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Subject: [BSE-L] Detection of infectious prions in urine (Soto et al Available online 13 August 2008.)
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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.
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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.
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."
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 ( firstname.lastname@example.org ).
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
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.
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. ...
CWD AND ENVIRONMENTAL FACTORS i.e. saliva, fecal shedding and fecal-oral transmission is likely
Tuesday, August 26, 2008 CWD Stakeholder Advisory Group Wednesday, August 22, 2007 11:31 AM
Chronic Wasting Disease CWD
Thursday, August 28, 2008 CWD TISSUE INFECTIVITY brain, lymph node, blood, urine, feces, antler velvet and muscle
Sunday, August 24, 2008 HAVE ANOTHER GLASS OF CWD PRIONS COURTESY Dane County Wisconsin Mike DiMaggio, solid waste manager
Thursday, August 28, 2008 cwd, feeding, and baiting piles