Excretion of transmissible spongiform encephalopathy infectivity in urine
Suggested citation for this article: Gregori L, Kovacs GG, Alexeeva I, Budka H, Rohwer RG.
Excretion of transmissible spongiform encephalopathy infectivity in urine.
Emerg Infect Dis. 2008 Sep; [Epub ahead of print]
Excretion of Transmissible Spongiform Encephalopathy Infectivity in Urine Luisa Gregori, Gabor G. Kovacs, Irina Alexeeva, Herbert Budka, and Robert G. Rohwer Author affiliations: Veterans Affairs Medical Center, Baltimore, Maryland, USA (L. Gregori, I. Alexeeva, R.G. Rohwer); University of Maryland, Baltimore (L. Gregori, R.G. Rohwer); and Medical University of Vienna, Vienna, Austria (G.G. Kovacs, H. Budka) The route of transmission of most naturally acquired transmissible spongiform encephalopathy (TSE) infections remains speculative. To investigate urine as a potential source of TSE exposure, we used a sensitive method for detection and quantitation of TSE infectivity. Pooled urine collected from 22 hamsters showing clinical signs of 263K scrapie contained 3.8 ± 0.9 infectious doses/mL of infectivity. Titration of homogenates of kidneys and urinary bladders from the same animals gave concentrations 20,000-fold greater. Histologic and immunohistochemical examination of these same tissues showed no indications of inflammatory or other pathologic changes except for occasional deposits of diseaseassociated prion protein in kidneys. Although the source of TSE infectivity in urine remains unresolved, these results establish that TSE infectivity is excreted in urine and may thereby play a role in the horizontal transmission of natural TSEs. The results also indicate potential risk for TSE transmission from human urine-derived hormones and other medicines.
Discussion Anticipating that the titer of scrapie infectivity in excreted urine would be low, we measured concentration by using limiting dilution titration, a method with which we have extensive experience quantitating TSE infectivity in blood and blood components. In a limiting dilution titration, all animals in the bioassay are inoculated with the highest concentration of inoculum that is tolerated by the intracranial (most efficient) route. Infectivity assorts randomly into the inoculated animals; provided that at least some, but not all, of the animals are infected, the concentration can be calculated from the Poisson distribution of the infections (1). The method is highly sensitive and far more precise than other methods of TSE titration. We considered concentrating the urine before bioassay, but to circumvent uncertainties about the recovery of endogenous infectivity, we decided to inject the urine as collected. We found TSE infectivity in the urine of hamsters that had no evidence of kidney or bladder inflammation. In contrast, Seeger et al. did not detect infectivity in the urine of scrapieinfected mice (11) unless the mice were also affected by nephritis, in which case they found low levels of infectivity. Whether the bioassay they used was capable of detecting infectivity at the Page 9 of 16 concentration we observed for hamsters is not clear. If it was not capable, then detection of infectivity in mice with nephritis implies a higher concentration of infectivity in urine excreted by a nephritic kidney. In another study, urine and feces from deer with chronic wasting disease failed to demonstrate infectivity when orally given to the same susceptible species (17). Although usually an inefficient route of inoculation, the oral route did successfully transmit chronic wasting disease infectivity in saliva. The authors identified several possible reasons for the unsuccessful transmission by excreta, including incubation time, genotype, or sample size. In our experiments, cross-contamination by feces can not be excluded as a source of infectivity. Although the metabolism cage effectively separated urine and feces, some contact is possible because of the anatomy of the hamster. Protein misfolding cyclic amplification uses sonication to generate PrPres and infectivity in vitro. Although we routinely disperse all samples by ultrasonication before injection, our conditions are much harsher than those used to generate PrPres de novo (18) and do not support protein misfolding cyclic amplification of PrPres, or presumably infectivity (L. Gregori and