Saturday, March 09, 2013

Chronic Wasting Disease in Bank Voles: Characterisation of the Shortest Incubation Time Model for Prion Diseases

Research Article Chronic Wasting Disease in Bank Voles: Characterisation of the Shortest Incubation Time Model for Prion Diseases

Michele Angelo Di Bari mail, * E-mail:

Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Romolo Nonno, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Joaquín Castilla, Affiliation: CIC bioGUNE and IKERBASQUE, Basque Foundation for Science, Derio and Bilbao, Bizkaia, Spain

X Claudia D'Agostino, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Laura Pirisinu, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Geraldina Riccardi, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Michela Conte, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Juergen Richt, Affiliation: National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, United States of America

Current address: Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United States of America

X Robert Kunkle, Affiliation: National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, United States of America

X Jan Langeveld, Affiliation: Department of Infection Biology, Central Veterinary Institute of Wageningen UR, Lelystad, Netherlands

X Gabriele Vaccari, Affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy

X Umberto Agrimi


In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ~35 days. Neuropathological and molecular characterisation of Bv109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrPSc (PrPres) in the same brain regions. Despite the low PrPres levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 108,4 i.c. ID50 infectious units per gram. Bv109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv109ICWD showed unique characteristics of “virulence”, low PrPres accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity.

Author Summary Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids and is expanding increasingly in the USA and Canada. Animal models are of key importance in the study of prion diseases but their development for CWD has long been hampered by its very inefficient transmission to wild-type mice. Significant progress was made following the generation of transgenic mice over-expressing cervid PrP. Here we show that the bank vole (Myodes glareolus), a wild rodent species that we demonstrated to be susceptible to many animal and human prion diseases, is also very susceptible to CWD from elk, mule deer and white-tailed deer. Adaptation of CWD to bank vole led to the isolation of a prion strain with peculiar characteristics: unprecedented short incubation and survival times, respectively of 25–28 and ~35 days, low PrPSc levels compared with other vole-adapted prion strains and high infectious titre. These features were all faithfully maintained upon the generation of this strain in vitro by protein misfolding cyclic amplification. The development of a model for prion diseases that led to disease in less than one month accumulating high infectious titres but low PrPSc levels, represents a significant tool for investigating the still unclear relationship between PrPSc, neurodegeneration and infectivity in prion diseases.

Citation: Di Bari MA, Nonno R, Castilla J, D'Agostino C, Pirisinu L, et al. (2013) Chronic Wasting Disease in Bank Voles: Characterisation of the Shortest Incubation Time Model for Prion Diseases. PLoS Pathog 9(3): e1003219. doi:10.1371/journal.ppat.1003219

Editor: David Westaway, University of Alberta, Canada

Received: August 31, 2012; Accepted: January 12, 2013; Published: March 7, 2013

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: This work was financially supported by the European Union (Neuroprion Network of Excellence, CT-2004-506579), the Alliance Biosecure Foundation (“Vole PMCA” research grant), the Italian Ministry of Health, a national grant from Spain (AGL2009-11553-C02-01) and from the Basque Government (PI2010-18). 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.



In the present study we investigated the susceptibility of Bv109I and Bv109M to seven CWD isolates from three deer species and found that these animal models are highly permissive to CWD, showing 100% attack rate and mean survival times between 156 and 281 d.p.i.. A deepened transmission and characterization study of CWD was carried out in Bv109I. The susceptibility of this model appeared comparable to that of transgenic mice expressing cervid PrP [6], [7], [9], [10], [11]. The reasons for such a high susceptibility are unclear but apparently not related to a different expression of PrPC. As a matter of fact, its expression level in Bv109I is comparable to that of mouse and hamster (Figure S5). The dramatic drop in survival time with vole-to-vole sub-passages suggests that CWD still encounters a high transmission barrier in Bv109I, implying that Bv109I permissiveness to CWD was not due to the absence of transmission barrier, as observed in transgenic mice expressing cervid PrP. Using Bv109M voles we have previously shown that this species is permissive to a variety of human and animal prion diseases. Studies aimed at investigating the molecular basis of the susceptibility of bank voles to foreign prions and their selective strain preferences suggested that two asparagine residues at positions 150 and 170, specific to vole PrP, might play a role [18], [22], [25]. Interestingly, cervid PrP also has asparagine at residue 170, suggesting that sequence identity at codon 170 might facilitate the transmission of CWD to Bv109I. This interpretation is supported by the relative ease of transmission of CWD to meadow voles [14], which also have asparagine at positions 150 and 170, as well as by studies in MoPrP170N, 174T transgenic mice [24], [26] and by in vitro amplification of CWD by PMCA [27].

A striking feature of Bv109I-adapted CWD was the short incubation time of less than one month. In an earlier work, we showed that bank voles and related rodent species have peculiarly short survival times after infection with adapted prions, and presumably support equally fast prion replication kinetics, possibly due to the previously mentioned 150N–170N PrP residues [22]. This was also observed in vitro using Bv109M brain homogenates as substrate for PMCA-driven prion replication [28]. Notwithstanding this, our previous and on-going studies with Bv109M have not shown evidence of ultra-fast strains such as Bv109ICWD, and the fastest strains observed so far in voles show incubation times of ~2 months [19, unpublished data). Transmission studies of CWD to Bv109M have not yet completed but we have observed that also CWD adapts to Bv109M with survival times longer than Bv109ICWD (60–100 d.p.i.) (unpublished data). The short survival time of Bv109ICWD is also unprecedented when compared with those found in transgenic mouse models, in which PrP overexpression greatly fosters prion diseases [6], [7], [8], [9], [10], [11]. Indeed, the fastest rodent models reported so far, i.e. Tga20 [29], Tg52NSE [30], Tg7 [31], Tg4053 [32] and Tg338 mice [33], express several-fold higher PrP levels compared with wild-type mice and have incubation periods at least twice as long as Bv109ICWD. Interestingly, it was recently shown that TgS3581 mice overexpressing vole PrP encoding for Isoleucine at position 109, undergo spontaneous prion disease and that it adapts to the same model with mean survival time of 35 days [34]. These findings suggest that the presence of Isoleucine at position 109 of the vole PrP plays a specific role in determining the short survival time of Bv109ICWD

In the present work we provide evidence that all the hallmarks of TSEs were recapitulated within one month in bank vole CWD. The finding that neurodegeneration and PrPSc deposition showed a discrete brain distribution, involving specific neuronal populations such as those in the medulla and thalamus, might suggest that Bv109ICWD replication primarily involves the so called clinical target areas (CTAs) which, once colonised by prions, trigger the clinical signs and death of the animals [35], [36]. A recent study showed that shorter time periods were needed to initiate the clinical phase when the 127S scrapie strain primarily targeted CTAs, as in intraperitoneally-inoculated Tg338 mice, compared with intracerebrally-inoculated mice [37]. This was accompanied by comparatively low levels of PrPres and infectivity in the brain of ip-inoculated mice. In Bv109ICWD we also observed unusually low levels of PrPres, compared with those observed in most of the vole-adapted prion strains (Figure 3). However, by endpoint titration we found unexpected high prion titres in Bv109ICWD, 108,4 i.c. ID50 U g−1, similar to those usually observed in standard hamster and mouse-adapted scrapie strains, whose incubation times are 3–10 times longer than Bv109ICWD. This implies that Bv109ICWD undergoes extraordinarily fast replication kinetics in Bv109I brain.

Several observations suggest that prion infectivity and toxicity might be uncoupled [38], [39], [40] and these observations are currently incorporated in a general model of prion replication and toxicity [41]. According to this model, neurotoxicity is mediated by a lethal PrP species, PrPL, which is distinct from PrPSc, but its formation is catalysed during the autocatalytic replication of PrPSc. Neurotoxicity may require a critical PrPL concentration to be reached, which would depend on the kinetics of prion propagation. The relative levels of toxicity and infectivity are governed by the ratio of the initial rate of PrPC conversion (which leads to the production of PrPL) to the rate of its maturation into PrPSc. Thus fast prion replication in Bv109ICWD might have triggered the production of high levels of PrPL in short time periods, leading to rapid disease onset and animal death. The low levels of PrPSc and the fast replication kinetics observed in Bv109ICWD are consistent with this interpretation.

A recent work showed that in mice inoculated with the RML scrapie strain the concentration of PrPC did not affect the overall level of prion infectious titres at terminal disease, while it was directly related to the incubation time, suggesting that the production of PrPL is directly proportional to PrPC concentrations [42]. Our observations with Bv109ICWD, i.e. the unusually short survival time and the high prion infectious titre in a model that does not overexpress PrPC, suggest that the kinetics of prion propagation and toxicity are governed by mechanisms that cannot be interpreted solely on the basis of the amount of available substrate (PrPC).

