CWD TISSUE INFECTIVITY brain, lymph node, blood, urine, feces, antler velvet and muscle

2007 Annual Report

1a.Objectives (from AD-416) To identify and differentiate typical and atypical case samples of CWD and Scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice.

1b.Approach (from AD-416) Tissue samples from deer, elk, sheep and goats with Transmissible Spongiform Encephalopathy (TSE) will be administered to mice by intracerebral injection. Multiple tissue types will be included, such as samples of brain, lymph node, blood, urine, feces, antler velvet and muscle. Mouse models used as recipient hosts will include both preexisting and recently created transgenic and inbred mouse lines. Recipient mouse phenotype will be evaluated by measuring clinical response, population disease rate, incubation time, and pathologic profile within the central nervous system (CNS). Pathologic profile of CNS lesion foci is assessed by evaluating anatomic localization, spongiform change, astrocytic gliosis, and deposition of protease resistant prion protein. BSL-1; 9-4-06. Documents SCA with U. of WA.

3.Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Washington. Additional details of research can be found in the report for the parent project 5348-32000-026-00D Transmissible Spongiform Encephalopathies: the role of genetics, strain variation, and environmental contamination in disease control. The purpose of this new SCA is to identify and differentiate typical and atypical case samples of CWD and scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice. There will be weekly interactions between the ADODR, ADRU scientists and University of Washington collaborators through personnel visits, conference calls and emails.


http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=411895&fy=2007



Thursday, July 24, 2008

Prion diseases are efficiently transmitted by blood transfusion in sheep

Submitted April 18, 2008 Accepted June 28, 2008


http://vcjdblood.blogspot.com/2008/07/prion-diseases-are-efficiently.html



DOI: 10.3201/eid1409.080259 Suggested citation for this article: Gregori L, Kovacs GG, Alexeeva I, Budka H, Rohwer RG.

Excretion of transmissible spongiform encephalopathy infectivity in urine.

Emerg Infect Dis. 2008 Sep; [Epub ahead of print]

Excretion of Transmissible Spongiform Encephalopathy Infectivity in Urine Luisa Gregori, Gabor G. Kovacs, Irina Alexeeva, Herbert Budka, and Robert G. Rohwer Author affiliations: Veterans Affairs Medical Center, Baltimore, Maryland, USA (L. Gregori, I. Alexeeva, R.G. Rohwer); University of Maryland, Baltimore (L. Gregori, R.G. Rohwer); and Medical University of Vienna, Vienna, Austria (G.G. Kovacs, H. Budka) The route of transmission of most naturally acquired transmissible spongiform encephalopathy (TSE) infections remains speculative. To investigate urine as a potential source of TSE exposure, we used a sensitive method for detection and quantitation of TSE infectivity. Pooled urine collected from 22 hamsters showing clinical signs of 263K scrapie contained 3.8 ± 0.9 infectious doses/mL of infectivity. Titration of homogenates of kidneys and urinary bladders from the same animals gave concentrations 20,000-fold greater. Histologic and immunohistochemical examination of these same tissues showed no indications of inflammatory or other pathologic changes except for occasional deposits of diseaseassociated prion protein in kidneys. Although the source of TSE infectivity in urine remains unresolved, these results establish that TSE infectivity is excreted in urine and may thereby play a role in the horizontal transmission of natural TSEs. The results also indicate potential risk for TSE transmission from human urine-derived hormones and other medicines.

snip...

Discussion Anticipating that the titer of scrapie infectivity in excreted urine would be low, we measured concentration by using limiting dilution titration, a method with which we have extensive experience quantitating TSE infectivity in blood and blood components. In a limiting dilution titration, all animals in the bioassay are inoculated with the highest concentration of inoculum that is tolerated by the intracranial (most efficient) route. Infectivity assorts randomly into the inoculated animals; provided that at least some, but not all, of the animals are infected, the concentration can be calculated from the Poisson distribution of the infections (1). The method is highly sensitive and far more precise than other methods of TSE titration. We considered concentrating the urine before bioassay, but to circumvent uncertainties about the recovery of endogenous infectivity, we decided to inject the urine as collected. We found TSE infectivity in the urine of hamsters t hat had no evidence of kidney or bladder inflammation. In contrast, Seeger et al. did not detect infectivity in the urine of scrapieinfected mice (11) unless the mice were also affected by nephritis, in which case they found low levels of infectivity. Whether the bioassay they used was capable of detecting infectivity at the Page 9 of 16 concentration we observed for hamsters is not clear. If it was not capable, then detection of infectivity in mice with nephritis implies a higher concentration of infectivity in urine excreted by a nephritic kidney. In another study, urine and feces from deer with chronic wasting disease failed to demonstrate infectivity when orally given to the same susceptible species (17). Although usually an inefficient route of inoculation, the oral route did successfully transmit chronic wasting disease infectivity in saliva. The authors identified several possible reasons for the unsuccessful transmission by excreta, including incubation time, geno type, or sample size. In our experiments, cross-contamination by feces can not be excluded as a source of infectivity. Although the metabolism cage effectively separated urine and feces, some contact is possible because of the anatomy of the hamster. Protein misfolding cyclic amplification uses sonication to generate PrPres and infectivity in vitro. Although we routinely disperse all samples by ultrasonication before injection, our conditions are much harsher than those used to generate PrPres de novo (18) and do not support protein misfolding cyclic amplification of PrPres, or presumably infectivity (L. Gregori and R.G. Rohwer, unpub. data). The kidney and bladder titers were far greater than expected compared with findings of historical studies in which, with only rare exceptions (19-21), most attempts at transmission have been unsuccessful. These titers cannot be explained by the infectivity in residual blood (10 ID/mL) (1,2). In addition, we observed PrPd in the kidn eys of scrapie-infected animals that had no indications of tissue inflammation. Heikenwalder et al. found PrPd staining within follicular infiltrates only in kidneys of mice affected by nephritis and not in control mice with noncomplicated scrapie (12). These data together with those by Seeger et al. (11) suggested that renal inflammation might be a prerequisite for TSE infectivity in renal tissue and its excretion in urine. In contrast, our results indicate that renal inflammation is not necessary for the deposition of PrPd in kidneys or for excretion of infectivity. One interpretation is that nephritis enhances the accumulation of PrPd at sites of inflammation, consistent with the excretion of higher levels of infectivity inferred above for this same condition (11). Two studies of scrapie in naturally and experimentally infected sheep reported PrPd depositions in the renal papillae (22) and in the intraepithelial cortex, medulla, and papillae (23). Similar to our findi ngs, both studies indicated that not all scrapie tissues examined were positive Page 10 of 16 for PrPd. In chronic wasting disease, PrPd staining was uniquely localized in the ectopic lymphoid follicle of the kidney of a whitetail deer (24). All studies indicated either no changes (23,24) or mild to no inflammatory changes of the kidney (23). Thus, our histologic and immunohistochemical results for scrapie-infected hamsters are consistent with results found for sheep and deer and suggest that under normal conditions TSE diseases do not have concomitant inflammatory changes in the kidney. That urine titer is similar to that of plasma suggests that urine infectivity may originate from blood (25), but how the infectivity would be excreted is not clear. In general, proteins >40 kDa are not excreted and smaller proteins crossing the glomeruli are reabsorbed in the renal tubule and returned to the blood. If TSE infectivity is particulate (>40 kDa), its presence in urine might i ndicate abnormalities in renal filtration, perhaps related to the accumulation of PrPd in the collecting tubules of the medulla. The accumulation of immunoglobulins in the urine of TSEinfected hamsters and humans may also indicate malfunction of the urinary system (9,26). Excretion of a small C-terminal fragment of the normal cellular form of the prion protein in urine of infected and noninfected animals has been reported (27), but PrPres or PrPd forms can only be inferred from the presence of infectivity. Nevertheless, excretion of proteins similar to PrPres or PrPd forms has been documented. Follicle-stimulating hormone is a glycosylated protein of 203 amino acids organized mostly as a â-sheet, which bears some remarkable similarities to â-rich forms of the prion protein. Follicle-stimulating and several similar hormones are excreted in urine at great enough concentration to be extracted commercially. Alternatively, TSE infectivity may be excreted by processes analogou s to those responsible for the low-level virurias that occur during infections of the nervous system by mumps, measles, and West Nile virus (28-30). To the extent that results from the hamster model can be generalized to other TSE infections (and it has so far proven highly predictive), then even the very low concentrations of infectivity measured here could result in substantial environmental contamination. Several liters of urine and several thousand doses of TSE infectivity may be excreted daily over the course of the illness; even higher titers might be excreted by an animal with nephritis. The high stability of TSE infectivity would account for its persistence in pasture years after infected animals are removed (31). Recent studies have shown that infectivity that is adsorbed and immobilized by soil minerals (32) can still infect hamsters by oral exposure 29 months later (33). Our study also Page 11 of 16 warns of a possible risk from TSE contamination to fertility hormones and other medicinal products extracted from human urine.

