithelium, vomeronasal organ, and olfactory mucosa of hamsters infected with transmissible mink encephalopathy (19) and ferrets infected with CWD (20). Although no instance of CWD transmission to humans has been detected, the present results emphasize the prudence of using impervious gloves during contact with saliva or blood of cervids that may be CWD-infected.

Environmental contamination by excreta from infected cervids has traditionally seemed the most plausible explanation for the dissemination of CWD (21). However, we could not detect PrPCWD in cohort 3 deer inoculated repeatedly with urine and feces from CWDþ deer and examined up to 18 months pi (Table 2). There are several reasons to view this negative finding cautiously, including small sample size, elective preclinical termination, and potential variation in individual susceptibility that may be associated with the 96 G/S polymorphism in the PRNP gene (7, 22). Although no genotype of white-tailed deer is resistant to CWD infection, PRNP genotypes S/S or G/S at codon 96 appear to have reduced susceptibility manifest by longer survival (7). Both deer in cohort 3 (urine and feces) were subsequently shown to be of the PRNP 96 G/S genotype. Thus, it is possible, although we think unlikely, that these deer had a prolonged incubation period (918 months pi) before the amplification of PrPCWD became detectable in tissues. Recent studies have shown that PrPres is poorly preserved after incubation with intestinal or fecal content (23, 24). Further research using cervid and surrogate cervid PrP transgenic mice (25) are indicated to continue to address the presence of infectious CWD prions in excreta of CWDþ deer and to provide a more substantial basis for reconsideration of the assumption that excreta are the chief vehicle for CWD dissemination and transmission.

The results reported here provide a plausible basis for the efficient transmission of CWD in nature. We demonstrate that blood and saliva in particular are able to transmit CWD to naBve deer and produce incubation periods consistent with those observed in naturally acquired infections (3, 26). The time from exposure to first detection of PrPCWD by tonsil biopsy was variable-as short as 3 months but as long as 18 months (likely underestimates due to sampling frequency). The results also reinforce a cautious view of the exposure risk presented by body fluids, excreta, and all tissues from CWDþ cervids. ...

SNIP...END

http://www.sciencemag.org/cgi/content/abstract/314/5796/133

http://www.sciencemag.org/

CWD AND ENVIRONMENTAL FACTORS i.e. saliva, fecal shedding and fecal-oral transmission is likely

http://p079.ezboard.com/fwolftracksproductionsfrm2.showMessage?topicID=592.topic

Tuesday, August 26, 2008 CWD Stakeholder Advisory Group Wednesday, August 22, 2007 11:31 AM

http://chronic-wasting-disease.blogspot.com/2008/08/cwd-stakeholder-advisory-group.html

Chronic Wasting Disease CWD

http://chronic-wasting-disease.blogspot.com/

Thursday, August 28, 2008 CWD TISSUE INFECTIVITY brain, lymph node, blood, urine, feces, antler velvet and muscle

http://chronic-wasting-disease.blogspot.com/2008/08/cwd-tissue-infectivity-brain-lymph-node.html

Sunday, August 24, 2008 HAVE ANOTHER GLASS OF CWD PRIONS COURTESY Dane County Wisconsin Mike DiMaggio, solid waste manager

http://chronic-wasting-disease.blogspot.com/2008/08/have-another-glass-of-cwd-prions.html

Thursday, August 28, 2008 cwd, feeding, and baiting piles

http://chronic-wasting-disease.blogspot.com/2008/08/cwd-feeding-and-baiting-piles.html

Tuesday, September 02, 2008
Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease

http://chronic-wasting-disease.blogspot.com/2008/09/fecal-transmission-of-aa-amyloidosis-in.html


Tuesday, September 02, 2008
Detection of infectious prions in urine (Soto et al Available online 13 August 2008.)

http://chronic-wasting-disease.blogspot.com/2008/09/detection-of-infectious-prions-in-urine.html


TSS
 

Kathy

Well-known member
if the agent were infectious... all animal exposed to them would react and this is one of the vital requirements of Koche's postulates.