The unique and easily distinguishable features of Bv109ICWD prompted us to pursue its in vitro propagation by saPMCA. Bv109ICWD PrPSc was indeed easily propagated in vitro, which allowed us to produce Bv109ICWDPMCA PrPSc, theoretically devoid of any PrPSc formed in vivo. Bv109ICWDPMCA was highly infectious and faithfully reproduced the peculiar phenotype of Bv109ICWD. These findings confirm that CWD prions can be generated in vitro, as already demonstrated by others using transgenic mice expressing cervid PrP [43], [44], [45], [46] and prairie voles [47]. Furthermore, given the unique characteristics of this strain, it is extremely unlikely that their faithful maintenance during saPMCA could have occurred by chance and our results represent a convincing confirmation of other studies that have already demonstrated the ability of PMCA to replicate prion strains faithfully [44], [45], [47], [48], [49].

Elk, mule deer and white-tailed deer are the species most affected by CWD. The homogeneous and peculiar phenotypes observed in Bv109I inoculated with CWD isolates from these three cervid species indicate that the same CWD strain was isolated from all species. Interestingly, Bv109ICWD was isolated not only from natural cases of disease in elk (CWD1, 2 and 4) and mule deer (CWD3), but also from white-tailed deer experimentally inoculated with CWD-affected white-tailed deer, mule deer and elk (CWD5, 7 and 8, respectively). Along with previous findings in transgenic mice expressing cervid PrP [6], [9], and in keeping with the ease of indirect horizontal transmission of CWD [50], these data suggest that the same CWD strain circulates among different cervid species and maintains its characteristics following interspecies transmission. Recently, a large transmission study with elk and mule deer isolates provided substantial evidence for two prevalent CWD prion strains and suggested that individual CWD inocula might contain mixtures of the two prion strains [11]. Interestingly, we found similar evidence in at least two of the seven inocula investigated, derived from elk and mule deer, although we were unable to stabilize two different Bv109I-adapted CWD strains. Indeed, on primary transmission of CWD2, CWD3 and CWD4 inocula we observed voles that developed clinical signs after unusually long times, showing a slightly different neuropathological profile from that of voles with shorter survival times. A sub-passage in Bv109I of two of these outliers induced a survival time of 150–160 d.p.i. on second passage, compared with 35–45 d.p.i. observed with all other sub-passages. Such a long survival time might have depended on a low infectious titre in the brain from outlier voles, although they showed levels of PrPres similar to the other voles of their groups. However this hypothesis is excluded when these results are compared with the survival times observed in the endpoint titration experiment, which showed that even 10−5 dilution of Bv109ICWD had a mean survival time <80 2="" a="" according="" addition="" also="" and="" as="" be="" before="" both="" bv109icwd="" by="" conformers="" converged="" cwd="" d.p.i..="" deviant="" div="" elimination="" evanescent="" extremely="" fast="" findings="" fitting="" following="" for="" in="" interpreted="" interspecies="" lesion="" less="" might="" nature="" neuropathological="" observed="" occurred="" of="" our="" outcompeted="" outlier="" outliers="" overall="" presence="" preserved="" prions="" profile="" progressive="" progressively="" propagated="" proposed="" quasi="" rapid="" second="" slightly="" species="" strain.="" strain="" strongly="" sub-passages="" suggest="" survival="" that="" the="" time="" to="" transmission.="" vole="" voles="" was="" which="" with="">

Here we demonstrate the high susceptibility of Bv109I to CWD, which adds Bv109I to the portfolio of animal models useful for the study of CWD strains. The unique properties of Bv109ICWD provide exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity. The short survival time of Bv109ICWD, coupled with its high infectious titre, offers useful advantages for titration studies, while its unique clinico-pathological phenotype makes Bv109ICWD one of the best options for studies aimed at investigating strain fidelity in experimental conditions.

J Gen Virol. 2012 January; 93(Pt 1): 212–221. doi: 10.1099/vir.0.035006-0 PMCID: PMC3352335

Evidence for distinct chronic wasting disease (CWD) strains in experimental CWD in ferrets

Matthew R. Perrott,1 Christina J. Sigurdson,2 Gary L. Mason,3 and Edward A. Hoover3


Chronic wasting disease (CWD) is an evolving prion disease of cervids (deer, elk and moose) that has been recognized in North America and Korea. Infection of non-cervid reservoir or transport species in nature is not reported. However, the ferret (Mustela putorius furo) is susceptible to CWD after experimental inoculation. Here, we report that infection of ferrets with either of two ferret CWD isolates by various routes of exposure has revealed biologically distinct strain-like properties distinguished by different clinical progression and survival period. The isolates of ferret CWD were also differentiated by the distribution of the infectious prion protein (PrPCWD) in the brain and periphery, and by the proteinase K sensitivity of PrPCWD. These findings suggest that diversity in prion conformers exists in CWD-infected cervids.


The results of these studies support the growing evidence for multiple strains of CWD prions.


Wednesday, September 08, 2010



CWD to cattle figures CORRECTION


I believe the statement and quote below is incorrect ;

"CWD has been transmitted to cattle after intracerebral inoculation, although the infection rate was low (4 of 13 animals [Hamir et al. 2001]). This finding raised concerns that CWD prions might be transmitted to cattle grazing in contaminated pastures."

Please see ;

Within 26 months post inoculation, 12 inoculated animals had lost weight, revealed abnormal clinical signs, and were euthanatized. Laboratory tests revealed the presence of a unique pattern of the disease agent in tissues of these animals. These findings demonstrate that when CWD is directly inoculated into the brain of cattle, 86% of inoculated cattle develop clinical signs of the disease.

" although the infection rate was low (4 of 13 animals [Hamir et al. 2001]). "

shouldn't this be corrected, 86% is NOT a low rate. ...

kindest regards,

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

Thank you!

Thanks so much for your updates/comments. We intend to publish as rapidly as possible all updates/comments that contribute substantially to the topic under discussion.

re-Prions David W. Colby1,* and Stanley B. Prusiner1,2 + Author Affiliations

1Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, California 94143 2Department of Neurology, University of California, San Francisco, San Francisco, California 94143 Correspondence:

Mule deer, white-tailed deer, and elk have been reported to develop CWD. As the only prion disease identified in free-ranging animals, CWD appears to be far more communicable than other forms of prion disease. CWD was first described in 1967 and was reported to be a spongiform encephalopathy in 1978 on the basis of histopathology of the brain. Originally detected in the American West, CWD has spread across much of North America and has been reported also in South Korea. In captive populations, up to 90% of mule deer have been reported to be positive for prions (Williams and Young 1980). The incidence of CWD in cervids living in the wild has been estimated to be as high as 15% (Miller et al. 2000). The development of transgenic (Tg) mice expressing cervid PrP, and thus susceptible to CWD, has enhanced detection of CWD and the estimation of prion titers (Browning et al. 2004; Tamgüney et al. 2006). Shedding of prions in the feces, even in presymptomatic deer, has been identified as a likely source of infection for these grazing animals (Williams and Miller 2002; Tamgüney et al. 2009b). CWD has been transmitted to cattle after intracerebral inoculation, although the infection rate was low (4 of 13 animals [Hamir et al. 2001]). This finding raised concerns that CWD prions might be transmitted to cattle grazing in contaminated pastures.


----- Original Message -----

From: David Colby To:

Cc: stanley@XXXXXXXX

Sent: Tuesday, March 01, 2011 8:25 AM

Subject: Re: FW: re-Prions David W. Colby1,* and Stanley B. Prusiner1,2 + Author Affiliations

Dear Terry Singeltary,

Thank you for your correspondence regarding the review article Stanley Prusiner and I recently wrote for Cold Spring Harbor Perspectives. Dr. Prusiner asked that I reply to your message due to his busy schedule. We agree that the transmission of CWD prions to beef livestock would be a troubling development and assessing that risk is important. In our article, we cite a peer-reviewed publication reporting confirmed cases of laboratory transmission based on stringent criteria. The less stringent criteria for transmission described in the abstract you refer to lead to the discrepancy between your numbers and ours and thus the interpretation of the transmission rate. We stand by our assessment of the literature--namely that the transmission rate of CWD to bovines appears relatively low, but we recognize that even a low transmission rate could have important implications for public health and we thank you for bringing attention to this matter.