Acknowledgments

snip...full text ;


http://www.cdc.gov/eid/content/14/9/pdfs/08-0259.pdf



http://chronic-wasting-disease.blogspot.com/2008/06/transmission-and-detection-of-prions-in.html


http://chronic-wasting-disease.blogspot.com/



Wednesday, August 13, 2008

Excretion of BSE and scrapie prions in stools from murine models

doi:10.1016/j.vetmic.2008.02.014 Copyright © 2008 Elsevier B.V. All rights reserved. Short communication


http://chronic-wasting-disease.blogspot.com/2008/08/excretion-of-bse-and-scrapie-prions-in.html



Sunday, August 24, 2008 HAVE ANOTHER GLASS OF CWD PRIONS COURTESY Dane County Wisconsin Mike DiMaggio, solid waste manager


http://chronic-wasting-disease.blogspot.com/2008/08/have-another-glass-of-cwd-prions.html



Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Diseases of Livestock

2007 Annual Report

1a.Objectives (from AD-416)


http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408808&showpars=true&fy=2007



The white-tailed deer in Michigan is recognized as the primary reservoir host of bovine TB. Once the disease is eliminated from the deer, the disease should die out in the non-cervid species. As long as bovine TB exists in the wild, free-ranging deer population, there will be some risk to local wildlife species that feed on bovine TB-infected deer carcasses or gut piles.


http://www.michigan.gov/emergingdiseases/0,1607,7-186-25804_25811-75908--,00.html



For this reason, when I asked the DEC medical folks why they didn't require hunters to remove the gut-pile from the woods in the special CWD management ...


http://www.nysrpa.org/encon/CWD.html



Friday, August 8, 2008 PS 76-59: White-tailed deer carcass decomposition and risk of chronic wasting disease exposure to scavenger communities in Wisconsin Chris S. Jennelle, Michael D. Samuel, Cherrie A. Nolden, and Elizabeth A. Berkley. University of Wisconsin

Background/Question/Methods

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy (TSE) afflicting members of the family Cervidae, and causes neurodegeneration and ultimately death. While there have been no reports of natural cross-species transmission of CWD outside this group, we addressed the role of white-tailed deer (Odocoileus virginianus) carcasses as environmental sources of CWD in Wisconsin. Our objectives were to estimate rates of deer carcass and gut pile decomposition in the environment, characterize vertebrate scavenger communities, and quantify the relative activity of scavengers to determine CWD exposure risk. We placed 40 disease-free deer carcasses and nine gut piles in the CWD-affected area of Wisconsin from September to April in 2003 through 2005. We used photos from remotely operated cameras to characterize scavenger communities and relative activity. We used Kaplan-Meier survival analysis and a generalized linear mixed model to quantify the driving factors and rate of carcass removal (decomposition) from the environment.

Results/Conclusions

We recorded 14 species of scavenging mammals (six visiting species), and eight species of scavenging birds (14 visiting species). Prominent scavengers included American crows (Corvus brachyrhynchos), raccoons (Procyon lotor), and Virginia opossums (Didelphis virginiana). We found no evidence that deer directly consumed conspecific remains, although they visited them frequently. Domestic dogs (Canis familiaris), cats (Felis catus), and cows (Bos spp.) either scavenged or visited carcass sites, which could increase exposure risk of CWD to humans and human food supplies. Deer carcasses persisted for a median of 18 to 101 days, while gut piles lasted for a median of three days. Habitat did not influence carcass decomposition, but mammalian and avian scavenger activity and higher temperatures (proxy for microbial and arthropod activity) were associated with greater rates of carcass removal. Infected deer carcasses serve as environmental sources of CWD prions to a wide variety of mammalian and avian scavengers. Such sources of infectious material likely influence the maintenance and spread of CWD (in particular), and should be considered in the dynamics of other disease systems as well. Prudence would dictate the use of preemptive management strategies, and we highlight strategies for carcass disposal to mitigate the influence of carcasses as environmental sources of infectious diseases.

See more of PS 76 - Latebreaking: Disease and Epidemiology See more of Latebreakers

See more of The 93rd ESA Annual Meeting (August 3 -- August 8, 2008)


http://eco.confex.com/eco/2008/techprogram/P14681.HTM



11. Disease control challenges posed by CWD

Evidence is building that indicates efficient horizontal transmission occurs in CWD, indeed a complicating aspect in disease control [91]. Potential transmission mechanisms range from spread via direct contact among animals to environmental exposure through grazing in areas contaminated by prion-infected secretions, excretions (saliva, urine, feces), tissues (placenta), or decomposed carcasses.

snip...see full text 19 pages ;


http://www.vetres.org/index.php?option=article&access=standard&Itemid=129&url=/articles/vetres/pdf/2008/04/v08092.pdf



http://chronic-wasting-disease.blogspot.com/



Title: Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer

Authors

Hamir, Amirali Miller, Janice - ARS RETIRED Kunkle, Robert Hall, S - USDA, APHIS, NVSL, PL Richt, Juergen

Submitted to: Veterinary Pathology Publication Type: Peer Reviewed Journal Publication Acceptance Date: February 20, 2007 Publication Date: July 1, 2007 Citation: Hamir, A.N., Miller, J.M., Kunkle, R.A., Hall, S.M., Richt, J.A. 2007. Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer. Veterinary Pathology. 44:487-493.

