Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse Bioassay Nicholas J. Haley1, Davis M. Seelig1, Mark D. Zabel1, Glenn C. Telling2, Edward A. Hoover1*
1 Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America, 2 Department of Molecular Biology and Genetics, University of Kentucky, Lexington, Kentucky, United States of America
Abstract Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrPCWD was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrPCWD levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373±3days in 2 of 9 urine-inoculated mice and 342±109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.
Citation: Haley NJ, Seelig DM, Zabel MD, Telling GC, Hoover EA (2009) Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse Bioassay. PLoS ONE 4(3): e4848. doi:10.1371/journal.pone.0004848
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received: November 8, 2008; Accepted: February 3, 2009; Published: March 18, 2009
Copyright: © 2009 Haley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH/NCRR Ruth L. Kirschstein Institutional T32 R07072-03 and NIH/NIAID NO1-AI-25491-02 (EAH, GCT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: Edward.Hoover@colostate.edu
Discussion The salient feature of chronic wasting disease is its facile transmission among its host species. Until recently, little was known regarding the mechanisms of this efficient transmissibility, however, we have previously demonstrated infectious prions in the saliva and blood of infected deer . By using intracerebral inoculation of concentrated urine in cervid PrP transgenic mice, we report the presence of infectious prions in urine from CWD-infected cervids, and confirm the phenomenon of prionsialia in these animals. The identification of CWD prions in bodily fluids described in the current report could portend infectivity in secretions and excretions in other prion diseases.
In contrast to the data presented here, oral inoculation of urine in cervid bioassays was unable to identify infectious prions in the urine of CWD+ deer . This result could have been due to necessarily limited observation period possible in those studies (18 months), or variations in source and recipient genotype , , route of inoculation , or the sensitivity of traditional PrPCWD detection assays , . The mule deer providing inoculum pools in prior studies were of an unreported genotype; the majority of the recipient deer were homozygous for glycine at residue 96, although a single animal was heterozygous; sharing both G96 and S96 alleles . Likewise, the inocula used in the present study were pooled from sources heterogeneous at codon 96 of the cervid prion gene. Transgenic mice used in bioassay studies, on the other hand, were uniformly homogenous for a glycine residue at this position , a polymorphism which is reported to be overrepresented in CWD-infected deer . As a result, it is possible that the genotypic background of either source or subject animals may have been a factor in susceptibility, though we are at present unable to draw any concrete conclusions regarding this relationship. While mouse genotype may have played a role in the outcome, it is also probable that cervid PrP transgenic mouse bioassay simply represents a more sensitive detection system for prions in excreta. Intracranial inoculation, reportedly a more sensitive route of prion exposure , , is more easily performed in mouse bioassay, a model which also permits extended incubation periods and inclusion of a greater number of test animals.
While our findings point to urine as an additional vehicle for CWD transmission, only 2 of 9 inoculated tg1536 mice were confirmed WB/IHC-positive for prion infection, with a third PrPCWD+ animal later identified by PMCA. This contrasts with 8 of 9 positive mice receiving saliva and infers a much lower concentration of prion infectivity in urine. The wide range of survival times in inoculated mice suggests relatively low levels of infectious prions and/or uneven distribution of infectious PrP moieties in the inocula . Differing [CerPrP] zygosity in tg1536 mice (homozygous vs. hemizygous) may also have played a role in this variation.
Using sPMCA, PrPCWD was repeatedly identified in test urine and spiked urine and saliva used as positive control, but was not detected in test saliva after three rounds of amplification. The reasons for our inability to identify PrPCWD in saliva – given the definitive bioassay findings – remain unknown, and we propose the presence of as-yet unidentified inhibitors such as mucin or salivary proteases which are thought to negatively affect other in vitro assays , .
The finding of PrPCWD in urine and saliva calls for the identification of the pathological processes and cellular associations of the prion protein involved in shedding. Previous studies have related renal pathology to prionuria , , a finding which corresponds to our identification of mild to moderate nephritis in those deer providing samples for the current study. It is plausible that renal pathology contributed to prionuria in each of these animals; as samples were pooled, however, we cannot identify specific animals in which it may have been occurring, nor can we accurately estimate the relative level of prionuria occurring in each donor as ultrastructural studies were not performed . While we have not yet identified pathologic prions in renal source tissues [Unpublished data], protease-resistant PrPCWD has been identified by immunostaining in renal tissue of prion-infected deer , sheep , hamsters and most intriguingly humans , foreshadowing the potential for prionuria in other transmissible spongiform encephalopathies. We continue to examine tissues from CWD+ deer in an effort to determine the pathogenesis and kinetics of CWD prion excretion and shedding.
Evidence for excretion and shedding of infectious prions is also accumulating in the scrapie system. PrPC-converting activity has been identified by sPMCA in the urine of scrapie-infected sheep, hamsters and mice , , , . Prion infectivity has also been demonstrated in the feces of hamsters orally infected with scrapie . Other studies point to infectious prions in the milk of scrapie-infected ewes , . As noted above, it remains unknown whether other prion diseases (e.g. Kuru, BSE, CJD, TME) may be transmitted by bodily fluids or excreta other than blood. Additional studies examining feces, milk, and other body fluids are therefore necessary in CWD and other prion diseases, studies currently underway in our laboratory.
As CWD transmission may model communicability of other TSE's, the transmissible nature of prion diseases may serve as a model for other protein-misfolding diseases. For example, feces, but not urine, from both mice and cheetahs affected with systemic amyloidosis A (SAA) was recently shown to induce SAA in a mouse model, although negative controls were not available in those studies . In light of the prionuria detected in CWD and in models of scrapie, further investigations of infectivity in body fluids in other protein folding diseases may be warranted in the event that prion diseases are not the only infectious proteinopathies.
In summary, we confirm prionsialia in CWD-affected deer by bioassay in cervidized mice and demonstrate for the first time infectious prions in the urine of these cervids by both bioassay and sPMCA. We are currently evaluating urine and saliva from individual animals in hopes of identifying predisposing factors, such as genotypic background and underlying pathology, which may contribute to prionuria and prionsialia. Concurrently, we have begun to explore the tissue origins and protease sensitivity of the infectious prions as well as the onset and duration of shedding in these bodily fluids.
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Monday, February 09, 2009
Exotic Meats USA Announces Urgent Statewide Recall of Elk Tenderloin Because It May Contain Meat Derived From An Elk Confirmed To Have CWD
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
Thursday, December 25, 2008
Elk meat recalled due to CWD Boulder County Health Department and Colorado Department of Public Health and Environment
Saturday, January 24, 2009
Research Project: Detection of TSE Agents in Livestock, Wildlife, Agricultural Products, and the Environment Location: 2008 Annual Report