R.G. Rohwer, unpub. data). The kidney and bladder titers were far greater than expected compared with findings of historical studies in which, with only rare exceptions (19-21), most attempts at transmission have been unsuccessful. These titers cannot be explained by the infectivity in residual blood (10 ID/mL) (1,2). In addition, we observed PrPd in the kidneys of scrapie-infected animals that had no indications of tissue inflammation. Heikenwalder et al. found PrPd staining within follicular infiltrates only in kidneys of mice affected by nephritis and not in control mice with noncomplicated scrapie (12). These data together with those by Seeger et al. (11) suggested that renal inflammation might be a prerequisite for TSE infectivity in renal tissue and its excretion in urine. In contrast, our results indicate that renal inflammation is not necessary for the deposition of PrPd in kidneys or for excretion of infectivity. One interpretation is that nephritis enhances the accumulation of PrPd at sites of inflammation, consistent with the excretion of higher levels of infectivity inferred above for this same condition (11). Two studies of scrapie in naturally and experimentally infected sheep reported PrPd depositions in the renal papillae (22) and in the intraepithelial cortex, medulla, and papillae (23). Similar to our findings, both studies indicated that not all scrapie tissues examined were positive Page 10 of 16 for PrPd. In chronic wasting disease, PrPd staining was uniqu ly localized in the ectopic lymphoid follicle of the kidney of a whitetail deer (24). All studies indicated either no changes (23,24) or mild to no inflammatory changes of the kidney (23). Thus, our histologic and immunohistochemical results for scrapie-infected hamsters are consistent with results found for sheep and deer and suggest that under normal conditions TSE diseases do not have concomitant inflammatory changes in the kidney. That urine titer is similar to that of plasma suggests that urine infectivity may originate from blood (25), but how the infectivity would be excreted is not clear. In general, proteins >40 kDa are not excreted and smaller proteins crossing the glomeruli are reabsorbed in the renal tubule and returned to the blood. If TSE infectivity is particulate (>40 kDa), its presence in urine might indicate abnormalities in renal filtration, perhaps related to the accumulation of PrPd in the collecting tubules of the medulla. The accumulation of immunoglobulins in the urine of TSEinfected hamsters and humans may also indicate malfunction of the urinary system (9,26). Excretion of a small C-terminal fragment of the normal cellular form of the prion protein in urine of infected and noninfected animals has been reported (27), but PrPres or PrPd forms can only be inferred from the presence of infectivity. Nevertheless, excretion of proteins similar to PrPres or PrPd forms has been documented. Follicle-stimulating hormone is a glycosylated protein of 203 amino acids organized mostly as a â-sheet, which bears some remarkable similarities to â-rich forms of the prion protein. Follicle-stimulating and several similar hormones are excreted in urine at great enough concentration to be extracted commercially. Alternatively, TSE infectivity may be excreted by processes analogous to those responsible for the low-level virurias that occur during infections of the nervous system by mumps, measles, and West Nile virus (28-30). To the extent that results from the hamster model can be generalized to other TSE infections (and it has so far proven highly predictive), then even the very low concentrations of infectivity measured here could result in substantial environmental contamination. Several liters of urine and several thousand doses of TSE infectivity may be excreted daily over the course of the illness; even higher titers might be excreted by an animal with nephritis. The high stability of TSE infectivity would account for its persistence in pasture years after infected animals are removed (31). Recent studies have shown that infectivity that is adsorbed and immobilized by soil minerals (32) can still infect hamsters by oral exposure 29 months later (33). Our study also Page 11 of 16 warns of a possible risk from TSE contamination to fertility hormones and other medicinal products extracted from human urine.