Interpretive Summary: This study reports findings assessing susceptibility of cattle to infection following direct surgical inoculation of the transmissible spongiform encephalopathy (TSE), chronic wasting disease (CWD, from white tailed deer) into the brain of 14 cattle. Three-month-old calves were inoculated with the CWD agent from white tailed deer. Two non-inoculated calves served as controls. 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. The findings also indicate that diagnostic techniques currently used for detection of bovine spongiform encephalopathy (BSE) would detect CWD in cattle should it ever cross the species barrier. Moreover, these findings confirm our earlier findings with CWD from mule deer, thus demonstrating a unique pattern of the CWD disease agent from deer when experimentally inoculated into cattle, further validating our ability to distinguish this form of cross-species TSE transmission from BSE in cattle. Technical Abstract: To compare clinicopathological findings of chronic wasting disease (CWD) from white-tailed deer (CWD**wtd) with other transmissible spongiform encephalopathies [transmissible spongiform encephalopathy (TSE), prion diseases) that have been shown to be experimentally transmissible to cattle [sheep scrapie, CWD of mule deer (CWD**md) and transmissible mink encephalopathy (TME)], 14 three-month-old calves were intracerebrally inoculated with the CWD**wtd agent. Two uninoculated calves served as controls. Within 26 months post inoculation (MPI), 12 inoculated animals had lost considerable weight and eventually became recumbent. Eleven of these had clinical signs of central nervous system (CNS) abnormality and all 12 were euthanized. Although microscopic lesions of spongiform encephalopathy (SE) were not seen in CNS tissues, PrP**res was detected by immunohistochemistry (IHC) and Western blot (WB). These findings demonstrate that when CWD**wtd is intracerebrally inoculated in cattle, 86% of inoculated cattle develop abnormal clinical signs and amplify PrP**res in their CNS tissues without evidence of morphologic lesions of SE. The latter has also been shown with other TSE agents (scrapie and CWD**md) similarly inoculated into cattle. These findings suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect CWD**wtd in cattle should it occur naturally. The absence of microscopic morphologic lesions and a unique IHC pattern of CWD**wtd in cattle, suggests that it should be possible to distinguish this form of cross-species transmission from BSE in cattle.


http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=194089



Title: Detection of PrP**CWD in retinal tissues in white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) with CWD

Authors

Spraker, Terry - COLORADO STATE UNIVERSITY Gidleweski, Thomas - APHIS-VS, FORT COLLINS Greenlee, Justin Keane, Delwyn - WISCONSIN DIAGNOSTIC LAB Hamir, Amirali O`rourke, Katherine

Submitted to: American Association of Veterinary Laboratory Diagnosticians Publication Type: Abstract Publication Acceptance Date: July 15, 2007 Publication Date: October 18, 2007 Citation: Spraker, T., Gidleweski, T., Greenlee, J., Keane, D., Hamir, A., Orourke, K. 2007. Detection of PrP**CWD in retinal tissues in white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) with CWD [abstract]. American Association of Veterinary Laboratory Diagnosticians 50th Annual Meeting. p. 47.

Technical Abstract: Introduction. Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging mule deer (Odocoileus hemionus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni). An abnormal isoform of a prion protein (PrP**CWD) that has been associated with CWD has been reported in numerous internal organs other than the brain and lymphoid tissues, including the retina of mule deer. The objective of this project was to investigate the possibility of PrP**CWD in the retina of white-tailed deer and Rocky Mountain elk with CWD. Materials and methods. Eyes from 80 captive white-tailed deer that were killed during a depopulation program were collected and placed in 10% neutral buffered formalin. None of these 80 white-tailed deer showed any clinical signs suggestive of CWD prior to euthanasia (gun shot); however 79% were positive for CWD by using immunohistochemical staining of the brain stem and head lymphoid tissues. Eyes from 7 captive and 2 research elk were collected and placed in Davison¿s fixative. Clinical signs typical of CWD were observed in 5 elk (3 with M/M genotype, 1 M/L and 1 L/L). The other 4 were non-clinical (3 with genotype M/M and 1 L/L), but did have a previous positive rectal biopsy. The globe from each animal was trimmed and embedded in a single paraffin block and sectioned at 5 µm. Tissue sections were stained with H&E and immunostained with Anti-Prion 99 and P4 for the detection of PrP**CWD. Results. Prion was only found in the retina, all other regions of the eye (cornea, lens, ciliary body, iris, choroid, sclera) were free of PrP**CWD in the deer. Examination of the eyes from the white-tailed deer revealed 4 to have detectable PrP**CWD within the retina. PrP**CWD was restricted to the inner and outer plexiform layers of these deer. Sections from all 9 elk had PrP**CWD in 8 of the 10 retinal layers and in the optic nerve. All other regions of the eye were free of PrP**CWD. The most prominent features in the elk were heavy PrP**CWD staining in the inner and outer plexiform layers with minimal intracytoplasmic staining in ganglion cells in the M/M and M/L elk. The 2 L/L elk had minimal PrP**CWD staining in the plexiform layers, but relatively heavy staining in the cytoplasm of ganglion cells and an unusual accumulation of PrP**CWD just inside outer limiting membrane layer. An occasional ganglion cell within the ganglion cell layer contained an intracytoplasmic vacuole in the M/M elk. Discussion/Conclusion. Deer and elk do have an abundance of PrP**CWD in retinal tissues and optic nerve (elk). This accumulation of PrP**CWD may affect vision especially in elk. Genotypes did result in different patterns of PrP**CWD accumulation in elk. The LL genotype at codon 132, which has a prolonged incubation period, had much less PrP**CWD in the retina, especially within the inner and outer plexiform layers. In addition, the LL elk seemed to have more intracytoplasmic staining within ganglion cells as compared to the MM and ML elk.


http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=220211



Research Project: Strain Typing of Chronic Wasting Disease (Cwd) and Scrapie by Intracerebral Inoculation into Transgenic and Inbred Mouse Lines Location: Animal Diseases Research

Project Number: 5348-32000-026-07 Project Type: Specific C/A

Start Date: Sep 28, 2007 End Date: Sep 27, 2012

Objective: To identify and differentiate typical and atypical case samples of CWD and Scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice.

Approach: Tissue samples from deer, elk, sheep and goats with Transmissible Spongiform Encephalopathy (TSE) will be administered to mice by intracerebral injection. Multiple tissue types will be included, such as samples of brain, lymph node, blood, urine, feces, antler velvet and muscle. Mouse models used as recipient hosts will include both preexisting and recently created transgenic and inbred mouse lines. Recipient mouse phenotype will be evaluated by measuring clinical response, population disease rate, incubation time, and pathologic profile within the central nervous system (CNS). Pathologic profile of CNS lesion foci is assessed by evaluating anatomic localization, spongiform change, astrocytic gliosis, and deposition of protease resistant prion protein. BSL-1; 9-4-06. Documents SCA with U. of WA.

Project Team

Knowles, Donald - Don Schneider, David O`rourke, Katherine

Project Annual Reports

FY 2007

Related National Programs

Animal Health (103)


http://genes.pp.ksu.edu/research/projects/projects.htm?ACCN_NO=411895



1: Vet Res. 2008 Apr 3;39(4):41

A prion disease of cervids: Chronic wasting disease

Sigurdson CJ.

The recent discovery of chronic wasting disease in cervids (CWD) beyond the borders of Colorado and Wyoming, as far east as New York and including two Canadian Provinces, has led to the emergence of CWD as a prion disease of domestic and international importance. The apparent ease of horizontal transmission, potentially via environmental contamination or by prion-containing saliva, creates enormous challenges for disease management. Ongoing studies of CWD interspecies transmission by exposure of domestic and non-domestic species directly or using transgenic mice have shed light on species barriers. Transgenic mice expressing cervid PrP have also proven useful for assessing the genetic influences of Prnp polymorphisms on CWD susceptibility. Accumulating evidence of CWD pathogenesis indicates that the misfolded prion protein, PrPSc, seems to be widely disseminated in many nonneural organs, and CWD infectivity has been recently detected in blood. This review highlights recent research findings in this disease of free-ranging wildlife.

snip...