Warm Regards, David Colby -- David Colby, PhD

Assistant Professor Department of Chemical Engineering University of Delaware



Sunday, August 19, 2012

Susceptibility of cattle to the agent of chronic wasting disease from elk after intracranial inoculation 2012


PO-081: Chronic wasting disease in the cat— Similarities to feline spongiform encephalopathy (FSE)

PO-081: Chronic wasting disease in the cat— Similarities to feline spongiform encephalopathy (FSE)

Thursday, May 31, 2012

CHRONIC WASTING DISEASE CWD PRION2012 Aerosol, Inhalation transmission, Scrapie, cats, species barrier, burial, and more


page 30,

*** Spraker suggested an interesting explanation for the occurrence of CWD. The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr. Bob Davis. At or about that time, allegedly, some scrapie work was conducted at this site. When deer were introduced to the pens they occupied ground that had previously been occupied by sheep.







Transmission Studies

Mule deer transmissions of CWD were by intracerebral inoculation and compared with natural cases resulted in a more rapidly progressive clinical disease with repeated episodes of synocopy ending in coma. One control animal became affected, it is believed through contamination of inoculam (?saline).

Further CWD transmissions were carried out by Dick Marsh into ferret, mink and squirrel monkey. Transmission occurred in all of these species with the shortest incubation period in the ferret.

2011 Annual Report


2011 Annual Report

In Objective 1, Assess cross-species transmissibility of transmissible spongiform encephalopathies (TSEs) in livestock and wildlife, numerous experiments assessing the susceptibility of various TSEs in different host species were conducted. Most notable is deer inoculated with scrapie, which exhibits similarities to chronic wasting disease (CWD) in deer suggestive of sheep scrapie as an origin of CWD.


4.Accomplishments 1. Deer inoculated with domestic isolates of sheep scrapie. Scrapie-affected deer exhibit 2 different patterns of disease associated prion protein. In some regions of the brain the pattern is much like that observed for scrapie, while in others it is more like chronic wasting disease (CWD), the transmissible spongiform encephalopathy typically associated with deer. This work conducted by ARS scientists at the National Animal Disease Center, Ames, IA suggests that an interspecies transmission of sheep scrapie to deer may have been the origin of CWD. This is important for husbandry practices with both captive deer, elk and sheep for farmers and ranchers attempting to keep their herds and flocks free of CWD and scrapie.

now, years later, see the latest studies here on scrapie and cwd ;

PO-039: A comparison of scrapie and chronic wasting disease in white-tailed deer

Justin Greenlee, Jodi Smith, Eric Nicholson US Dept. Agriculture; Agricultural Research Service, National Animal Disease Center; Ames, IA USA

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of these experiments was to determine susceptibility of white-tailed deer (WTD) to scrapie and to compare the resultant clinical signs, lesions, and molecular profiles of PrPSc to those of chronic wasting disease (CWD). We inoculated WTD intracranially (IC; n = 5) and by a natural route of exposure (concurrent oral and intranasal (IN); n = 5) with a US scrapie isolate. All deer were inoculated with a 10% (wt/vol) brain homogenate from sheep with scrapie (1ml IC, 1 ml IN, 30 ml oral). All deer inoculated by the intracranial route had evidence of PrPSc accumulation. PrPSc was detected in lymphoid tissues as early as 7 months-post-inoculation (PI) and a single deer that was necropsied at 15.6 months had widespread distribution of PrPSc highlighting that PrPSc is widely distributed in the CNS and lymphoid tissues prior to the onset of clinical signs. IC inoculated deer necropsied after 20 months PI (3/5) had clinical signs, spongiform encephalopathy, and widespread distribution of PrPSc in neural and lymphoid tissues. The results of this study suggest that there are many similarities in the manifestation of CWD and scrapie in WTD after IC inoculation including early and widespread presence of PrPSc in lymphoid tissues, clinical signs of depression and weight loss progressing to wasting, and an incubation time of 21-23 months. Moreover, western blots (WB) done on brain material from the obex region have a molecular profile similar to CWD and distinct from tissues of the cerebrum or the scrapie inoculum. However, results of microscopic and IHC examination indicate that there are differences between the lesions expected in CWD and those that occur in deer with scrapie: amyloid plaques were not noted in any sections of brain examined from these deer and the pattern of immunoreactivity by IHC was diffuse rather than plaque-like. After a natural route of exposure, 100% of WTD were susceptible to scrapie. Deer developed clinical signs of wasting and mental depression and were necropsied from 28 to 33 months PI. Tissues from these deer were positive for PrPSc by IHC and WB. Similar to IC inoculated deer, samples from these deer exhibited two different molecular profiles: samples from obex resembled CWD whereas those from cerebrum were similar to the original scrapie inoculum. On further examination by WB using a panel of antibodies, the tissues from deer with scrapie exhibit properties differing from tissues either from sheep with scrapie or WTD with CWD. Samples from WTD with CWD or sheep with scrapie are strongly immunoreactive when probed with mAb P4, however, samples from WTD with scrapie are only weakly immunoreactive. In contrast, when probed with mAb’s 6H4 or SAF 84, samples from sheep with scrapie and WTD with CWD are weakly immunoreactive and samples from WTD with scrapie are strongly positive. This work demonstrates that WTD are highly susceptible to sheep scrapie, but on first passage, scrapie in WTD is differentiable from CWD.

White-tailed Deer are Susceptible to Scrapie by Natural Route of Infection

Jodi D. Smith, Justin J. Greenlee, and Robert A. Kunkle; Virus and Prion Research Unit, National Animal Disease Center, USDA-ARS

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. Previous experiments demonstrated that white-tailed deer are susceptible to sheep-derived scrapie by intracranial inoculation. The purpose of this study was to determine susceptibility of white-tailed deer to scrapie after a natural route of exposure. Deer (n=5) were inoculated by concurrent oral (30 ml) and intranasal (1 ml) instillation of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. Non-inoculated deer were maintained as negative controls. All deer were observed daily for clinical signs. Deer were euthanized and necropsied when neurologic disease was evident, and tissues were examined for abnormal prion protein (PrPSc) by immunohistochemistry (IHC) and western blot (WB). One animal was euthanized 15 months post-inoculation (MPI) due to an injury. At that time, examination of obex and lymphoid tissues by IHC was positive, but WB of obex and colliculus were negative. Remaining deer developed clinical signs of wasting and mental depression and were necropsied from 28 to 33 MPI. Tissues from these deer were positive for scrapie by IHC and WB. Tissues with PrPSc immunoreactivity included brain, tonsil, retropharyngeal and mesenteric lymph nodes, hemal node, Peyer’s patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by potential natural routes of inoculation. In-depth analysis of tissues will be done to determine similarities between scrapie in deer after intracranial and oral/intranasal inoculation and chronic wasting disease resulting from similar routes of inoculation.

see full text ;

Envt.11: Swine Are Susceptible to Chronic Wasting Disease by Intracerebral Inoculation

Justin Greenlee,† Robert Kunkle and Jodi Smith National Animal Disease Center, ARS, USDA; Ames, IA USA †Presenting author; Email:

Transmissible spongiform encephalopathies (TSEs, prion diseases) are chronic neurodegenerative diseases that occur in humans, cattle, sheep, goats, cervids and a number of laboratory animal models. There is no evidence of the natural occurrence of any form of TSE in the pig, but pigs have been shown to be susceptible to Bovine Spongiform Encephalopathy (BSE) infection by multiple-route parenteral challenge. However, pigs orally exposed at eight weeks of age to large amounts of brain from cattle clinically affected with BSE did not support infection after seven years of observation. In the US, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine, mink and poultry still occurs. Although unlikely, the potential for swine to have access to TSE-contaminated feedstuffs exists. The potential for swine to serve as a host for the agent of chronic wasting disease (CWD) is unknown. The purpose of this study was to perform intracerebral inoculation of the CWD agent to determine the potential of swine as a host for the CWD agent and their clinical susceptibility. This study utilized 26 swine randomly divided into controls (n = 6) and intracranial inoculates (n = 20). CWD inoculum was a pooled 10% (w/v) homogenate derived from three white-tailed deer clinically ill with CWD from three different sources (elk, white-tailed deer, mule deer) and was given by a single intracranial injection of 0.75 ml. Necropsies were done on ten animals at six months post inoculation (PI), at approximately the time the pigs were expected to reach market weight. Additional pigs have been necropsied due to intercurrent disease (primarily lameness) over the course of the study (29–64 months). Samples collected at necropsy were examined for spongiform change after routine staining (hematoxylin and eosin) and for immunoreactivity to prion protein (PrPSc) by immunohistochemistry. Further, brain samples from at least two regions were tested by western blot. No results suggestive of spongiform encephalopathy were obtained from animals necropsied at six months PI, but positive results after an incubation period of only six months would be uncharacteristic. A single animal was positive for CWD by IHC and WB at 64 months PI. Two inoculated pigs and one control pig remain alive, so it is not possible to determine the attack rate of CWD in swine at this time. However, lack of positive results in pigs necropsied at 29–56 months PI and the long incubation of the single positive case suggest that swine are unlikely to be affected by CWD if inoculated by a natural route.