In this study, we attempted to detect prions in urine of experimentally infected animals. PrPSc was detected in 80% of the animals studied

Overview
Auburn University is seeking a licensee or development partner for naturally occurring novel metal clusters that exhibit protein scavenging properties. Proteon nucleating centers (PNCs) consist of 1- to 2-nm nanoparticles that contain 40–300 non-ionic metal atoms.

These nanoclusters have been shown to scavenge misfolded proteins to form proteons: clusters of up to 100,000 protein molecules with metal centers. Thus, PNCs have tremendous potential in the diagnosis and treatment of diseases associated with misfolded proteins, including prion-related diseases, neurological diseases, blood diseases and cancer.

Advantages
• Naturally occurring in blood, indicating inherent biocompatibility
• Scavenge misfolded proteins, suggesting numerous medical
applications
• Lethal to cultured cancer cells (view images | Quicktime movie)
• Can be isolated and purified through simple methods
• Multiple production methods exist

Description
Unfolding and subsequent aggregation of proteins is a common phenomenon linked to many human disorders. In investigating a possible mechanism by which excess hemoglobin release may be controlled in blood plasma in the disease state, it was discovered that human blood contains particles (“proteons”) that may reinforce hemoglobin scavenging.
It was later determined that these proteons consist of a small core of metal atoms (PNCs) that have scavenged misfolded proteins to form a protein shell around the metal center.

PNCs have been found and isolated from humans and numerous animal species.

This activity against misfolded proteins suggests numerous potential medical applications.

Proteins that have misfolded and/or aggregated have been tied to many disorders, some of which do not currently have appropriate therapies. Such disorders include prion-related diseases (e.g., Bovine spongiform encephalopathy), neurological diseases including Alzheimer’s and Parkinson’s, and blood disorders such as sickle cell anemia and some
autoimmune disorders. PNCs and proteons could also be used to collect misfolded proteins in a patient, allowing for a means to diagnose and measure progression of disease states.

Additionally, PNCs were found to be unexpectedly pro-apoptotic when added to cultured animal cells, showing much greater effectiveness against cancer cells than non-cancerous cells. This, along with their natural occurrence, suggests that PNCs could have significant
effectiveness in cancer therapy. Preliminary studies also indicate PNCs could have applications in entirely different medical areas as well as non-medical areas.

Status
• A U.S. Patent has been issued (7,138,255). Several non-provisional U.S. applications (including 20050142611) and one EU patent application are pending
• Proteons and PNCs have been isolated and well characterized
• Activity against cancer cells has been demonstrated in vitro

Partnership Opportunities
• Available for exclusive, field of use, research or non-exclusive licensing
• Joint development opportunities include collaboration, funded research or joint venture

Link: http://ott.auburn.edu/overview/PNC-Misfolded-Proteins.pdf

All prions are proteons, but not all proteons are prions!
 

flounder

Well-known member
Kathy said:
if the agent were infectious... all animal exposed to them would react and this is one of the vital requirements of Koche's postulates.

In this study, we attempted to detect prions in urine of experimentally infected animals. PrPSc was detected in 80% of the animals studied


SNIP...

http://ott.auburn.edu/overview/PNC-Misfolded-Proteins.pdf

All prions are proteons, but not all proteons are prions!



Definition of Koch's postulates

Koch's postulates: In 1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria for judging whether a given bacteria is the cause of a given disease. Koch's criteria brought some much-needed scientific clarity to what was then a very confused field.

Koch's postulates are as follows:

The bacteria must be present in every case of the disease.
The bacteria must be isolated from the host with the disease and grown in pure culture.
The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host.
The bacteria must be recoverable from the experimentally infected host.
However, Koch's postulates have their limitations and so may not always be the last word. They may not hold if:

The particular bacteria (such as the one that causes leprosy) cannot be "grown in pure culture" in the laboratory.
There is no animal model of infection with that particular bacteria.
A harmless bacteria may cause disease if:

It has acquired extra virulence factors making it pathogenic.
It gains access to deep tissues via trauma, surgery, an IV line, etc.
It infects an immunocompromised patient.