3. CWD prion spread and target organs

Collectively, CWD pathogenesis studies have revealed extensive deposition of PrPSc in the central nervous system (CNS) and extraneural tissues (Fig. 1). The only other natural prion diseases that even approach this degree of systemic involvement are variant Creutzfeldt-Jakob disease (vCJD) in humans, sheep scrapie, and transmissible mink encephalopathy [22, 23, 30, 61, 62]. In mule deer, PrPSc is detectable in the retropharyngeal lymph node within only 6 weeks following an oral exposure [76]. In a further study of the kinetics of prion

1
http://www.aphis .usda.gov/vs/nahps/cwd/cwd-distribution.html





5

infection in mule deer, Fox et al. showed that PrPSc is widely distributed in lymphoid tissues by 3 months post-oral exposure when it is first detected in brain [17]. By 9 months, PrPSc was detected in the myenteric and submucosal plexi throughout the gastrointestinal tract and in the vagus nerve, and by 16 months, PrPSc deposits were detectable throughout the brain and spinal cord. The Prnp genotype seemed to impact the infection kinetics in that mule deer that were SF heterozygous at codon 225 showed a delay in PrPSc spread; PrPSc was not

detectable in the brain until 16 months post-inoculation which was 13 months later than the 225SS deer. Perhaps the 225F allele confers a dominant negative effect on the kinetics of this CWD strain, as has been described in sheep, where the 171R allele has been shown to have a dominant negative effect on prion susceptibility [20, 33]. CWD pathogenesis seems to vary between deer and elk: PrPSc levels have been found to be lower in lymphoid tissues of elk compared to deer [66]. In a report of 226 CWD-infected elk, 28 had no PrPSc in lymphoid tissues despite having PrPSc in brain [81]. In addition to lymphoid tissues, PrPSc or infectivity has been detected in other non-CNS tissues, including pancreas [17, 77], adrenal gland [17, 77], and skeletal muscle [2]. Recently PrPSc was described in cardiac muscle from 7 of 16 (44%) white-tailed deer and from 12 of 17 (71%) elk [35]. This is the first report of PrPSc in cardiac muscle in any TSE. The cellular and molecular mechanisms of systemic prion spread are under investigation in many laboratories. A recent report showed that blood from CWD-infected deer contained infectivity and could transmit prion disease via a blood transfusion [50]. This finding recapitulates indirect findings of blood infectivity in vCJD affected humans [61] and experimental transfusion studies of scrapie sick sheep [32], and indicates that prion transport

throughout the body may include the blood as a potential vehicle.

snip...

6. CWD strains among deer and elk

Prion strains, such as those seen in sheep scrapie, show distinct incubation periods in differentially susceptible inbred mice and lesions target discrete brain regions [11, 18, 19]. CWD in deer and elk has been considered a single disease entity, and western blot glycoform patterns of PrPSc are similar among deer and elk [67]. However, some new data indicate otherwise, suggesting that conformational variants, or strains, may exist. In a study by Raymond et al., Syrian golden hamsters were infected with mule deer or elk CWD, but with an incomplete attack rate; only 2 of 7 and 0 of 7 hamsters developed terminal disease, respectively. Indeed, second and third passage of the mule deer derived strain resulted in a short incubation period of only 85-89 days, whereas the elk-derived strain led to an incubation period of 408-544 days. Surprisingly, when mule deer CWD was first passaged in hamster PrP expressing transgenic mice and then into hamsters, a slowly replicating strain

with distinct clinical disease and PrPSc deposition patterns in brain ensued. Therefore two different strains could be passaged from a single mule deer CWD isolate, a rapid and a slowly replicating strain with differing disease phenotypes [70]. Alternatively, these strains could have been generated upon interspecies transmission [6].

We have also observed two strains arising from a single CWD-infected mule deer upon

passage in transgenic mice overexpressing murine PrP. Here, mice developed different

PrPSc aggregate morphologies in brain, either dense, congophilic plaques or fine, diffuse aggregates which could be selectively passaged [78]. LaFauci et al. have reported that elk PrP expressing transgenic mice developed phenotypically divergent diseases when inoculated with either mule deer or elk CWD, which was also suggestive of different strains [44]. In each of these studies, it is not clear whether mule deer and elk possess heterogeneous PrP aggregates (strain mixture), or whether the strains may have developed in the new host. However, Safar et al. have reported differing conformational characteristics for PrPSc from CWD-infected white-tailed deer and elk directly, using a conformation dependent immunoassay (CDI) [71], which supports the existence of CWD strains. The possible existence of CWD strains is perhaps not entirely surprising, considering that there are genetic Prnp differences among deer and elk that could influence PrPSc conformation [34, 36, 58].

7

7. Interspecies CWD transmission

Wild predators and scavengers are presumably feeding on CWD-infected carcasses.

Skeletal muscle has been shown to harbor CWD prion infectivity [2], underscoring that other species will almost certainly be exposed to CWD through feeding. However, CWD has not been successfully transmitted by oral inoculation to species outside of the cervid family, suggestive of a strong species barrier for heterologous PrP conversion. Ferrets (family Mustelidae) can be infected with deer CWD after intracerebral (ic) but not oral exposure [5, 80]. Raccoons resisted even ic infection for up to 2 years thus far [24]. Mountain lion (Puma concolor) susceptibility to experimental feeding of CWD prions is currently under investigation (M. Miller and L. Wolfe, personal communication).

Could wild rodents colonizing CWD- or scrapie-infected pastures serve as an environmental reservoir of prion infectivity? Interestingly, bank voles (Clethrionomys glareolus), are readily infected with CWD and sheep scrapie by intracerebral inoculation ([64]; U. Agrimi, unpublished data) and are considered as a potential reservoir for sheep scrapie [64]. Many vole species occur in North America [65, 83] and further research may determine whether voles enhance CWD or scrapie spread through environmental contamination.

Given that environmental contamination with CWD prions likely occurs [55], domestic

ruminants may be exposed to CWD through common grazing areas. However, sheep and

cattle appear to be poorly susceptible to mule deer CWD: ic inoculation with mule deer CWD succeeded to infect only 2 of 8 sheep [28]; likewise cattle have not been infected after cograzing with CWD-infected mule deer, or after a direct oral exposure (over 6 years) (M. Miller, personal communication). Even direct ic inoculation led to CWD infection in only 5 of 13 cattle (38%) after 2-5 years [26]. In contrast, cattle are highly susceptible to white-tailed deer CWD with 12 of 14 animals developing neurologic disease and PrPSc by only 22 months post-ic inoculation (+/-0.5 months) [29]. Further studies are planned to determine whether

cattle are susceptible to white-tailed deer prions after an oral exposure (J. Richt, personal communication). The differential susceptibility of cattle to CWD from mule deer versus whitetailed deer suggests that CWD strains exist, and that CWD may differentially cross species barriers depending on the strain. Nevertheless, to date, natural CWD infections have been detected only in cervids.

Is the converse true, are cervids susceptible to sheep scrapie? Only one study has been performed on cervid susceptibility to sheep scrapie by the ic route, and showed that 3 of 6 elk developed neurologic signs, spongiform encephalopathy and PrPSc in brain [25]. Further experiments to address this question may be interesting since sheep scrapie is considered a possible source for CWD in North America [89, 91].