PO-041: Susceptibility of domestic cats to CWD infection

Amy Nalls, Jeanette Hayes-Klug, Kelly Anderson, Davis Seelig, Kevin Carnes, Susan Kraft, Edward Hoover, Candace Mathiason

Colorado State University; Fort Collins, CO USA

Domestic and non-domestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE); very likely due to consumption of bovine spongiform encephalopathy (BSE) contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of domestic cats to CWD infection experimentally. Groups of n = 5 cats each were inoculated either intracerebrally (IC) or orally (PO) with CWD-infected deer brain homogenate.

Between 40 and 43 months two IC-inoculated cats developed slowly progressive symptoms including weight loss, anorexia, polydipsia, patterned motor behaviors, and ataxia”’ultimately mandating euthanasia. PrPCWD was detected in the brains of these animals by western blot, immunohistochemistry (IHC), and quaking-induced conversion (RT-QuIC) assays. No clinical signs of TSE were detected in the remaining primary passage cats at 86 months pi. Feline-adapted CWD (FelCWD) was sub-passaged into groups (n = 4 or 5) of cats by IC, PO, and IP/SQ routes.

All 5 IC inoculated cats developed symptoms of disease 20–24 months pi (approximately half the incubation period of primary passage). Additional symptoms in these animals included increasing aggressiveness and hyper responsiveness. FelCWD was demonstrated in the brains of all the affected cats by western blot and IHC. Currently, 3 of 4 IP/SQ, and 1 of 4 PO inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. Magnetic resonance imaging (MRI) has been performed on 11 cats (6 clinically ill, 2 asymptomatic, and 3 age-matched negative controls). Abnormalities were detected in 4 of 6 clinically ill cats and included multifocal signal changes consistent with inflammation, ventricular size increases, more prominent sulci, and white matter tract cavitation.

These results demonstrate that CWD can be transmitted and adapted to the domestic cat, and raise the potential for cervid-to-feline transmission in nature.

PO-055: Transgenetic modeling of the CWD species barrier to humans

Eri Saijo,1 Sehun Kim,2 Claudio Soto,3 Glenn Telling2 1University of Kentucky College of Medicine; Fort Collins, CO USA; 2Department of Microbiology, Immunology and Pathology; Colorado State University; Fort Collins, CO USA ; 3Department of Neurology; University of Texas Houston Medical School; Houston, TX USA

Our recent studies raise significant concerns about the zoonotic potential of CWD. First, we showed that tissues consumed by humans derived from deer or elk with CWD, including skeletal muscle and antler velvet, harbor infectious prions. In other studies, cervid PrPSc converted human PrPC after CWD prions were stabilized by successive passages in vitro or in vivo.

We also identified at least two distinct strains of CWD, referred to as CWD1 and CWD2, the host-range properties of which are currently undefined. Other studies showed that codon 129, and the corresponding 132 residue in elk, significantly influenced the transmission of BSE and CWD prions respectively in transgenic (Tg) mouse models.

We inoculated Tg mice expressing human PrP encoding either methionine (M) or valine (V) at codon 129 with deer or elk CWD prions that previously produced disease in Tg mice expressing deer PrP with characteristics typical of CWD1 and CWD2 strains, as well as CWD prions that had been passaged multiple times in Tg mice expressing deer PrP.

While most Tg mice remained free of signs of prion disease for >260 days, small numbers of inoculated mice developed multiple, progressive neurological signs, that were consistent with prion disease. However, examination of brain materials from diseased mice failed to confirm the presence of protease-resistant human PrP. These preliminary results are consistent with a significant species barrier in humans to these CWD strains, and indicate that the 129 polymorphism does not modulate susceptibility.

PO-056: PrPCWD profiling of white-tailed deer (Odocoileus virginianus) with different Prnp genotypes following experimental oral infection

Camilo Duque Velasquez,1 Allen Herbst,1 Chad Johnson,2 Judd Aiken,1 Debbie McKenzie1 1Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, AB Canada; 2Department of Soil Science; University of Wisconsin; Madison, WI USA

Chronic wasting disease (CWD) affects captive and free-ranging cervid populations in North America and farmed cervids of South Korea. CWD and scrapie are the only prion diseases in which the transmission occurs horizontally. The cervid Prnp gene is polymorphic at various positions. The effect of these changes on infection is influenced by the sequence and structure compatibility between the host and the infectious source. Prion strains have been described virtually in every prion disease and strongly impact disease characteristics (clinical symptoms, neuropathological profiles, incubation periods, species tropism as well as biochemical and biophysical properties of the abnormally folded prion protein). Prion protein sequence differences can result, upon subsequent infection, in the generation of novel strains as documented in sheep scrapie. We have previously shown that Prnp polymorphisms influence susceptibility to CWD in free-ranging white-tailed deer. In CWD-positive deer populations, alleles Q95G96 (wt) were over-represented compared to the H95G96 and Q95S96 alleles. Experimental oral infection of white-tailed deer with known Prnp genotypes (with inoculum from CWDpositive wt/wt deer) confirmed the link between prion protein primary sequence and the progression of disease. Heterozygous interference occurred in varying degrees as evidenced by the difference in the extension of the incubation period as an effect of alleles Q95S96 and H95G96. Interestingly, the biochemical profiles of the wt/Q95S96 and wt/H95G96 isolates resemble the wt/wt, differing only in the amount of protease resistant PrPCWD present suggesting that the wt allele is preferentially converted. PrPCWD profiling revealed differences between deer with at least one wt allele and the H95G96/Q95S96 deer suggesting that this PrPCWD is unique compared to the PrPCWD from wt/wt animals.

PO-057: Host factors influence prion strain adaptation

Crystal Meyerett Reid, Mark Zabel Colorado State University; Fort Collins, CO USA

Background. Chronic Wasting disease (CWD) is one of many prion-mediated diseases known as transmissible spongiform encephalopathies (TSEs). There is ever-increasing biological and biochemical evidence that prion pathogenesis is caused by the conversion of the normal host protein (PrPC) into an abnormal disease causing conformation (PrPRES). How prions encipher heritable strain properties without nucleic acid remains unclear. Previously we have shown that host factors have contributed to the adaptation of an original deer CWD prion strain to transgenic mice.

Materials and Methods. We assessed strain differences using biological and biochemical assays and found that amplified cervid prions and serial-passaged cervid prions were significantly different than that of the original cervid strain. It is possible that prion mutation and adaptation can broaden the host range. Previous reports, however, indicate that there is a strict species barrier preventing CWD infection in wildtype mice.

Results. Here we show the generation of a mouse-adapted strain of CWD upon serial passage into transgenic cervidized mice and then subsequent passage into wildtype mice. All wildtype mice remained non-clinical upon first passage but became completely susceptible after second passage with similar incubation times to those of mice terminally ill from a mouse adapted scrapie strain. Inoculation of our mouse adapted CWD strain back into cervidized mice delayed progression to terminal disease.

Conclusion. We conclude that prion strain adaptation and mutation is highly dependent upon host factors and host encoded PrPC primary sequence. Upon serial passage the adapted prion strain shares more characteristics with prion strains from the new host rather than the original species.

OR-12: Chronic wasting disease transmission and pathogenesis in cervid and non-cervid Species

Edward A. Hoover, Candace K. Mathiason, Nicholas J. Haley, Timothy D. Kurt, Davis M. Seelig, Nathaniel D. Denkers, Amy V. Nalls, Mark D. Zabel, and Glenn C. Telling

Prion Research Program, Department of Microbiology, Immunology, and Pathology; Colorado State University; Fort Collins, CO USA

Since its recognition as a TSE in the late 1970s, chronic wasting disease (CWD) of cervids has been distinguished by its facile spread and is now recognized in 18 states, 2 Canadian provinces, and South Korea. The efficient horizontal spread of CWD reflects a prion/host relationship that facilitates efficient mucosal uptake, peripheral lymphoid amplification, and dissemination by exploiting excretory tissues and their products, helping to establish indirect/environmental and well as direct (e.g., salivary) transmission. Recent studies from our group also support the likelihood of early life mother to offspring and aerosol CWD prion transmission. Studies of cervid CWD exposure by natural routes indicate that incubation period for detection of overt infection, while still uncertain, may be much longer than originally thought.

