Wednesday, June 18, 2008


Wasting disease found in 24 more deer

Erika Beauchesne Edmonton Journal

Wednesday, June 18, 2008

Twenty-four more cases of chronic wasting disease have been found in Alberta's wild deer, the Sustainable Resource Development of Alberta announced Tuesday.

The results, from a 2007-08 testing program, bring the province's count of the disease up to 53 cases.

CWD affects the central nervous system and causes infected animals to slowly waste away. Evidence suggests it does not affect humans. Darcy Whiteside, spokesperson for Sustainable Resource and Development, said the department has consulted with communities such as Provost, Oyen and Wainwright, where many of the diseased deer have been detected.

"The next step is really making hunters aware there are opportunities here," he said, referring to an ongoing hunting program the government implemented in 2005 when the first diseased deer was found 30 kilometers southeast of Oyen.

The program aims to reduce deer populations and track the disease.

"It's a contagious disease, so any animal we can take away from the population is a plus," he said, adding the province was seeing excessive growth in the number of wild deer even before CDW.

But Whiteside said the new numbers are not huge. "We're still seeing very low percentages," he said. "There were more deer tested this year."


Public Significance

This disease poses significant economic problems for farmers of elk and deer. CWD was unintentionally introduced into farmed elk populations from live wild elk and deer taken from affected areas in the U.S. It was then TRANSLOCATED to farms in various states as well as SASKATCHEWAN AND KOREA. The source of the infection on farms in Alberta is not known. The economics of trade in live elk and their products (primarily antler velvet) have been seriously affected. IN ADDITION, the association with BSE has let to possible PUBLIC HEALTH CONCERNS. ...



8. Human susceptibility to CWD

Millions of North Americans hunt deer and elk (U.S. Department of theInterior, Census Bureau), and there is no doubt that people have beenexposed to CWD through venison consumption, particularly in light of recentdata showing CWD prions in muscle [2]. Human susceptibility to CWD or toother newly emerging animal TSE [9, 14] is still unclear, although we can besomewhat reassured in that there have been no large scale outbreaks of humanTSE cases in Colorado and Wyoming, where CWD has existed for decades [51].Up until approximately 10 years ago, autopsies were not performed on suspecthuman TSE cases in many states due to biosafety concerns, therefore thediagnosis of potential new TSE strains has been hampered. This indicatesthat clinical TSE diagnoses in humans were not confirmed, nor was any straintyping done to look for the appearance of potentially subtle or unusualpathological or biochemical phenotypes of a new TSE strain. Fortunately, theautopsy rate for suspect cases is improving. At the National Prion DiseasePathology Surveillance Center at Case Western Reserve University (Cleveland,Ohio), Creutzfeldt-Jakob disease (CJD) suspect cases are studied andclassified by CJD subtype. Thus far,


*** twenty-seven CJD patients who regularly consumed venison were reportedto the Surveillance Center***,

however there have been no unusual or novel prion subtypes that mightindicate the appearance of a new prion strain [7, 41]. Other indirectstudies of human susceptibility to CWD also suggest that the risk is low. Inbiochemical conversion studies, Raymond et al. [68] showed that theefficiency of CWD to convert recombinant human PrP into amyloid fibrils waslow, but similar to that of both BSE and scrapie fibrils to do the same.These results suggest that there is a molecular incompatibility in theconversion of human PrPC by CWD, sheep scrapie, or BSE, and that crossspecies infections in humans may be rare events. To determine whether commonPrPSc strain features may link CWD and CJD, histopathology and the PrPScbiochemical characteristics from deer and elk were compared with that ofhumans with sporadic CJD (sCJD) cases that are methionine homozygous atcodon 129 of the Prnp gene by Xie et al. [96], although strain featuresincluding histologic profile, target organs, and glycoform patterns will notnecessarily remain the same upon crossing species barriers [6, 5, 8, 57].The PrPSc form is cleaved by proteinase-K (PK) at different sites dependingon the conformation of the protein and may aid determination of whether thePrPSc conformation is similar. By western blot (SDS-PAGE) of elk CWD, theunglycosylated PK-resistant PrPSc migrated at 21 kDa, similar to sCJD (MM1subtype) and the PK cleavage site was the same, occurring at residues 78 and82 as assessed by N-terminal sequencing. Conformational stability wasevaluated by measuring the PrPSc stability under partially denaturingconditions and also showed no significant difference between elk CWD andsCJD MM1 PrPSc. However, elk CWD and human sCJD MM1 strains exhibiteddistinct glycoform patterns by two dimensional gel electrophoresis,suggesting that the strains differed. Future studies may utilize luminescentconjugated polymers, which were recently shown to distinguish naturally- andexperimentally-derived prion strains [79]. To study elk-human prion speciesbarriers, Kong et al. inoculated elk CWD into transgenic mice expressingeither human PrP or elk PrP. Whereas the elk PrP expressing mice developeddisease after only 118-142 days post-inoculation, human PrP expressing mice(129M) did not develop any features of TSE after more than 657 or more than756 days [41]. In accordance with these results, Tamgüney et al. alsoreported that human PrP overexpressing mice were not susceptible to 9 CWDisolates from mule deer, white-tailed deer, and elk [84]. However, mice havea limited lifespan and further passages may be necessary to detect lowlevels of prion infectivity that may be present subclinically. Although indirect evidence is accumulating that there may be a robust species barrier forCWD transmission to humans, one report indicates nonhuman primatesusceptibility to CWD. Intracerebral inoculation of squirrel monkeys(Saimiri sciureus) demonstrated a positive CWD transmission [49]. Amongnon-human primates, however, the Prnp sequence of the new world monkeys arethe most distant from humans [72], and therefore may not indicate that humanprion conversion would occur by CWD.