8. Human susceptibility to CWD

Millions of North Americans hunt deer and elk (U.S. Department of the Interior, Census Bureau), and there is no doubt that people have been exposed to CWD through venison consumption, particularly in light of recent data showing CWD prions in muscle [2]. Human susceptibility to CWD or to other newly emerging animal TSE [9, 14] is still unclear, although we can be somewhat reassured in that there have been no large scale outbreaks of human TSE cases in Colorado and Wyoming, where CWD has existed for decades [51]. Up until approximately 10 years ago, autopsies were not performed on suspect human TSE cases in many states due to biosafety concerns, therefore the diagnosis of potential new TSE strains has been hampered. This indicates that clinical TSE diagnoses in humans were not confirmed, nor was any strain typing done to look for the appearance of potentially subtle or unusual pathological or biochemical phenotypes of a new TSE strain. Fortunately, the

autopsy rate for suspect cases is improving. At the National Prion Disease Pathology

Surveillance Center at Case Western Reserve University (Cleveland, Ohio), Creutzfeldt-Jakob disease (CJD) suspect cases are studied and classified by CJD subtype. Thus far,

8

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

Surveillance Center***,

however there have been no unusual or novel prion subtypes that might indicate the appearance of a new prion strain [7, 41]. Other indirect studies of human susceptibility to CWD also suggest that the risk is low. In biochemical conversion studies, Raymond et al. [68] showed that the efficiency of CWD to

convert recombinant human PrP into amyloid fibrils was low, but similar to that of both BSE and scrapie fibrils to do the same. These results suggest that there is a molecular incompatibility in the conversion of human PrPC by CWD, sheep scrapie, or BSE, and that cross species infections in humans may be rare events.

To determine whether common PrPSc strain features may link CWD and CJD, histopathology and the PrPSc biochemical characteristics from deer and elk were compared with that of humans with sporadic CJD (sCJD) cases that are methionine homozygous at codon 129 of the Prnp gene by Xie et al. [96], although strain features including histologic profile, target organs, and glycoform patterns will not necessarily remain the same upon crossing species barriers [6, 5, 8, 57]. The PrPSc form is cleaved by proteinase-K (PK) at different sites depending on the conformation of the protein and may aid determination of whether the PrPSc conformation is similar. By western blot (SDS-PAGE) of elk CWD, the unglycosylated

PK-resistant PrPSc migrated at 21 kDa, similar to sCJD (MM1 subtype) and the PK cleavage site was the same, occurring at residues 78 and 82 as assessed by N-terminal sequencing. Conformational stability was evaluated by measuring the PrPSc stability under partially denaturing conditions and also showed no significant difference between elk CWD and sCJD MM1 PrPSc. However, elk CWD and human sCJD MM1 strains exhibited distinct glycoform patterns by two dimensional gel electrophoresis, suggesting that the strains differed. Future studies may utilize luminescent conjugated polymers, which were recently shown to distinguish naturally- and experimentally-derived prion strains [79].

To study elk-human prion species barriers, Kong et al. inoculated elk CWD into transgenic mice expressing either human PrP or elk PrP. Whereas the elk PrP expressing mice developed disease 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 than 756 days [41].

In accordance with these results, Tamgüney et al. also reported that human PrP

overexpressing mice were not susceptible to 9 CWD isolates from mule deer, white-tailed deer, and elk [84]. However, mice have a limited lifespan and further passages may be necessary to detect low levels of prion infectivity that may be present subclinically. Although indirect evidence is accumulating that there may be a robust species barrier for CWD transmission to humans, one report indicates nonhuman primate susceptibility to CWD. Intracerebral inoculation of squirrel monkeys (Saimiri sciureus) demonstrated a positive CWD transmission [49]. Among non-human primates, however, the Prnp sequence of the new world monkeys are the most distant from humans [72], and therefore may not indicate that human prion conversion would occur by CWD.

snip...

11. Disease control challenges posed by CWD

Evidence is building that indicates efficient horizontal transmission occurs in CWD, indeed a complicating aspect in disease control [91]. Potential transmission mechanisms range from spread via direct contact among animals to environmental exposure through grazing in areas contaminated by prion-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]. An additional experiment by Miller and colleagues showed that CWD-infected carcasses allowed to decay naturally in confined pastures can lead to CWD infections in captive deer, demonstrating the potential for

environmental contamination to spread infection [55]. Modelling studies have provided further

10

support that environmental contamination is likely playing a significant role in transmitting CWD [56, 53]. Additionally, infectious prions have been demonstrated to bind soil particles and remain infectious to animals by both intracerebral and oral exposure routes [38, 37]. Prion infectivity has been recovered from soil more than two years after experimental exposure to prions, suggesting the soil may serve as a reservoir for CWD prions [75]. Taken together, these results indicate that there may even be multiple sources for CWD exposure, perhaps through direct contact and environmental routes.

Significant challenges to CWD eradication exist in free-ranging cervids. Infected deer and elk range over a broad geographic region, and even previously surmised geographic barriers such as the Continental Divide have proven passable by infected animals. Ridding the environment of CWD-contaminated soil or even CWD-infected carcasses is not possible.

Moreover, the available ante-mortem diagnostic tests for surveillance are laborious and impractical for large numbers of free-ranging animals [74, 88, 95]. Therefore for a wildlife manager, this disease is costly to survey and difficult to control.

12. Conclusion

CWD in cervids is efficiently transmitted, likely more than any other TSE in animals or humans. Therefore, it is unlikely that this TSE can be eradicated, but perhaps through an improved understanding of transmission routes, biological factors influencing pathogenesis, and the molecular basis of CWD prion conversion, a targeted strategy for interrupting disease spread may be developed.

Acknowledgements

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

snip...see full text 19 pages ;


http://www.vetres.org/index.php?option=article&access=standard&Itemid=129&url=/a



Elk with a long incubation prion disease phenotype have a unique PrPd profile.

CELLULAR, MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE

Neuroreport. 18(18):1935-1938, December 3, 2007. O'Rourke, Katherine I. a; Spraker, Terry R. c; Zhuang, Dongyue a; Greenlee, Justin J. b; Gidlewski, Thomas E. d; Hamir, Amir N. b Abstract: The transmissible spongiform encephalopathies (TSEs) invariably result in fatal neurodegeneration and accumulation of PrPd, an abnormal form of the host prion protein PrPc, encoded by the PRNP gene. A naturally occurring polymorphism (methionine/valine) at PRNP codon 129 is associated with variation in relative disease susceptibility, incubation time, clinical presentation, neuropathology, and/or PrPd biochemical characteristics in a range of human TSEs. A methionine/leucine polymorphism at the corresponding site in the Rocky Mountain elk PRNP gene is associated with variation in relative susceptibility and incubation time in the cervid TSE chronic wasting disease. We now report that elk lacking the predisposing 132-methionine allele develop chronic wasting disease after a long incubation period and display a novel PrPd folding pattern.