Several non-cervid species can be infected by CWD experimentally (e.g., ferrets, voles, cats) with consequent species-specific disease phenotypes. The species-adapted prions so generated can be transmitted by mucosal, i.e., more natural, routes. Whether non-cervid species sympatric with deer/elk can be infected in nature, however, remains unknown. In vitro CWD prion amplification studies, in particular sPMCA, can foreshadow in vivo susceptibility and suggest the importance of the PrPC rigid loop region in species barrier permissiveness. Trans-species CWD amplification appears to broaden the host range/strain characteristics of the resultant prions. The origins of CWD remain unknown, however, the existence of multiple CWD prion strains/ quasi-species, the mechanisms of prion shedding/dissemination, and the relationship between sheep scrapie and CWD merit further investigation.

PO-060: Transmission of chronic wasting disease from mother to offspring

Candace Mathiason, Amy Nalls, Stephenie Fullaway, Kelly Anderson, Jeanette Hayes-Klug, Nicholas Haley, Edward Hoover

Colorado State University; Fort Collins, CO USA

To investigate the role mother to offspring transmission plays in chronic wasting disease (CWD) we have developed a cervid model employing the Reeve’s muntjac deer (Muntiacus reevesi). Eight muntjac doe were orally inoculated with CWD and tested PrPCWD lymphoid positive by 4 mo post infection. Twelve fawns were born to these eight CWD-infected doe, 3 were born viable, 6 were born non-viable, and 3 were harvested as fetuses (1 each from first, second or third trimester of pregnancy) from CWDinfected doe euthanized at end-stage disease. The viable fawns have been monitored for CWD infection by immunohistochemistry (IHC) performed on serial tonsil and rectal lymphoid tissue biopsies. One fawn that was IHC PrPCWD positive at 40 d of age is now, at 28 mo of age, showing early clinical signs associated with CWD infection. Moreover, CWD prions have been detected by sPMCA in placenta, brain, spleen and mesenteric lymphoid tissue harvested from 5 full-term non-viable fawns, and in fetal placenta and brain tissue harvested in utero from the second and third trimester fetuses. Additional tissues and pregnancy related fluids from doe and offspring are being analyzed for CWD prions. In summary, using the muntjac deer model we have demonstrated CWD clinical disease in an offspring born to a CWD-infected doe, and in utero transmission of CWD from mother to offspring. These studies provide basis to further investigate the mechanisms of maternal transfer of prions.

PO-081: Chronic wasting disease in the cat— Similarities to feline spongiform encephalopathy (FSE)

Davis Seelig, Amy Nalls, Maryanne Flasik, Victoria Frank, Candace Mathiason, Edward Hoover Colorado State University; Fort Collins, CO USA

Background and Introduction. Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids with an as yet to be fully defined host range. Moreover, the risk that CWD poses to feline predators and scavangers, through crossspecies consumption and subsequent transmission, is unknown. Previous and ongoing studies in our laboratory evaluating the susceptibility of domestic cats (Felis catus) to CWD (Mathiason et. al., NeuroPrion 2011, Nalls et. al., NeuroPrion 2012) have documented the susceptibility of domestic cats to CWD following intracerebral (IC) inoculation. However, many of the pathologic features of feline-adapted CWD, including the neural and systemic patterns of PrPCWD accumulation and neuropathology, remain unknown.

The chief objectives of this work were:

(1) to design a sensitive, enhanced immunohistochemical (E-IHC) protocol for the detection of CWD prions (PrPCWD) in feline tissues;

(2) to document the systemic distribution of PrPCWD in CWD-infected cats through E-IHC;

(3) to utilize single and multiple-label immunostaining and laser scanning confocal microscopy (LSCM) to provide insights into the subcellular patterns of PrPCWD accumulation and neuropathologic features of CWD-infected cats; and

(4) to compare feline CWD to the other known feline TSE Materials and Methods. Periodate-lysine-paraformaldehyde (PLP)-fixed, paraffin-embedded (PLP-PE) from terminal, IC-inoculated (n = 9) and sham-inoculated (n = 2), 1st and 2nd passage, CWD-infected cats were examined by E-IHC for the presence of PrPCWD and its association with markers of cell phenotype and organelles.

Results. The most sensitive E-IHC technique for the detection of PrPCWD in feline tissues incorporated a combination of slide pretreatment with proteinase-K (PK) in concert with tyramide signal amplification (TSA). With this protocol, we identified PrPCWD deposits throughout the CNS, which, in the 1st passage cats was primarily restricted to the obex, but increased in distribution and severity upon 2nd passage to include a number of midbrain nuclei, cortical gray matter, the thalamus and hypothalamus, and the hippocampus. Peripheral PrPCWD deposits were detected only in the 2nd passage cats, and included the enteric nervous system, the Peyer’s patches, and the retropharyngeal and mesenteric lymph nodes. PrPCWD was not detected in the sham-inoculated cats.

Moreover, using multi-label analysis, intracellular PrPCWD aggregates were seen in association with neurofilament heavy chain (NFH)-positive neurons and GFAP-positive astrocytes. In addition, large aggregates of intracellular PrPCWD were identified within LAMP1-positive lysosomes.

Conclusions. Feline PrPCWD is present in CNS neurons, astrocytes and LAMP-1-positive lysosomes. The morphologic overlap between the PrPCWD deposits in feline CWD and BSE-origin feline spongiform encephalopathy (FSE), implicates the importance of the host as a key determinant in the development of prion neuropathology and suggest a signature for detection of potential spontaneous feline prion disease.

PO-099: Estimating the risk of CWD transmission to humans—An interim report of a comprehensive study in non-human primates

Ann-Christin Schmaedicke DPZ; Goettingen, Germany

Chronic Wasting Disease (CWD) is a transmissible prion disease that occurs primarily among North American cervid species. CWD has emerged as a prion disease in captive as well as freeranging cervids with rising incidence. Continuous consumption of cervid-derived products in conjunction with increasing CWD prevalence suggests a risk for human exposure to CWD prions.

Although surveillance data collected in the North American population did not provide epidemiological evidence for CWD transmissions to humans it remains unclear whether a biological risk for such transmissions exists or can be excluded. In order to gauge the potential transmissibility of CWD to humans, a comprehensive CWD risk assessment in cynomolgus macaques has been initiated. To test for the CWD interspecies transmission to humans, we used a primate species previously shown to mimic the BSE susceptibility of humans. In addition, this species is phylogenetically close to humans with a homologous amino acid sequence of the prion protein. When we assessed the genotype of macaque PrP, the homozygosity for methionine at codon 129 was confirmed in all animals.

The zoonotic potential of CWD is evaluated by challenging groups of animals via different inoculation routes. General transmissibility of CWD to macaques is tested by intracerebral (i.c.) inoculation of brain homogenate from CWD-infected white-tailed deer (WTD) and elk. This was either done by direct injection of 10 mg CWDWTD to two animals (> 880 dpi) or by surgical implantation of CWD-WTD or CWD-elk contaminated steel wires in two animals (> 750 dpi) or three animals (> 400 dpi), respectively.

To address the risk of hunters while field dressing carcasses, we inoculated two animals with CWD-WTD by dermal scarification (> 920 dpi). To simulate human consumption of CWDinfected food products, we orally challenged three animals with 10 g CWD-WTD brain (> 820 dpi) and three animals with 3 kg CWD muscle tissue from different cervid species (> 950 dpi) by repeated feeding. Mock-inoculated macaques are co-housed in the same cage allowing exposure to saliva and faeces of CWDinoculated macaques.

To date, all inoculated animals remain asymptomatic. Cerebrospinal fluid and blood from all animals were and are going to be collected bimonthly, processed and stored in a repository. Analysis of these samples using ultrasensitive prion detection methods has been initiated. Oral challenge of 50 mg BSE to cynomolgus macaques can lead to fatal disease only after more than 5 y of incubation time (1952 dpi).

Thus, to estimate whether CWD could be transmitted to macaques or not, we assume that observation of challenged animals would be necessary for at least 8–10 y.

PO-248: TSE infectivity survives burial for five years with little reduction in titer

Allister Smith, Robert Somerville, Karen Fernie The Roslin Institute and R(D)SVS; University of Edinburgh; Edinburgh, UK

BSE infected animals, BSE-contaminated materials and other sources of TSE (prion) infection, such as carcasses from scrapie infected sheep, CWD infected deer and cadavers of individuals infected with CJD may all end up in the environment through burial or other methods of disposal. They may continue to act as a reservoir of TSE infectivity if cattle or other susceptible animals were to be exposed to these sources in the future. In order to address these concerns, we performed two large scale demonstration experiments under field conditions which were designed to mimic some of the ways by which TSE infected materials may have been disposed of. The project examined the fate of TSE infectivity over a period of five years in two scenarios; when the infectivity was contained within bovine heads and when the infectivity was buried without any containment. Two soil types were compared: a sandy loam and a clay loam. We used the 301V TSE strain which was derived by serial passage of BSE in VM mice.