***Not all people infected by a bacteria may develop disease-subclinical infection is usually more common than clinically obvious infection.

Despite such limitations, Koch's postulates are still a useful benchmark in judging whether there is a cause-and-effect relationship between a bacteria (or any other type of microorganism) and a clinical disease.
 

Kathy

Well-known member
A harmless bacteria may cause disease if:

It has acquired extra virulence factors making it pathogenic.


One way of acquiring extra virulence factors is demonstrated by, in the case of yeast prions, control of the growing medium. By exposing the yeast to inadequate amounts of require minerals for good health, and imposing other minerals capable of replacing the required ones.

Also, exposing the yeast to other factors such as energy (various sources including gamma, beta, and alpha radiation) UV both external and internal.

see link: http://www.fil.lt/directions/radbio.htm

partial quote:

The recent two years have been dedicated to investigation of yeast prions ? proteins with altered conformation. Prions are the aggregated proteins which have lost their functions due to abnormal folding mutations. The analysis of the dynamics of proteins aggregation and it dependence on the RAS/cAMP pathway activity is performed. The environmental factors which have influence on prionization and/or prion elimination from the cells are being studied. At this point the apoptosis process ? programmed death of the cell - is under investigation. Apoptosis is important for cancer cells treatment. It was pointed out, that prions provide adaptability for yeast cells and prevent apoptosis.

The study of the several phenomena related to the radiation effect on behavior of yeast systems is previewed by exposing it to ionizing radiation (α, b, g, X-ray and UV).

* Investigation of the different radiation kind and dose effect on cell vital regulation processes including cell sensitivity analysis at different stages of the cell cycle.

* Radiation effect on prionization, prion elimination in the yeast cells. The lethality of the prionic protein.

* The apoptosis phenomena.

The external or internal irradiation of the sample (yeast) by desired ionizing source is foreseen. It could be a strong α source such as 239Pu, b - 90Sr and g or X-ray sources depending on the desirable dose level. The first step of the research consists of irradiation of synchronized yeast cells by X-rays. Irradiation (depending on the dose) can be concentrated on different parts of the cell (nucleus or cytoplasm) and at different stages of the cell cycle. Cell is most sensitive to irradiation at the cell cycle division stage. Viability of the cell is observed control parameter.

For detailed calculation of the deposited ionizing radiation dose in the sample (cell and its surroundings) the available simulation codes are used. A simulation model of the ionizing source and the sample allows one to obtain the exact radiation dose rate received by the yeast (nucleus and cytoplasm) during the irradiation time. Calculations and the observation of the radiation-induced perturbation of normal physiological processes, along with the biological system responses, will help to understand the damage level: free radicals production, breakup of chemical bonds, new chemical bonds production or damage molecules that regulate vital cell processes.
 

Shaft

Well-known member
Koch's postulates are still a useful benchmark in judging whether there is a cause-and-effect relationship between a bacteria (or any other type of microorganism) and a clinical disease.

Quite true TSS. However Kathy, a prion is not a bacteria nor is it a microorganism nor is it 'alive' even to the degree that a virus or viroid is arguably 'alive'. A prion is a protein, whether glycosylated or not, bonded to one or more metal ions or not, in crystalline form ot not. It is not alive in any sense of the word, either scientific or vernacular. Koch's postulates are worse than useless benchmarks when discussing prions and prion diseases. Sort of like employing Koch's postulates to validate or not the presence of heavy metal poisoning, for example.
 

Kathy

Well-known member
The question is not that I choose Koche's postulates to define the prions virulence. I only say this, as Mark Purdey did, because of the insistance that TSE are "infections" "infectious". This is a term that does not/should not apply to toxins which replicate themselves, or release a toxoid.

Viruses are not alive.