11. Disease control challenges posed by CWD

Evidence is building that indicates efficient horizontal transmission occursin CWD, indeed a complicating aspect in disease control [91]. Potentialtransmission mechanisms range from spread via direct contact among animalsto environmental exposure through grazing in areas contaminated byprion-infected secretions, excretions (saliva, urine, feces), tissues(placenta), or decomposed carcasses. Recently, in a breakthrough finding,saliva from CWD infected deer was shown to transmit prion disease [50]. Anadditional experiment by Miller and colleagues showed that CWD-infectedcarcasses allowed to decay naturally in confined pastures can lead to CWDinfections in captive deer, demonstrating the potential for environmentalcontamination to spread infection [55]. Modelling studies have providedfurther


support that environmental contamination is likely playing a significantrole in transmitting CWD [56, 53]. Additionally, infectious prions have beendemonstrated to bind soil particles and remain infectious to animals by bothintracerebral and oral exposure routes [38, 37]. Prion infectivity has beenrecovered from soil more than two years after experimental exposure toprions, suggesting the soil may serve as a reservoir for CWD prions [75].Taken together, these results indicate that there may even be multiplesources for CWD exposure, perhaps through direct contact and environmentalroutes. Significant challenges to CWD eradication exist in free-rangingcervids. Infected deer and elk range over a broad geographic region, andeven previously surmised geographic barriers such as the Continental Dividehave proven passable by infected animals. Ridding the environment ofCWD-contaminated soil or even CWD-infected carcasses is not possible.Moreover, the available ante-mortem diagnostic tests for surveillance arelaborious and impractical for large numbers of free-ranging animals [74, 88,95]. Therefore for a wildlife manager, this disease is costly to survey anddifficult to control.

12. Conclusion

CWD in cervids is efficiently transmitted, likely more than any other TSE inanimals or humans. Therefore, it is unlikely that this TSE can beeradicated, but perhaps through an improved understanding of transmissionroutes, biological factors influencing pathogenesis, and the molecular basisof CWD prion conversion, a targeted strategy for interrupting disease spreadmay be developed.


I thank Drs. Michael Miller, Jason Bartz and Mathias Heikenwalder forcritical review of the manuscript.

snip...see full text 19 pages ;

Subject: Species barriers for chronic wasting disease by in vitro conversion of prion protein Date: November 3, 2007 at 10:57 am PST

Species barriers for chronic wasting disease by in vitro conversion of prion protein

Li Li a, Michael B. Coulthart b, Aru Balachandran c, Avi Chakrabartty d, Neil R. Cashman a,* a Brain Research Centre, Division of Neurology, Department of Medicine, University of British Columbia and Vancouver Coastal Health, UBC Hospital, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 b Prion Diseases Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Man., Canada R3E 3R2 Q1 c National Reference Laboratory for Scrapie and CWD, Animal Diseases Research Institute, Canadian Food Inspection Agency, 3851 Fallowfield Road, Nepean, Ont., Canada K2H 8P9 d University Health Network, Department of Medical Biophysics, University of Toronto, Toronto, Ont., Canada M5G 1L7 Received 6 October 2007


Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrPCWD of elk can readily induce the conversion of normal cervid PrP (PrPC) molecules to a protease-resistant form, but is less efficient in converting the PrPC of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrPCWD-induced conversion can be greatly enhanced in all species. Our results dem- onstrate 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. Moreover, partial denaturation of substrate PrPC can apparently overcome the structural barriers between more distant species.


Although Syrian hamsters were initially deemed resistant to CWD [19], a recent publication demonstrates that CWD can be transmitted and adapted to hamster [20].