TSE infectivity was recovered from all the heads exhumed annually for five years from both types of soil, with little reduction in the amount of infectivity throughout the period of the experiment. Small amounts of infectivity were found in the soil immediately surrounding the heads, but not in samples remote from them. Similarly there was no evidence of significant lateral movement of infectivity from the buried bolus. However large amounts of TSE infectivity were recovered at the site of burial of both boluses. There was limited vertical upward movement of infectivity from the bolus buried in clay soil and downward movement from the bolus buried in sandy soil.

Now that these experiments are completed we conclude that TSE infectivity is likely to survive burial for long periods of time with minimal loss of infectivity and restricted movement from the site of burial. These experiments emphasize that the environment is a viable reservoir for retaining large quantities of TSE infectivity, and reinforce the importance of risk assessment when disposing of this type of infectious material.

Chronic Wasting Disease Susceptibility of Four North American Rodents

Chad J. Johnson1*, Jay R. Schneider2, Christopher J. Johnson2, Natalie A. Mickelsen2, Julia A. Langenberg3, Philip N. Bochsler4, Delwyn P. Keane4, Daniel J. Barr4, and Dennis M. Heisey2 1University of Wisconsin School of Veterinary Medicine, Department of Comparative Biosciences, 1656 Linden Drive, Madison WI 53706, USA 2US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison WI 53711, USA 3Wisconsin Department of Natural Resources, 101 South Webster Street, Madison WI 53703, USA 4Wisconsin Veterinary Diagnostic Lab, 445 Easterday Lane, Madison WI 53706, USA *Corresponding author email:

We intracerebrally challenged four species of native North American rodents that inhabit locations undergoing cervid chronic wasting disease (CWD) epidemics. The species were: deer mice (Peromyscus maniculatus), white-footed mice (P. leucopus), meadow voles (Microtus pennsylvanicus), and red-backed voles (Myodes gapperi). The inocula were prepared from the brains of hunter-harvested white-tailed deer from Wisconsin that tested positive for CWD. Meadow voles proved to be most susceptible, with a median incubation period of 272 days. Immunoblotting and immunohistochemistry confirmed the presence of PrPd in the brains of all challenged meadow voles. Subsequent passages in meadow voles lead to a significant reduction in incubation period. The disease progression in red-backed voles, which are very closely related to the European bank vole (M. glareolus) which have been demonstrated to be sensitive to a number of TSEs, was slower than in meadow voles with a median incubation period of 351 days. We sequenced the meadow vole and red-backed vole Prnp genes and found three amino acid (AA) differences outside of the signal and GPI anchor sequences. Of these differences (T56-, G90S, S170N; read-backed vole:meadow vole), S170N is particularly intriguing due its postulated involvement in "rigid loop" structure and CWD susceptibility. Deer mice did not exhibit disease signs until nearly 1.5 years post-inoculation, but appear to be exhibiting a high degree of disease penetrance. White-footed mice have an even longer incubation period but are also showing high penetrance. Second passage experiments show significant shortening of incubation periods. Meadow voles in particular appear to be interesting lab models for CWD. These rodents scavenge carrion, and are an important food source for many predator species. Furthermore, these rodents enter human and domestic livestock food chains by accidental inclusion in grain and forage. Further investigation of these species as potential hosts, bridge species, and reservoirs of CWD is required.

please see ;

Title: Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation


Hamir, Amirali Kunkle, Robert Cutlip, Randall - ARS RETIRED Miller, Janice - ARS RETIRED Williams, Elizabeth - UNIV OF WYOMING, LARAMIE Richt, Juergen

Submitted to: Journal of Veterinary Diagnostic Investigation Publication Type: Peer Reviewed Journal Publication Acceptance Date: June 20, 2006 Publication Date: November 1, 2006 Citation: Hamir, A.N., Kunkle, R.A., Cutlip, R.C., Miller, J.M., Williams, E.S., Richt, J.A. 2006. Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation. Journal of Veterinary Diagnostic Investigation. 18(6):558-565.

Interpretive Summary: Chronic wasting disease (CWD) has been identified in captive and free ranging deer and elk since 1967. To determine the transmissibility of CWD to sheep and to provide information about the disease and tests for detection of CWD in sheep, 8 lambs were inoculated with brain suspension from mule deer naturally affected with CWD. Two other lambs were kept as controls. Only 1 sheep developed clinical disease at 35 months after inoculation. The study was terminated at 72 months after the inoculation. At that time one other sheep was found to be positive for the disease. It is proposed that the host's genetic makeup may play a role in transmission of the disease to domestic sheep. Impact. This is the first study which shows that it is possible to transmit CWD to a small number of sheep. Technical Abstract: Chronic wasting disease (CWD) has been identified in captive and free-ranging cervids since 1967. To determine the transmissibility of CWD to sheep and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of CWD in sheep, 8 Suffolk lambs (4 QQ and 4 QR at codon 171 of prion protein (PRNP) gene) were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD (CWD**md). Two other lambs (1 QQ and 1 QR at codon 171 of PRNP gene) were kept as non-inoculated controls. Within 36 months post inoculation (MPI), 2 animals became recumbent and were euthanized. However, only 1 sheep (euthanized at 35 MPI) had shown clinical signs that were consistent with those of scrapie. Microscopic lesions of spongiform encephalopathy (SE) were seen in this sheep and its tissues were positive for the abnormal prion protein (PrPres) by immunohistochemistry and Western blot. Retrospective examination of the PRNP genotype of this animal revealed that it was heterozygous (AV) at codon 136. In the next 24 months, 3 other sheep were euthanized because of conditions unrelated to TSE. The remaining 3 sheep remained non-clinical at the termination of the study (72 MPI) and were euthanized at that time. One of these 3 revealed SE and its tissues were positive for PrPres. These findings demonstrate that it is possible to transmit CWD**md agent to sheep via the intracerebral route. However, the host genotype may play a significant part in successful transmission and incubation period of this agent.

Chronic wasting disease: Fingerprinting the culprit in risk assessments

Volume 6, Issue 1 January/February/March 2012 Pages 17 - 22

Keywords: Fourier transform-infrared (FT-IR) spectroscopy, chronic wasting disease (CWD), prion, prion protein (PrP), prion typing, protein misfolding cyclic amplification (PMCA), risk assessment, seeding activity, strains, transmissible spongiform encephalopathies (TSE)

Authors: Martin L. Daus and Michael Beekes View affiliations Hide affiliations Martin L. Daus

P24 -Transmissible Spongiform Encephalopathies;

Robert Koch-Institut; Berlin, Germany Michael Beekes Corresponding author: P24 -Transmissible Spongiform Encephalopathies; Robert Koch-Institut; Berlin, Germany

Abstract: Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains, and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose.


Exposure of humans to CWD prions

Chronic wasting disease is a TSE in white-tailed deer, mule deer, Rocky Mountain elk and moose. Over the past years this disease has shown a sustained spread in captive as well as free-ranging cervids in North America.6,7 The increasingly frequent and widespread 5 occurrence of affected animals is likely to augment the exposure of humans to the CWD agent. Prion infectivity or TSE-associated prion protein have been detected in the central and peripheral nervous system, in a variety of lymphoid tissues as well as in heart muscle, blood, saliva, feces and urine of CWD-infected cervids7. Also, infectious CWD agent was found in antler velvet of elk and in skeletal muscles of mule deer with chronic wasting disease.8,9 Thus, particularly persons processing cervid carcasses, users of medicinal products made from antler velvet and consumers of venison may be exposed to an elevated risk for contamination with CWD prions.

Recently, PrPTSE and its proteinaceous seeding activity could be directly demonstrated, for the first time, in skeletal muscles of CWD-infected cervids.10 The animals examined in this study were farmed and free-ranging WTD for which no clinical signs of CWD had been recognized. However, they had been officially confirmed positive for CWD based on the detection of PrPTSE in brain tissue or lymph nodes and were thus apparently in a state of pre or subclinical infection. Muscles from such clinically inconspicuous carrier animals appear more likely to enter the human food chain than meat from cervids that show symptoms of CWD. Whether this may provide a relevant mode for the inadvertent foodborne transmission of CWD prions is still unclear. Yet, the presence and seeding activity of PrPTSE in skeletal muscles of pre- or subclinically infected WTD reinforced the need to comprehensively assess whether humans are susceptible to zoonotic CWD infections.