(C) 2007 Lippincott Williams & Wilkins, Inc.


http://www.neuroreport.com/pt/re/neuroreport/abstract.00001756-200712030-00012.htm;jsessionid=L2nZpnpJQkMxcnsp2J2210J46CXBQSl7nPhwGfLyGyn2J5p0bTQR!231517226!181195629!8091!-1



Transmission and Detection of Prions in Feces

Jiri G. Safar,1,2 Pierre Lessard,1 Gültekin Tamgüney,1,2 Yevgeniy Freyman,1 Camille Deering,1 Frederic Letessier,1 Stephen J. DeArmond,1,3 and Stanley B. Prusiner1,2,4

1Institute for Neurodegenerative Diseases, Departments of 2Neurology, 3Pathology, and 4Biochemistry and Biophysics, University of California, San Francisco, San Francisco

In chronic wasting disease (CWD) in cervids and in scrapie in sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as the mode of transmission of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of 140 days and a rate of prion infection of 80%-100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrPSc) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored 60 ng/g PrPSc and prion titers of 106.6 ID50/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Received 9 October 2007; accepted 15 November 2007; electronically published 27 May 2008.

(See the editorial commentary by Bosque and Tyler, on pages 8-9.)

Potential conflicts of interest: none reported.

Financial support: National Institutes of Health (grants AG02132, AG010770, NS22786, and NS14069); G. Harold and Leila Y. Mathers Foundation; Sherman Fairchild Foundation.

Reprints or correspondence: Dr. Stanley B. Prusiner, 513 Parnassus Ave., HSE-774, San Francisco, CA 94143-0518 (mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:stanley@ind.ucsf.edu).


http://www.journals.uchicago.edu/doi/abs/10.1086/588193



LIVE TESTING

Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa Lorenzo González1, Robert Horton, Drew Ramsay, Reet Toomik, Valerie Leathers, Quentin Tonelli, Mark P. Dagleish, Martin Jeffrey and Linda Terry Correspondence: 1Corresponding Author: Lorenzo González, Veterinary Laboratories Agency, Pentlands Science Park, Bush Loan, PENICUIK, Midlothian EH26 0PZ, UK, e-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:l.gonzalez@vla.defra.gsi.gov.uk

In recent publications, it was shown that disease-associated prion protein (PrPd) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrPd can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrPd could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.


http://jvdi.org/cgi/content/abstract/20/2/203



Detection of PrPCWD in postmortem rectal lymphoid tissues in Rocky mountain elk (Cervus elaphus nelsoni) infected with chronic wasting disease Terry R. Spraker1, Thomas L. Gidlewski, Aru Balachandran, Kurt C. VerCauteren, Lynn Creekmore and Randy D. Munger

Correspondence: 1Corresponding Author: Terry R Spraker, Colorado State University Diagnostic Laboratory, Colorado State University, 300 W Drake Rd, Fort Collins, CO 80526, e-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:Terry.Spraker@colostate.edu

Preclinical diagnostic tests for transmissible spongiform encephalopathies have been described for mule deer (Odocoileus hemionus), using biopsy tissues of palatine tonsil, and for sheep, using lymphoid tissues from palatine tonsil, third eyelid, and rectal mucosa. The utility of examining the rectal mucosal lymphoid tissues to detect chronic wasting disease (CWD) was investigated in Rocky Mountain elk (Cervus elaphus nelsoni), a species for which there is not a live-animal diagnostic test. Postmortem rectal mucosal sections were examined from 308 elk from two privately owned herds that were depopulated. The results of the postmortem rectal mucosal sections were compared to immunohistochemical staining of the brainstem, retropharyngeal lymph nodes, and palatine tonsil. Seven elk were found positive using the brainstem (dorsal motor nucleus of the vagus nerve), retropharyngeal lymph nodes, and palatine tonsil. Six of these elk were also found positive using postmortem rectal mucosal sections. The remaining 301 elk in which CWD-associated abnormal isoform of the prion protein (PrPCWD) was not detected in the brainstem and cranial lymphoid tissues were also found to be free of PrPCWD when postmortem rectal mucosal sections were examined. The use of rectal mucosal lymphoid tissues may be suitable for a live-animal diagnostic test as part of an integrated management strategy to limit CWD in elk.


http://jvdi.org/cgi/content/abstract/18/6/553



PrPCWD in rectal lymphoid tissue of deer (Odocoileus spp.) Lisa L. Wolfe1, Terry R. Spraker2, Lorenzo González3, Mark P. Dagleish4, Tracey M. Sirochman1,5, Jeremy C. Brown6, Martin Jeffrey3 and Michael W. Miller1

1 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA 2 Colorado State University Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 80523, USA 3 Veterinary Laboratories Agency – Lasswade, Pentlands Science Park, Penicuik EH26 0PZ, UK 4 Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, UK 5 Department of Molecular Biology, University of Wyoming, 1000 E. University Avenue, Laramie, WY 82071, USA 6 Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070, USA

Correspondence Lisa L. Wolfe mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:lisa.wolfe@state.co.us

The utility of rectal lymphoid tissue sampling for the diagnosis of chronic wasting disease (CWD) infections in mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) was evaluated. CWD-associated prion protein (PrPCWD) deposits were observed in the rectal mucosa from 19 orally inoculated mule deer by 381 days post-inoculation (p.i.); similarly, 45 out of 50 naturally infected mule deer had PrPCWD in their rectal mucosa. In orally inoculated white-tailed deer, the presence of glycine (G) or serine (S) at codon 96 of the native PrP (denoted 96GG, 96GS or 96SS) appeared to influence the temporal patterns of PrPCWD deposition: nine out of 11 infected 96GG individuals had PrPCWD in their rectal mucosa by 342 days p.i., whereas only three out of seven infected 96GS individuals had PrPCWD in their rectal mucosa by 381 days p.i. and none of three 96SS individuals had PrPCWD in their rectal mucosa by 751 days p.i. These findings support further evaluation of rectal mucosa sampling in CWD surveillance.


http://vir.sgmjournals.org/cgi/content/abstract/88/7/2078



http://vir.sgmjournals.org/cgi/content/full/88/7/2078



CJD QUESTIONNAIRE

example=antler velvet nutritional supplements. _any_ nutritional supplements??? name/ ...


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



http://cjdquestionnaire.blogspot.com/


http://www.regulations.gov/fdmspublic/ContentViewer?objectId=09000064801f3413&disposition=attachment&contentType=msw8





2007 Annual Report

1a.Objectives (from AD-416) To identify and differentiate typical and atypical case samples of CWD and Scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice.

1b.Approach (from AD-416) Tissue samples from deer, elk, sheep and goats with Transmissible Spongiform Encephalopathy (TSE) will be administered to mice by intracerebral injection. Multiple tissue types will be included, such as samples of brain, lymph node, blood, urine, feces, antler velvet and muscle. Mouse models used as recipient hosts will include both preexisting and recently created transgenic and inbred mouse lines. Recipient mouse phenotype will be evaluated by measuring clinical response, population disease rate, incubation time, and pathologic profile within the central nervous system (CNS). Pathologic profile of CNS lesion foci is assessed by evaluating anatomic localization, spongiform change, astrocytic gliosis, and deposition of protease resistant prion protein. BSL-1; 9-4-06. Documents SCA with U. of WA.

3.Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Washington. Additional details of research can be found in the report for the parent project 5348-32000-026-00D Transmissible Spongiform Encephalopathies: the role of genetics, strain variation, and environmental contamination in disease control. The purpose of this new SCA is to identify and differentiate typical and atypical case samples of CWD and scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice. There will be weekly interactions between the ADODR, ADRU scientists and University of Washington collaborators through personnel visits, conference calls and emails.



http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=411895&fy=2007



Thursday, July 24, 2008

Prion diseases are efficiently transmitted by blood transfusion in sheep

Submitted April 18, 2008 Accepted June 28, 2008


http://vcjdblood.blogspot.com/2008/07/prion-diseases-are-efficiently.html



DOI: 10.3201/eid1409.080259 Suggested citation for this article: Gregori L, Kovacs GG, Alexeeva I, Budka H, Rohwer RG.