Viruses are carriers of DNA or RNA which then are attached to the cells DNA to induce the production of the proteins necessary to build new virus-like particles. If the cell factory is devoid of any key component of the virus manufacturing process - the cell can avert replication.

Viruses are nucleated upon an agent, usually a metal particle which has scavanged (bound with) the DNA or RNA fragment and proteins. The presence of the nucleating agent aids in the attachment of the fragments to the living cell.
 

flounder

Well-known member
Kathy said:
The question is not that I choose Koche's postulates to define the prions virulence. I only say this, as Mark Purdey did, because of the insistance that TSE are "infections" "infectious". This is a term that does not/should not apply to toxins which replicate themselves, or release a toxoid.

Viruses are not alive.

Viruses are carriers of DNA or RNA which then are attached to the cells DNA to induce the production of the proteins necessary to build new virus-like particles. If the cell factory is devoid of any key component of the virus manufacturing process - the cell can avert replication.

Viruses are nucleated upon an agent, usually a metal particle which has scavanged (bound with) the DNA or RNA fragment and proteins. The presence of the nucleating agent aids in the attachment of the fragments to the living cell.



kathy 1st wrote ;



Kathy said:
if the agent were infectious... all animal exposed to them would react and this is one of the vital requirements of Koche's postulates.



:? :???: :roll:



postulating prions in 2008 ;



The Nature of the Prion Although formulated a century ago, Koch’s postulates remain the bedrock of microbiology. According to Koch, three conditions must be met to identify a microbe as the causative agent of any given infection: (a) The microorganism must be detectable in all diseased tissues, (b) its isolation and growth must be achieved in pure culture, and (c) the culture-derived microorganisms must be able to induce disease after experimental infection of a subject, from which a further round of reisolation of the microorganism should be possible. Although Koch’s work was performed long before contemporary molecular biology, his postulates continue to serve remarkably well in defining conventional viral and bacterial agents. However, as prions are thought to be infectious proteins that amplify in a self-catalytic misfolding process, their microbiological culture sensu strictiori is not possible. Therefore whether Koch’s postulates can be meaningfully applied to prion disease is questionable. Furthermore, Koch’s postulates account for the influence of host susceptibility, which is of utmost importance in prion disease. Prion disease development depends on the presence of PrPC on host cells, and the species-specific amino acid sequence and polymorphism of codon 129 are important. Alternate postulates for infectious proteinaceous agents have recently been suggested (Walker et al. 2006), but it remains to be seen whether they will garner universal acceptance. In the prion field, researchers generally accept that a reasonable surrogate for Koch’s second postulate be fulfilled by the generation of synthetic prions in vitro, i.e., the recovery of perpetually transmissible infectivity from prion protein produced recombinantly or chemically from defined constituents. Major progress toward this end has been made in recent days. Purified PrPSc was used to generate PK-resistant PrP (PrPres) in a cell-free system that could even reflect two typical features of prions: species barrier and strain specificity (Bessen et al. 1995, Kocisko et al. 1995).


www.annualreviews.org • The Prion’s Elusive Reason for Being 445


Wednesday, September 03, 2008 Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers

http://chronic-wasting-disease.blogspot.com/2008/09/accelerated-high-fidelity-prion.html


Copper deficiency in the young bovine results in dramatic decreases in brain copper concentration but does not alter brain prion protein biology

L. R. Legleiter, J. W. Spears and H. C. Liu Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh, NC

[email protected]