Transmissibility to humans

The current state of epidemiological research suggests a rather robust barrier for the transmission of CWD to humans. Particularly, the surveillance of human prion diseases in areas with a long history of endemic CWD such as Colorado and Wyoming did not reveal evidence for zoonotic transmissions of the disease to cervid hunters or consumers of meat from elk and deer.6,11 However, as discussed by Belay et al.,6 the intensity of human exposure to CWD prions may increase due to a further spread and rising prevalence of the disease in cervids. Therefore, and with the generally long latency periods of human prion diseases in mind, previous epidemiological findings cannot be readily extrapolated. Until recently, experimental studies that pursued biochemical approaches or used transgenic mice to ascertain the susceptibility of humans to CWD infections consistently seemed to corroborate current epidemiological findings: CWD-infected cervid brain tissue did not seed the conversion of PrPC 133 into PrPres in PMCA assays using brain homogenate from macaques or transgenic mice expressing human PrPC as test substrate12 , and transgenic mice overexpressing human PrPC were resistant to infection after intracerebral challenge with CWD prions from mule deer.13 However, a study published by Barria et al.14 in March 2011 found that cervid PrPTSE can seed the conversion of human PrPC into PrPres by PMCA when the CWD agent has been previously passaged in vitro or in vivo. Specifically, this was demonstrated for CWD prions from naturally affected mule deer either passaged by serial PMCA using deer PrPC as conversion substrate or in transgenic mice expressing cervid PrPC. The authors of this study pointed out that CWD prions may undergo a gradual process of change and adaptation via successive passages in the cervid population. They concluded that the reported findings, if corroborated by infectivity assays, may imply “that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading”.


Volume 18, Number 3—March 2012

Samuel E. Saunders1, Shannon L. Bartelt-Hunt, and Jason C. Bartz

Author affiliations: University of Nebraska-Lincoln, Omaha, Nebraska, USA (S.E. Saunders, S.L. Bartelt-Hunt); Creighton University, Omaha (J.C. Bartz)


Occurrence, Transmission, and Zoonotic Potential of Chronic Wasting Disease


Most epidemiologic studies and experimental work have suggested that the potential for CWD transmission to humans is low, and such transmission has not been documented through ongoing surveillance (2,3). In vitro prion replication assays report a relatively low efficiency of CWD PrPSc-directed conversion of human PrPc to PrPSc (30), and transgenic mice overexpressing human PrPc are resistant to CWD infection (31); these findings indicate low zoonotic potential. However, squirrel monkeys are susceptible to CWD by intracerebral and oral inoculation (32). Cynomolgus macaques, which are evolutionarily closer to humans than squirrel monkeys, are resistant to CWD infection (32). Regardless, the finding that a primate is orally susceptible to CWD is of concern.


Intraspecies and interspecies passage of the CWD agent may also increase the risk for zoonotic CWD transmission. The CWD prion agent is undergoing serial passage naturally as the disease continues to emerge. In vitro and in vivo intraspecies transmission of the CWD agent yields PrPSc with an increased capacity to convert human PrPc to PrPSc (30). Interspecies prion transmission can alter CWD host range (38) and yield multiple novel prion strains (3,28). The potential for interspecies CWD transmission (by cohabitating mammals) will only increase as the disease spreads and CWD prions continue to be shed into the environment. This environmental passage itself may alter CWD prions or exert selective pressures on CWD strain mixtures by interactions with soil, which are known to vary with prion strain (25), or exposure to environmental or gut degradation.

Given that prion disease in humans can be difficult to diagnose and the asymptomatic incubation period can last decades, continued research, epidemiologic surveillance, and caution in handling risky material remain prudent as CWD continues to spread and the opportunity for interspecies transmission increases. Otherwise, similar to what occurred in the United Kingdom after detection of variant CJD and its subsequent link to BSE, years of prevention could be lost if zoonotic transmission of CWD is subsequently identified,...



Volume 18, Number 3—March 2012 Synopsis Occurrence, Transmission, and Zoonotic Potential of Chronic Wasting Disease


Prevalence and Surveillance

Originally recognized only in southeastern Wyoming and northeastern Colorado, USA, CWD was reported in Canada in 1996 and Wisconsin in 2001 and continues to be identified in new geographic locations (Figure 1, panel A). CWD has been identified in free-ranging cervids in 15 US states and 2 Canadian provinces and in ≈100 captive herds in 15 states and provinces and in South Korea (Figure 1, panel B).


CWD surveillance programs are now in place in almost all US states and Canadian provinces (Figure 2, panel A). More than 1,060,000 free-ranging cervids have reportedly been tested for CWD (Figure 2, panel B) and ≈6,000 cases have been identified (Figure 2, panel C) according to data from state and provincial wildlife agencies.


Testing of captive cervids is routine in most states and provinces, but varies considerably in scope from mandatory testing of all dead animals to voluntary herd certification programs or mandatory testing of only animals suspected of dying of CWD.


Long-term effects of CWD on cervid populations and ecosystems remain unclear as the disease continues to spread and prevalence increases. In captive herds, CWD might persist at high levels and lead to complete herd destruction in the absence of human culling. Epidemiologic modeling suggests the disease could have severe effects on free-ranging deer populations, depending on hunting policies and environmental persistence (8,9). CWD has been associated with large decreases in free-ranging mule deer populations in an area of high CWD prevalence (Boulder, Colorado, USA) (5). In addition, CWD-infected deer are selectively preyed upon by mountain lions (5), and may also be more vulnerable to vehicle collisions (10). Long-term effects of the disease may vary considerably geographically, not only because of local hunting policies, predator populations, and human density (e.g., vehicular collisions) but also because of local environmental factors such as soil type (11) and local cervid population factors, such as genetics and movement patterns (S.E. Saunders, unpub. data).


Controlling the spread of CWD, especially by human action, is a more attainable goal than eradication. Human movement of cervids has likely led to spread of CWD in facilities for captive animals, which has most likely contributed to establishment of new disease foci in free-ranging populations (Figure 1, panel A). Thus, restrictions on human movement of cervids from disease-endemic areas or herds continue to be warranted. Anthropogenic factors that increase cervid congregation such as baiting and feeding should also be restricted to reduce CWD transmission. Appropriate disposal of carcasses of animals with suspected CWD is necessary to limit environmental contamination (20), and attractive onsite disposal options such as composting and burial require further investigation to determine contamination risks. The best options for lowering the risk for recurrence in facilities for captive animals with outbreaks are complete depopulation, stringent exclusion of free-ranging cervids, and disinfection of all exposed surfaces. However, even the most extensive decontamination measures may not be sufficient to eliminate the risk for disease recurrence (20; S.E. Saunders et al. unpub. data)

full text ;




Zoonotic Potential of CWD: Experimental Transmissions to Non-Human Primates

Emmanuel Comoy,1,† Valérie Durand,1 Evelyne Correia,1 Aru Balachandran,2 Jürgen Richt,3 Vincent Beringue,4 Juan-Maria Torres,5 Paul Brown,1 Bob Hills6 and Jean-Philippe Deslys1

1Atomic Energy Commission; Fontenay-aux-Roses, France; 2Canadian Food Inspection Agency; Ottawa, ON Canada; 3Kansas State University; Manhattan, KS USA; 4INRA; Jouy-en-Josas, France; 5INIA; Madrid, Spain; 6Health Canada; Ottawa, ON Canada

†Presenting author; Email:

The constant increase of chronic wasting disease (CWD) incidence in North America raises a question about their zoonotic potential. A recent publication showed their transmissibility to new-world monkeys, but no transmission to old-world monkeys, which are phylogenetically closer to humans, has so far been reported. Moreover, several studies have failed to transmit CWD to transgenic mice overexpressing human PrP. Bovine spongiform encephalopathy (BSE) is the only animal prion disease for which a zoonotic potential has been proven. We described the transmission of the atypical BSE-L strain of BSE to cynomolgus monkeys, suggesting a weak cattle-to-primate species barrier. We observed the same phenomenon with a cattleadapted strain of TME (Transmissible Mink Encephalopathy). Since cattle experimentally exposed to CWD strains have also developed spongiform encephalopathies, we inoculated brain tissue from CWD-infected cattle to three cynomolgus macaques as well as to transgenic mice overexpressing bovine or human PrP. Since CWD prion strains are highly lymphotropic, suggesting an adaptation of these agents after peripheral exposure, a parallel set of four monkeys was inoculated with CWD-infected cervid brains using the oral route. Nearly four years post-exposure, monkeys exposed to CWD-related prion strains remain asymptomatic. In contrast, bovinized and humanized transgenic mice showed signs of infection, suggesting that CWD-related prion strains may be capable of crossing the cattle-to-primate species barrier. Comparisons with transmission results and incubation periods obtained after exposure to other cattle prion strains (c-BSE, BSE-L, BSE-H and cattle-adapted TME) will also be presented, in order to evaluate the respective risks of each strain.