Excretion of transmissible spongiform encephalopathy infectivity in urine.

Emerg Infect Dis. 2008 Sep; [Epub ahead of print]

Excretion of Transmissible Spongiform Encephalopathy Infectivity in Urine Luisa Gregori, Gabor G. Kovacs, Irina Alexeeva, Herbert Budka, and Robert G. Rohwer Author affiliations: Veterans Affairs Medical Center, Baltimore, Maryland, USA (L. Gregori, I. Alexeeva, R.G. Rohwer); University of Maryland, Baltimore (L. Gregori, R.G. Rohwer); and Medical University of Vienna, Vienna, Austria (G.G. Kovacs, H. Budka) The route of transmission of most naturally acquired transmissible spongiform encephalopathy (TSE) infections remains speculative. To investigate urine as a potential source of TSE exposure, we used a sensitive method for detection and quantitation of TSE infectivity. Pooled urine collected from 22 hamsters showing clinical signs of 263K scrapie contained 3.8 ± 0.9 infectious doses/mL of infectivity. Titration of homogenates of kidneys and urinary bladders from the same animals gave concentrations 20,000-fold greater. Histologic and immunohistochemical examination of these same tissues showed no indications of inflammatory or other pathologic changes except for occasional deposits of diseaseassociated prion protein in kidneys. Although the source of TSE infectivity in urine remains unresolved, these results establish that TSE infectivity is excreted in urine and may thereby play a role in the horizontal transmission of natural TSEs. The results also indicate potential risk for TSE transmission from human urine-derived hormones and other medicines.

snip...

Discussion Anticipating that the titer of scrapie infectivity in excreted urine would be low, we measured concentration by using limiting dilution titration, a method with which we have extensive experience quantitating TSE infectivity in blood and blood components. In a limiting dilution titration, all animals in the bioassay are inoculated with the highest concentration of inoculum that is tolerated by the intracranial (most efficient) route. Infectivity assorts randomly into the inoculated animals; provided that at least some, but not all, of the animals are infected, the concentration can be calculated from the Poisson distribution of the infections (1). The method is highly sensitive and far more precise than other methods of TSE titration. We considered concentrating the urine before bioassay, but to circumvent uncertainties about the recovery of endogenous infectivity, we decided to inject the urine as collected. We found TSE infectivity in the urine of hamsters t hat had no evidence of kidney or bladder inflammation. In contrast, Seeger et al. did not detect infectivity in the urine of scrapieinfected mice (11) unless the mice were also affected by nephritis, in which case they found low levels of infectivity. Whether the bioassay they used was capable of detecting infectivity at the Page 9 of 16 concentration we observed for hamsters is not clear. If it was not capable, then detection of infectivity in mice with nephritis implies a higher concentration of infectivity in urine excreted by a nephritic kidney. In another study, urine and feces from deer with chronic wasting disease failed to demonstrate infectivity when orally given to the same susceptible species (17). Although usually an inefficient route of inoculation, the oral route did successfully transmit chronic wasting disease infectivity in saliva. The authors identified several possible reasons for the unsuccessful transmission by excreta, including incubation time, geno type, or sample size. In our experiments, cross-contamination by feces can not be excluded as a source of infectivity. Although the metabolism cage effectively separated urine and feces, some contact is possible because of the anatomy of the hamster. Protein misfolding cyclic amplification uses sonication to generate PrPres and infectivity in vitro. Although we routinely disperse all samples by ultrasonication before injection, our conditions are much harsher than those used to generate PrPres de novo (18) and do not support protein misfolding cyclic amplification of PrPres, or presumably infectivity (L. Gregori and R.G. Rohwer, unpub. data). The kidney and bladder titers were far greater than expected compared with findings of historical studies in which, with only rare exceptions (19-21), most attempts at transmission have been unsuccessful. These titers cannot be explained by the infectivity in residual blood (10 ID/mL) (1,2). In addition, we observed PrPd in the kidn eys of scrapie-infected animals that had no indications of tissue inflammation. Heikenwalder et al. found PrPd staining within follicular infiltrates only in kidneys of mice affected by nephritis and not in control mice with noncomplicated scrapie (12). These data together with those by Seeger et al. (11) suggested that renal inflammation might be a prerequisite for TSE infectivity in renal tissue and its excretion in urine. In contrast, our results indicate that renal inflammation is not necessary for the deposition of PrPd in kidneys or for excretion of infectivity. One interpretation is that nephritis enhances the accumulation of PrPd at sites of inflammation, consistent with the excretion of higher levels of infectivity inferred above for this same condition (11). Two studies of scrapie in naturally and experimentally infected sheep reported PrPd depositions in the renal papillae (22) and in the intraepithelial cortex, medulla, and papillae (23). Similar to our findi ngs, both studies indicated that not all scrapie tissues examined were positive Page 10 of 16 for P Pd. In chronic wasting disease, PrPd staining was uniquely localized in the ectopic lymphoid follicle of the kidney of a whitetail deer (24). All studies indicated either no changes (23,24) or mild to no inflammatory changes of the kidney (23). Thus, our histologic and immunohistochemical results for scrapie-infected hamsters are consistent with results found for sheep and deer and suggest that under normal conditions TSE diseases do not have concomitant inflammatory changes in the kidney. That urine titer is similar to that of plasma suggests that urine infectivity may originate from blood (25), but how the infectivity would be excreted is not clear. In general, proteins >40 kDa are not excreted and smaller proteins crossing the glomeruli are reabsorbed in the renal tubule and returned to the blood. If TSE infectivity is particulate (>40 kDa), its presence in urine might i ndicate abnormalities in renal filtration, perhaps related to the accumulation of PrPd in the collecting tubules of the medulla. The accumulation of immunoglobulins in the urine of TSEinfected hamsters and humans may also indicate malfunction of the urinary system (9,26). Excretion of a small C-terminal fragment of the normal cellular form of the prion protein in urine of infected and noninfected animals has been reported (27), but PrPres or PrPd forms can only be inferred from the presence of infectivity. Nevertheless, excretion of proteins similar to PrPres or PrPd forms has been documented. Follicle-stimulating hormone is a glycosylated protein of 203 amino acids organized mostly as a â-sheet, which bears some remarkable similarities to â-rich forms of the prion protein. Follicle-stimulating and several similar hormones are excreted in urine at great enough concentration to be extracted commercially. Alternatively, TSE infectivity may be excreted by processes analogou s to those responsible for the low-level virurias that occur during infections of the nervous system by mumps, measles, and West Nile virus (28-30). To the extent that results from the hamster model can be generalized to other TSE infections (and it has so far proven highly predictive), then even the very low concentrations of infectivity measured here could result in substantial environmental contamination. Several liters of urine and several thousand doses of TSE infectivity may be excreted daily over the course of the illness; even higher titers might be excreted by an animal with nephritis. The high stability of TSE infectivity would account for its persistence in pasture years after infected animals are removed (31). Recent studies have shown that infectivity that is adsorbed and immobilized by soil minerals (32) can still infect hamsters by oral exposure 29 months later (33). Our study also Page 11 of 16 warns of a possible risk from TSE contamination to fertility hormones and other medicinal products extracted from human urine.