Abstract

A manganese (Mn) for copper (Cu) substitution on cellular prion proteins (PrPc) in the brain that results in biochemical changes to PrPc has been implicated in the pathogenesis of transmissible spongiform encephalopathies. Recent research in the mature bovine does _NOT_ support this theory. The present study tested this hypothesis using progeny from gestating cows receiving Cu-deficient diets or Cu-deficient diets coupled with high dietary Mn. Copper-adequate cows (n = 39) were assigned randomly to treatments: 1) control (adequate in Cu and Mn), 2) Cu-deficient (-Cu), and 3) Cu-deficient plus high dietary Mn (-Cu+Mn). Cows assigned to treatments -Cu and -Cu+Mn received no supplemental Cu and were supplemented with molybdenum (Mo) to further induce Cu deficiency. The -Cu+Mn treatment also received 500 mg supplemental Mn/kg dietary DM. Calves were weaned at 180 d and maintained on the same treatments as their respective dams for 260 d. Copper-deficient calves (-Cu and -Cu+Mn) had decreased (P = 0.001) brain (obex) Cu and tended to have increased (P = 0.09) obex Mn relative to controls. Obex Mn/Cu ratios were substantially increased (P < 0.001) in calves receiving -Cu and -Cu+Mn treatments compared to controls and were higher (P < 0.001) in -Cu+Mn calves than in -Cu calves. Obex prion protein characteristics, including proteinase K degradability, superoxide dismutase (SOD)-like activity, and glycoform distributions, were largely unaffected. Obex tissue antioxidant capacity was not compromised by perturbations in brain metals, but Cu-deficient calves tended to have decreased (P = 0.06) Cu/Zn SOD activity and increased (P = 0.06) Mn SOD activity. Although obex copper was decreased due to Cu deficiency and Mn increased due to exposure to high dietary Mn, the obex metal imbalance had minimal effects on PrPc functional characteristics in the calves.

http://jas.fass.org/cgi/content/abstract/jas.2007-0403v1

Subject: FATEPriDE KEY FINDINGS ORGANOPHOSPHATE NO RELATIONSHIP TO CAUSE TSE Date: May 3, 2007 at 8:41 am PST

KEY FINDINGS

Organophosphate Studies

6. Studies using phosmet (an organophosphate pesticide) were carried out throughout the project. No relationship between this compound and the potential to cause a TSE were identified. In studies with oral dosing of rats, it was shown that PrP expression levels increased in the brain but there was no association between this and formation of proteinase K (PK) resistant PrP.

snip...

12. A model of seed protein aggregation and fibril formation was established using PrP charged with Mn2+. PrP-Mn2+ was found to form small circular aggregates able to catalyse further protein aggregation and fibrilisation of PrP. This model unlike other published models (for example those of Baskakov et al.1) does not require the presence of denaturants and is not an autocatalytic process (i.e. the substrate of the reaction did not aggregate). The results suggest that Mn2+ may play a role in the formation of prion seeds

__although further studies showed that this material was not infectious in mouse bioassay.__

snip...

24. The project also generated information concerning the relation of TSEs to environmental factors: • __Potentially no role for organophosphates in TSEs.__ • Increased Mn in the diet results in higher PrP levels in the brain. • No conclusion is yet possible in terms of the relationship between environmental trace element concentrations and the geographical occurrence of TSEs (classical scrapie or BSE). • Some confirmation was provided that in some specific farms occurrence of classical scrapie correlates with high Mn levels.

http://www.seac.gov.uk/papers/97-4.pdf

a) As regards the involvement of organophosphates in the origin of BSE, no new scientific information providing evidence or supporting the hypothesis by valid data became available after the adoption of the last opinion of the SSC on this issue. Consequently there is no reason for modifying the existing opinions.

b) Regarding the possibility of OP poisoning, the European legislation for registration of plant protection products and veterinary medicines – addressed in the enquiries – provide the basis for safe use of registered compounds and their formulations. Regarding the alleged intoxication cases reported and OP exposure it must be concluded that safety measures may not have been strictly followed.