Pathological Prion Protein (PrPTSE) in Skeletal Muscles of Farmed and Free Ranging White-Tailed Deer Infected with Chronic Wasting Disease

Martin L. Daus,1,† Johanna Breyer,2 Katjs Wagenfuehr,1 Wiebke Wemheuer,2 Achim Thomzig,1 Walter Schulz-Schaeffer2 and Michael Beekes1 1Robert Koch Institut; P24 TSE; Berlin, Germany; 2Department of Neuropathology, Prion and Dementia Research Unit, University Medical Center Göttingen; Göttingen, Germany

†Presenting author; Email:

Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE) occurring in cervids in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report a direct biochemical demonstration of pathological prion protein PrPTSE and of PrPTSE-associated seeding activity in skeletal muscles of CWD-infected cervids. The presence of PrPTSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrPTSE was revealed by protein misfolding cyclic amplification (PMCA). The concentration of PrPTSE in skeletal muscles of CWD-infected WTD was estimated to be approximately 2000- to 10000-fold lower than in brain tissue. Tissue-blot-analyses revealed that PrPTSE was located in muscle- associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrPTSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans.

now, let’s see what the authors said about this casual link, personal communications years ago. see where it is stated NO STRONG evidence. so, does this mean there IS casual evidence ????

“Our conclusion stating that we found no strong evidence of CWD transmission to humans”

From: TSS (


Date: September 30, 2002 at 7:06 am PST

From: "Belay, Ermias"


Cc: "Race, Richard (NIH)" ; ; "Belay, Ermias"

Sent: Monday, September 30, 2002 9:22 AM


Dear Sir/Madam,

In the Archives of Neurology you quoted (the abstract of which was attached to your email), we did not say CWD in humans will present like variant CJD.

That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091). Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.

Ermias Belay, M.D. Centers for Disease Control and Prevention

-----Original Message-----


Sent: Sunday, September 29, 2002 10:15 AM

To:;; ebb8@CDC.GOV


Sunday, November 10, 2002 6:26 PM ......snip........end..............TSS

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

Sigurdson CJ.


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


full text ;

CWD ongoing experiment on humans, long term $$$

Monday, November 14, 2011

WYOMING Creutzfeldt Jakob Disease, CWD, TSE, PRION REPORTING 2011

Wednesday, November 16, 2011

Wisconsin Creutzfeldt Jakob Disease, CWD, TSE, PRION REPORTING 2011

Sunday, November 13, 2011



Thursday, May 26, 2011

Travel History, Hunting, and Venison Consumption Related to Prion Disease Exposure, 2006-2007 FoodNet Population Survey

Journal of the American Dietetic Association Volume 111, Issue 6 , Pages 858-863, June 2011.

NOR IS THE FDA recalling this CWD positive elk meat for the well being of the dead elk ;

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


October 1994

Mr R.N. Elmhirst Chairman British Deer Farmers Association Holly Lodge Spencers Lane BerksWell Coventry CV7 7BZ

Dear Mr Elmhirst,


Thank you for your recent letter concerning the publication of the third annual report from the CJD Surveillance Unit. I am sorry that you are dissatisfied with the way in which this report was published.

The Surveillance Unit is a completely independant outside body and the Department of Health is committed to publishing their reports as soon as they become available. In the circumstances it is not the practice to circulate the report for comment since the findings of the report would not be amended. In future we can ensure that the British Deer Farmers Association receives a copy of the report in advance of publication.

The Chief Medical Officer has undertaken to keep the public fully informed of the results of any research in respect of CJD. This report was entirely the work of the unit and was produced completely independantly of the the Department.

The statistical results reqarding the consumption of venison was put into perspective in the body of the report and was not mentioned at all in the press release. Media attention regarding this report was low key but gave a realistic presentation of the statistical findings of the Unit. This approach to publication was successful in that consumption of venison was highlighted only once by the media ie. in the News at one television proqramme.

I believe that a further statement about the report, or indeed statistical links between CJD and consumption of venison, would increase, and quite possibly give damaging credence, to the whole issue. From the low key media reports of which I am aware it seems unlikely that venison consumption will suffer adversely, if at all.

please see ;

Wednesday, May 30, 2012

PO-248: TSE infectivity survives burial for five years with little reduction in titer

Friday, February 08, 2013

*** Behavior of Prions in the Environment: Implications for Prion Biology

Friday, November 09, 2012

*** Chronic Wasting Disease CWD in cervidae and transmission to other species

Sunday, November 11, 2012

*** Susceptibilities of Nonhuman Primates to Chronic Wasting Disease November 2012

Friday, December 14, 2012

Susceptibility Chronic Wasting Disease (CWD) in wild cervids to Humans 2005 - December 14, 2012


Volume 3, Number 8 01 August 2003


Tracking spongiform encephalopathies in North America

Xavier Bosch

My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost my mom to hvCJD (Heidenhain variant CJD) and have been searching for answers ever since. What I have found is that we have not been told the truth. CWD in deer and elk is a small portion of a much bigger problem. 49-year-old Singeltary is one of a number of people who have remained largely unsatisfied after being told that a close relative died from a rapidly progressive dementia compatible with spontaneous Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of documents on transmissible spongiform encephalopathies (TSE) and realised that if Britons could get variant CJD from bovine spongiform encephalopathy (BSE), Americans might get a similar disorder from chronic wasting disease (CWD)the relative of mad cow disease seen among deer and elk in the USA. Although his feverish search did not lead him to the smoking gun linking CWD to a similar disease in North American people, it did uncover a largely disappointing situation. Singeltary was greatly demoralised at the few attempts to monitor the occurrence of CJD and CWD in the USA. Only a few states have made CJD reportable. Human and animal TSEs should be reportable nationwide and internationally, he complained in a letter to the Journal of the American Medical Association (JAMA 2003; 285: 733). I hope that the CDC does not continue to expect us to still believe that the 85% plus of all CJD cases which are sporadic are all spontaneous, without route or source. Until recently, CWD was thought to be confined to the wild in a small region in Colorado. But since early 2002, it has been reported in other areas, including Wisconsin, South Dakota, and the Canadian province of Saskatchewan. Indeed, the occurrence of CWD in states that were not endemic previously increased concern about a widespread outbreak and possible transmission to people and cattle. To date, experimental studies have proven that the CWD agent can be transmitted to cattle by intracerebral inoculation and that it can cross the mucous membranes of the digestive tract to initiate infection in lymphoid tissue before invasion of the central nervous system. Yet the plausibility of CWD spreading to people has remained elusive. Getting data on TSEs in the USA from the government is like pulling teeth, Singeltary argues. You get it when they want you to have it, and only what they want you to have.


-----Original Message-----

Sent: Wednesday, March 06, 2013 8:19 AM


Dear Mr. Singeltary

Thank you for your message and comments. Please be informed that I have forwarded your suggestions to the responsible unit.

With kind regards Joanna Kniaz-Hawrot


Unit 04 - Communication Health & Consumers Directorate-General European Commission

-----Original Message-----

From: []

Sent: Friday, February 22, 2013 5:55 PM



Title: Mr.

First name : Terry Last name : Singeltary

Country of residence: USA Category :

Citizen Address: p.o. box 42

Postcode : 77518

City : Bacliff Country: Texas

Theme : Animal Health Subject : USA BSE GBR ASSESSMENT FRAUD Question (max 2000chars) :

I urge you to NOT to list the USA as BSE GBR II, and to immediately list the USA BSE GBR risk assessment to BSE GBR IV, for the following reasons, but for one, the USA has more documented TSE prion disease in wild animals and livestock animals than any other country in the world, excluding zoo animals, cats, and dogs, because the USA is NOT looking, all of which at one time have been fed back to food producing livestock animals, and to humans. ...the clock is ticking.

thank you,

with kindest regards, terry

please see why, and full text source refererence here ;

Wednesday, February 20, 2013

World Organization for Animal Health Recommends United States' BSE Risk Status Be Upgraded

Statement from Agriculture Secretary Tom Vilsack:

Thursday, February 14, 2013

The Many Faces of Mad Cow Disease Bovine Spongiform Encephalopathy BSE and TSE prion disease

Thursday, February 21, 2013

National Prion Disease Pathology Surveillance Center Cases Examined January 16, 2013


Reply language : English


kind regards,


Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518


Post a Comment

Subscribe to Post Comments [Atom]

<< Home