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Wednesday, June 11, 2008
Transmission and Detection of Prions in Feces
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
P04.61 Survival of PrPSc during Simulated Wastewater Treatment Processes
Pedersen, J1; Hinckley, G1; McMahon, K2; McKenzie, D3; Aiken, JM3 1University of Wisconsin, Soil Science/Civil and Environmental Engineering, USA; 2University of Wisconsin, Civil and Environmental Engineering, USA; 3University of Wisconsin, Comparative Biosciences, USA
Concern has been expressed that prions could enter wastewater treatment systems through sewer and/or septic systems (e.g., necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material. Prions are highly resistant to degradation and many disinfection procedures raising concern that they could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and survival of PrPSc during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. We establish that PrPSc can be efficiently extracted from activated and anaerobic digester sludges with 1% sodium dodecyl sulfate, 10% sodium undecyl sulfate, and 1% sodium N-lauryl sarcosinate. Activated sludge digestion does not result in significant degradation of PrPSc. The protein partitions strongly to the activated sludge solids and is expected to enter biosolids treatment processes. A large fraction of PrPSc survived simulated mesophilic anaerobic sludge digestion. Our results suggest that if prions were to enter municipal waste water treatment systems, most of the agent would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. Land application of biosolids containing prions could represent a route for their unintentional introduction into the environment. Our results argue for excluding inputs of prions to municipal wastewater treatment facilities that would result in unacceptable risk of prion disease transmission via contaminated biosolids.
Oral Transmissibility of Prion Disease Is Enhanced by Binding to Soil Particles
Christopher J. Johnson1,2, Joel A. Pedersen3, Rick J. Chappell4, Debbie McKenzie2, Judd M. Aiken1,2*
Soil may serve as an environmental reservoir for prion infectivity and contribute to the horizontal transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) of sheep, deer, and elk. TSE infectivity can persist in soil for years, and we previously demonstrated that the disease-associated form of the prion protein binds to soil particles and prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. …
In conclusion, our results provide compelling support for the hypothesis that soil serves as a biologically relevant reservoir of TSE infectivity. Our data are intriguing in light of reports that naïve animals can contract TSEs following exposure to presumably low doses of agent in the environment [5,79]. We find that Mte enhances the likelihood of TSE manifestation in cases that would otherwise remain subclinical (Figure 3B and 3C), and that prions bound to soil are orally infectious (Figure 5). Our results demonstrate that adsorption of TSE agent to inorganic microparticles and certain soils alter transmission efficiency via the oral route of exposure. full text is here:
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.
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Thursday, April 03, 2008 A prion disease of cervids: Chronic wasting disease 2008 1: Vet Res. 2008 Apr 3;39(4):41
A prion disease of cervids: Chronic wasting disease
*** twenty-seven CJD patients who regularly consumed venison were reported to the Surveillance Center***,
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Quantifying the Species Barrier in Chronic Wasting Disease by a Novel in vitro Conversion Assay
Li, L1; Coulthart, MB2; Balachandran, A3; Chakrabartty, A4; Cashman, NR1 1University of British Columbia, Brain Research Centre, Canada; 2Public Health Agency of Canada, National Microbiology Laboratory, Canada; 3Animal Diseases Research Institute, Canada Food Inspection Agency, National Reference Laboratory for Scrapie and CWD, Canada; 4Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Canada
Background: Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Although the risk of CWD crossing the species barrier and causing human disease is still unknown, however, definite bovine spongiform encephalopathy (BSE) transmission to humans as variant CJD (vCJD), it would seem prudent to limit the exposure of humans to CWD.
Aim: In view of the fact that BSE can be readily transmitted to non-bovid species, it is important to establish the species susceptibility range of CWD.
Methods: In vitro conversion system was performed by incubation of prions with normal brain homogenates as described before, and protease K (PK) resistant PrP was determined by immunoblotting with 6H4 monoclonal prion antibody.
Results: Our results demonstrate that PrPC from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Interestingly, hamster shows a high conversion ratio by PrPCWD. Moreover, partial denaturation of substrate PrPC can apparently overcome the structural barriers between more distant species.
Conclusions: Our work correctly predicted the transmission of CWD to a wild moose. We find a species barrier for prion protein conversion between cervids and other species, however, this barrier might be overcome if the PrPC substrate has been partially denatured in a cellular environment. Such an environment might also promote CWD transmission to non-cervid species, *** including humans. Acid/GdnHCl-treated brain PrPC was a superior substrate for the in vitro conversion than PrPC treated at physiological pH. This has implications for the process by which the prion protein is converted in disease.
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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@example.com
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Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518