References

Brown, D.R., Qin, K., Herms, J.W., Madlung, A., Manson, J., Strome, R., Fraser, P.E., Kruck, T., von Bohlen, A., Schulz- Schaeffer, W., Giese, A., Westaway, D. and Kretzschmar, H. (1997) The Cellular Prion Protein Binds Copper In Vivo, Nature, 390, 684-7. Purdey, M. (2000) Ecosystems Supporting Clusters of Sporadic TSEs Demonstrate Excesses of the Radical- Generating Divalent Cation Manganese and Deficiencies of Antioxidant Co-Factors Cu, Se, Fe, Zn Medical Hypotheses, 54, 278-306. Scientific Steering Committee, 1998. Opinion on possible links between BSE and Organophosphates. Adopted on 25-26 June 1998 Scientific Steering Committee, 2001. Opinion on Hypotheses on the origin and transmission of BSE. Adopted on 29-30 November 2001.

http://europa.eu.int/comm/food/fs/sc/ssc/out356_en.pdf

OP'S MEETING WITH PURDEY

http://www.bseinquiry.gov.uk/files/yb/1994/02/09001001.pdf

P04.27

Experimental BSE Infection of Non-human Primates: Efficacy of the Oral Route

Holznagel, E1; Yutzy, B1; Deslys, J-P2; Lasmézas, C2; Pocchiari, M3; Ingrosso, L3; Bierke, P4; Schulz-Schaeffer, W5; Motzkus, D6; Hunsmann, G6; Löwer, J1 1Paul-Ehrlich-Institut, Germany; 2Commissariat à l´Energie Atomique, France; 3Instituto Superiore di Sanità, Italy; 4Swedish Institute for Infectious Disease control, Sweden; 5Georg August University, Germany; 6German Primate Center, Germany

Background:

In 2001, a study was initiated in primates to assess the risk for humans to contract BSE through contaminated food. For this purpose, BSE brain was titrated in cynomolgus monkeys.

Aims:

The primary objective is the determination of the minimal infectious dose (MID50) for oral exposure to BSE in a simian model, and, by in doing this, to assess the risk for humans. Secondly, we aimed at examining the course of the disease to identify possible biomarkers.

Methods:

Groups with six monkeys each were orally dosed with lowering amounts of BSE brain: 16g, 5g, 0.5g, 0.05g, and 0.005g. In a second titration study, animals were intracerebrally (i.c.) dosed (50, 5, 0.5, 0.05, and 0.005 mg).

Results:

In an ongoing study, a considerable number of high-dosed macaques already developed simian vCJD upon oral or intracerebral exposure or are at the onset of the clinical phase. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of biomarkers.

Conclusions:

Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate. The difference in the incubation period between 5 g oral and 5 mg i.c. is only 1 year (5 years versus 4 years). However, there are rapid progressors among orally dosed monkeys that develop simian vCJD as fast as intracerebrally inoculated animals.

The work referenced was performed in partial fulfilment of the study “BSE in primates“ supported by the EU (QLK1-2002-01096).

http://www.prion2007.com/pdf/Prion%20Book%20of%20Abstracts.pdf

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

Risk of oral infection with bovine spongiform encephalopathy agent in primates

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

snip...

BSE bovine brain inoculum

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

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

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

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

PrPres biochemical detection

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

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

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

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

Published online January 27, 2005

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

It is clear that the designing scientists must

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

levels right down to 1 gram triggered infection.

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

6. It also appears to me that Mr Bradley’s answer (that it would take less than say 100 grams) was probably given with the benefit of hindsight; particularly if one considers that later in the same answer Mr Bradley expresses his surprise that it could take as little of 1 gram of brain to cause BSE by the oral route within the same species. This information did not become available until the "attack rate" experiment had been completed in 1995/96. This was a titration experiment designed to ascertain the infective dose. A range of dosages was used to ensure that the actual result was within both a lower and an upper limit within the study and the designing scientists would not have expected all the dose levels to trigger infection. The dose ranges chosen by the most informed scientists at that time ranged from 1 gram to three times one hundred grams. It is clear that the designing scientists must have also shared Mr Bradley’s surprise at the results because all the dose levels right down to 1 gram triggered infection.

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

2003D-0186 Guidance for Industry: Use of Material From Deer and Elk In Animal Feed

EMC 7 Terry S. Singeltary Sr. Vol #: 1

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: [email protected]

snip...

Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus ) Christina J. Sigurdson1, Elizabeth S. Williams2, Michael W. Miller3, Terry R. Spraker1,4, Katherine I. O'Rourke5 and Edward A. Hoover1

Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523- 1671, USA1 Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, University of Wyoming, Laramie, WY 82070, USA 2 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA3 Colorado State University Veterinary Diagnostic Laboratory, 300 West Drake Road, Fort Collins, CO 80523-1671, USA4 Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, 337 Bustad Hall, Washington State University, Pullman, WA 99164-7030, USA5

Author for correspondence: Edward Hoover.Fax +1 970 491 0523. e-mail [email protected]

Mule deer fawns (Odocoileus hemionus) were inoculated orally with a brain homogenate prepared from mule deer with naturally occurring chronic wasting disease (CWD), a prion-induced transmissible spongiform encephalopathy. Fawns were necropsied and examined for PrP res, the abnormal prion protein isoform, at 10, 42, 53, 77, 78 and 80 days post-inoculation (p.i.) using an immunohistochemistry assay modified to enhance sensitivity. PrPres was detected in alimentary-tract-associated lymphoid tissues (one or more of the following: retropharyngeal lymph node, tonsil, Peyer's patch and ileocaecal lymph node) as early as 42 days p.i. and in all fawns examined thereafter (53 to 80 days p.i.). No PrPres staining was detected in lymphoid tissue of three control fawns receiving a control brain inoculum, nor was PrPres detectable in neural tissue of any fawn. PrPres-specific staining was markedly enhanced by sequential tissue treatment with formic acid, proteinase K and hydrated autoclaving prior to immunohistochemical staining with monoclonal antibody F89/160.1.5. These results indicate that CWD PrP res can be detected in lymphoid tissues draining the alimentary tract within a few weeks after oral exposure to infectious prions and may reflect the initial pathway of CWD infection in deer. The rapid infection of deer fawns following exposure by the most plausible natural route is consistent with the efficient horizontal transmission of CWD in nature and enables accelerated studies of transmission and pathogenesis in the native species.

snip...

These results indicate that mule deer fawns develop detectable PrP res after oral exposure to an inoculum containing CWD prions. In the earliest post-exposure period, CWD PrPres was traced to the lymphoid tissues draining the oral and intestinal mucosa (i.e. the retropharyngeal lymph nodes, tonsil, ileal Peyer's patches and ileocaecal lymph nodes), which probably received the highest initial exposure to the inoculum. Hadlow et al. (1982) demonstrated scrapie agent in the tonsil, retropharyngeal and mesenteric lymph nodes, ileum and spleen in a 10-month-old naturally infected lamb by mouse bioassay. Eight of nine sheep had infectivity in the retropharyngeal lymph node. He concluded that the tissue distribution suggested primary infection via the gastrointestinal tract. The tissue distribution of PrPres in the early stages of infection in the fawns is strikingly similar to that seen in naturally infected sheep with scrapie. These findings support oral exposure as a natural route of CWD infection in deer and support oral inoculation as a reasonable exposure route for experimental studies of CWD.

snip...

http://vir.sgmjournals.org/cgi/content/full/80/10/2757

Subject: MAD DEER/ELK DISEASE AND POTENTIAL SOURCES Date: Sat, 25 May 2002 18:41:46 -0700 From: "Terry S. Singeltary Sr." Reply-To: BSE-L To: BSE-L

8420-20.5% Antler Developer For Deer and Game in the wild Guaranteed Analysis Ingredients / Products Feeding Directions

snip...

_animal protein_

http://www.surefed.com/deer.htm

BODE'S GAME FEED SUPPLEMENT #400 A RATION FOR DEER NET WEIGHT 50 POUNDS 22.6 KG.

snip...

_animal protein_

http://www.bodefeed.com/prod7.htm

J Infect Dis. 2004 Aug 1;190(3):653-60.