Substrate denaturation and human health

We confirm with multiple species that acid/GdnHCl- treated brain PrPC is a superior substrate for in vitro con- version than untreated PrPC, possibly by overcoming con- formational barriers in partial denaturation of substrate PrPC. PrP conversion in scrapie-infected neuroblastoma cells is believed to occur in endosomes, a low-pH and reducing environment [26]. The non-ruminant stomach possesses a low pH lumen, and PrPC is expressed in this organ [27]. Such acidic (denaturing) organ or cellular organellar environments might also promote CWD trans- mission to non-cervid species, including humans.


This work was supported by the Canadian Institutes of Health Research (Institute of Infection and Immunity, Safe Food and Water program) and PrioNet Canada.

[20] G.J. Raymond, L.D. Raymond, K.D. Meade-White, A.G. Hughson, C. Favara, D. Gardner, E.S. Williams, M.W. Miller, R.E. Race, B. Caughey, Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains, J. Virol. 81 (2007) 4305–4314.

2007 Elsevier Inc. All rights reserved.

Please cite this article in press as: L. Li et al., Species barriers for chronic wasting disease by in vitro..., Biochem. Biophys. Res. Commun. (2007), doi:10.1016/j.bbrc.2007.10.087

Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains

Gregory J. Raymond1, Lynne D. Raymond1, Kimberly D. Meade-White1, Andrew G. Hughson1, Cynthia Favara1, Donald Gardner2, Elizabeth S. Williams3§, Michael W. Miller4, Richard E. Race1*, and Byron Caughey1*

Running title: CWD transmission to rodent species

Laboratory of Persistent Viral Diseases1, and Rocky Mountain Veterinary Branch2, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, MT 59840; Department of Veterinary Sciences, University of Wyoming, Laramie, WY 820703; Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526-20974. §deceased *corresponding authors: Byron Caughey, Rocky Mountain Labs, 903 S. 4th St, Hamilton, MT 59840, USA;; Tel: (406) 363-9264; FAX: (406) 363-9286 Richard Race, Rocky Mountain Labs, 903 S. 4th St, Hamilton, MT 59840, USA;; Tel: (406) 363-9358; FAX: (406) 363-9286

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer and elk were inoculated intracerebrally into various rodent species to assess their susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters; transgenic mice expressing the Syrian golden hamster prion protein; and, RML Swiss and C57 BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian and Armenian hamsters had limited susceptibility to certain of the CWD inocula as evidenced by incomplete attack rates and long incubation periods. With serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized as isolates with either short (85-89 days) or long (408-544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained, or diverged into, at least two distinct transmissible spongiform encephalopathy strains.


Differences in PrP-res glycoform patterns analyzed from several CWD- affected deer and elk have also suggested that CWD in mule deer may be more heterogeneous than in elk (19). Curiously, however, this apparent strain difference was not manifested when the identical mule deer CWD inoculum was serially passaged through only one recipient species. Serial passage in Sg hamsters yielded only the fast isolate (Table 1 and Figure 3), while passage first through the Tg (haPrP) mice then into Sg hamsters yielded only the slow isolate (Table 2 and Figure 3). With this in mind, it is important to consider other possible explanations for these results. One possibility is that CWD might be able to undergo a stochastic change into a more rapid and aggressive strain in Sg hamsters, and that this happened to occur after the mule deer CWD inoculations. A similar emergence of both fast and slow strains has been observed upon inoculation of TME into Sg hamsters (5). These strains developed even when a clonal isolate of the TME inoculum was used, suggesting that they arose in the recipient Sg hamsters rather than in the mink source (1). Finally, although extensive precautions were taken, we cannot formally prove that inadvertent contamination of the mule deer CWD inoculum with hamster-derived 263K strain did not occur which potentially could yield short- incubation-period passages in Sg hamsters (Table 1). However, the incubation period observed with the CWD passages (85-89 d) were significantly longer than 263K incubation periods observed in our lab (70-75 d) and no mock-infected

controls became sick during their lifespan. Also, we saw no 263K-like infectivity develop in the highly susceptible Tg (haPrP) mice even though we used the identical primary inoculum for both recipient species. Interestingly, the similarity of the Sg hamster-adapted CWD fast isolate and 263K might be due to a common origin since there is circumstantial evidence that CWD arose from cervid exposure to sheep scrapie, which was also the origin of the 263K strain in hamsters (14). Furthermore, the Hyper strain derived from TME inoculations has 263K-like strain characteristics in Sg hamsters (5). Thus, it would appear that both CWD and TME transmissions into Sg hamsters can result in divergent fast and slow strains.


Transmissible Mink Encephalopathy TME

Terry S. Singeltary Sr.
P.O. Box 42
Bacliff, Texas USA 77518

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