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.

http://www.ncbi.nlm.nih.gov/

10,000,000+ LBS. of PROHIBITED BANNED MAD COW FEED I.E. MBM IN COMMERCE USA 2007

Date: March 21, 2007 at 2:27 pm PST RECALLS AND FIELD CORRECTIONS: VETERINARY MEDICINES -- CLASS II ___________________________________ PRODUCT Bulk cattle feed made with recalled Darling’s 85% Blood Meal, Flash Dried, Recall # V-024-2007 CODE Cattle feed delivered between 01/12/2007 and 01/26/2007 RECALLING FIRM/MANUFACTURER Pfeiffer, Arno, Inc, Greenbush, WI. by conversation on February 5, 2007. Firm initiated recall is ongoing. REASON Blood meal used to make cattle feed was recalled because it was cross-contaminated with prohibited bovine meat and bone meal that had been manufactured on common equipment and labeling did not bear cautionary BSE statement. VOLUME OF PRODUCT IN COMMERCE 42,090 lbs. DISTRIBUTION WI

___________________________________ PRODUCT Custom dairy premix products: MNM ALL PURPOSE Pellet, HILLSIDE/CDL Prot-Buffer Meal, LEE, M.-CLOSE UP PX Pellet, HIGH DESERT/ GHC LACT Meal, TATARKA, M CUST PROT Meal, SUNRIDGE/CDL PROTEIN Blend, LOURENZO, K PVM DAIRY Meal, DOUBLE B DAIRY/GHC LAC Mineral, WEST PIONT/GHC CLOSEUP Mineral, WEST POINT/GHC LACT Meal, JENKS, J/COMPASS PROTEIN Meal, COPPINI – 8# SPECIAL DAIRY Mix, GULICK, L-LACT Meal (Bulk), TRIPLE J – PROTEIN/LACTATION, ROCK CREEK/GHC MILK Mineral, BETTENCOURT/GHC S.SIDE MK-MN, BETTENCOURT #1/GHC MILK MINR, V&C DAIRY/GHC LACT Meal, VEENSTRA, F/GHC LACT Meal, SMUTNY, A-BYPASS ML W/SMARTA, Recall # V-025-2007 CODE The firm does not utilize a code - only shipping documentation with commodity and weights identified. RECALLING FIRM/MANUFACTURER Rangen, Inc, Buhl, ID, by letters on February 13 and 14, 2007. Firm initiated recall is complete. REASON Products manufactured from bulk feed containing blood meal that was cross contaminated with prohibited meat and bone meal and the labeling did not bear cautionary BSE statement. VOLUME OF PRODUCT IN COMMERCE 9,997,976 lbs. DISTRIBUTION ID and NV

END OF ENFORCEMENT REPORT FOR MARCH 21, 2007

http://www.fda.gov/bbs/topics/enforce/2007/ENF00996.html

Subject: MAD COW FEED RECALL USA SEPT 6, 2006 1961.72 TONS IN COMMERCE AL, TN, AND WV Date: September 6, 2006 at 7:58 am PST

snip... see listings and references of enormous amounts of banned mad cow protein 'in commerce' in 2006 and 2005 ;

see full text ;

Friday, April 25, 2008

Substances Prohibited From Use in Animal Food or Feed [Docket No. 2002N-0273] (Formerly Docket No. 02N-0273) RIN 0910-AF46

http://madcowfeed.blogspot.com/2008/04/substances-prohibited-from-use-in.html

SPECIFIED RISK MATERIALS

http://madcowspontaneousnot.blogspot.com/2008/02/specified-risk-materials-srm.html

Scientific Report of the European Food Safety Authority on the Assessment of the Geographical BSE Risk (GBR) of the United States of America (USA) Question number: EFSA-Q-2003-083

EFSA concludes that the current GBR level of USA is III, i.e. it is likely but not confirmed that domestic cattle are (clinically or pre-clinically) infected with the BSE-agent. As long as there are no significant changes in rendering or feeding, the stability remains extremely/very unstable. Thus, the probability of cattle to be (pre-clinically or clinically) infected with the BSE-agent persistently increases.

http://www.efsa.europa.eu/EFSA/Scientific_Document/sr03_biohaz02_usa_report_v2_en1,0.pdf



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