Acknowledgments

snip...full text ;


http://www.cdc.gov/eid/content/14/9/pdfs/08-0259.pdf



http://chronic-wasting-disease.blogspot.com/2008/06/transmission-and-detection-of-prions-in.html



http://chronic-wasting-disease.blogspot.com/



Wednesday, August 13, 2008

Excretion of BSE and scrapie prions in stools from murine models

doi:10.1016/j.vetmic.2008.02.014 Copyright © 2008 Elsevier B.V. All rights reserved. Short communication


http://chronic-wasting-disease.blogspot.com/2008/08/excretion-of-bse-and-scrapie-prions-in.html



Sunday, August 24, 2008

HAVE ANOTHER GLASS OF CWD PRIONS COURTESY Dane County Wisconsin Mike DiMaggio, solid waste manager


http://chronic-wasting-disease.blogspot.com/2008/08/have-another-glass-of-cwd-prions.html



Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Diseases of Livestock

2007 Annual Report

1a.Objectives (from AD-416)


http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408808&showpars=true&fy=2007



The white-tailed deer in Michigan is recognized as the primary reservoir host of bovine TB. Once the disease is eliminated from the deer, the disease should die out in the non-cervid species. As long as bovine TB exists in the wild, free-ranging deer population, there will be some risk to local wildlife species that feed on bovine TB-infected deer carcasses or gut piles.


http://www.michigan.gov/emergingdiseases/0,1607,7-186-25804_25811-75908--,00.html



For this reason, when I asked the DEC medical folks why they didn't require hunters to remove the gut-pile from the woods in the special CWD management ...


http://www.nysrpa.org/encon/CWD.html



Friday, August 8, 2008 PS 76-59: White-tailed deer carcass decomposition and risk of chronic wasting disease exposure to scavenger communities in Wisconsin Chris S. Jennelle, Michael D. Samuel, Cherrie A. Nolden, and Elizabeth A. Berkley. University of Wisconsin

Background/Question/Methods

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy (TSE) afflicting members of the family Cervidae, and causes neurodegeneration and ultimately death. While there have been no reports of natural cross-species transmission of CWD outside this group, we addressed the role of white-tailed deer (Odocoileus virginianus) carcasses as environmental sources of CWD in Wisconsin. Our objectives were to estimate rates of deer carcass and gut pile decomposition in the environment, characterize vertebrate scavenger communities, and quantify the relative activity of scavengers to determine CWD exposure risk. We placed 40 disease-free deer carcasses and nine gut piles in the CWD-affected area of Wisconsin from September to April in 2003 through 2005. We used photos from remotely operated cameras to characterize scavenger communities and relative activity. We used Kaplan-Meier survival analysis and a generalized linear mixed model to quantify the driving factors and rate of carcass removal (decomposition) from the environment.

Results/Conclusions

We recorded 14 species of scavenging mammals (six visiting species), and eight species of scavenging birds (14 visiting species). Prominent scavengers included American crows (Corvus brachyrhynchos), raccoons (Procyon lotor), and Virginia opossums (Didelphis virginiana). We found no evidence that deer directly consumed conspecific remains, although they visited them frequently. Domestic dogs (Canis familiaris), cats (Felis catus), and cows (Bos spp.) either scavenged or visited carcass sites, which could increase exposure risk of CWD to humans and human food supplies. Deer carcasses persisted for a median of 18 to 101 days, while gut piles lasted for a median of three days. Habitat did not influence carcass decomposition, but mammalian and avian scavenger activity and higher temperatures (proxy for microbial and arthropod activity) were associated with greater rates of carcass removal. Infected deer carcasses serve as environmental sources of CWD prions to a wide variety of mammalian and avian scavengers. Such sources of infectious material likely influence the maintenance and spread of CWD (in particular), and should be considered in the dynamics of other disease systems as well. Prudence would dictate the use of preemptive management strategies, and we highlight strategies for carcass disposal to mitigate the influence of carcasses as environmental sources of infectious diseases.

See more of PS 76 - Latebreaking: Disease and Epidemiology See more of Latebreakers

See more of The 93rd ESA Annual Meeting (August 3 -- August 8, 2008)


http://eco.confex.com/eco/2008/techprogram/P14681.HTM



11. Disease control challenges posed by CWD

Evidence is building that indicates efficient horizontal transmission occurs in CWD, indeed a complicating aspect in disease control [91]. Potential transmission mechanisms range from spread via direct contact among animals to environmental exposure through grazing in areas contaminated by prion-infected secretions, excretions (saliva, urine, feces), tissues (placenta), or decomposed carcasses.

snip...see full text 19 pages ;


http://www.vetres.org/index.php?option=article&access=standard&Itemid=129&url=/articles/vetres/pdf/2008/04/v08092.pdf



http://chronic-wasting-disease.blogspot.com/



Title: Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer

Authors

Hamir, Amirali Miller, Janice - ARS RETIRED Kunkle, Robert Hall, S - USDA, APHIS, NVSL, PL Richt, Juergen

Submitted to: Veterinary Pathology Publication Type: Peer Reviewed Journal Publication Acceptance Date: February 20, 2007 Publication Date: July 1, 2007 Citation: Hamir, A.N., Miller, J.M., Kunkle, R.A., Hall, S.M., Richt, J.A. 2007. Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer. Veterinary Pathology. 44:487-493.

Interpretive Summary: This study reports findings assessing susceptibility of cattle to infection following direct surgical inoculation of the transmissible spongiform encephalopathy (TSE), chronic wasting disease (CWD, from white tailed deer) into the brain of 14 cattle. Three-month-old calves were inoculated with the CWD agent from white tailed deer. Two non-inoculated calves served as controls. 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. The findings also indicate that diagnostic techniques currently used for detection of bovine spongiform encephalopathy (BSE) would detect CWD in cattle should it ever cross the species barrier. Moreover, these findings confirm our earlier findings with CWD from mule deer, thus demonstrating a unique pattern of the CWD disease agent from deer when experimentally inoculated into cattle, further validating our ability to distinguish this form of cross-species TSE transmission from BSE in cattle. Technical Abstract: To compare clinicopathological findings of chronic wasting disease (CWD) from white-tailed deer (CWD**wtd) with other transmissible spongiform encephalopathies [transmissible spongiform encephalopathy (TSE), prion diseases) that have been shown to be experimentally transmissible to cattle [sheep scrapie, CWD of mule deer (CWD**md) and transmissible mink encephalopathy (TME)], 14 three-month-old calves were intracerebrally inoculated with the CWD**wtd agent. Two uninoculated calves served as controls. Within 26 months post inoculation (MPI), 12 inoculated animals had lost considerable weight and eventually became recumbent. Eleven of these had clinical signs of central nervous system (CNS) abnormality and all 12 were euthanized. Although microscopic lesions of spongiform encephalopathy (SE) were not seen in CNS tissues, PrP**res was detected by immunohistochemistry (IHC) and Western blot (WB). These findings demonstrate that when CWD**wtd is intracerebrally inoculated in cattle, 86% of inoculated cattle develop abnormal clinical signs and amplify PrP**res in their CNS tissues without evidence of morphologic lesions of SE. The latter has also been shown with other TSE agents (scrapie and CWD**md) similarly inoculated into cattle. These findings suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect CWD**wtd in cattle should it occur naturally. The absence of microscopic morphologic lesions and a unique IHC pattern of CWD**wtd in cattle, suggests that it should be possible to distinguish this form of cross-species transmission from BSE in cattle.


http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=194089



Title: Detection of PrP**CWD in retinal tissues in white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) with CWD

Authors

Spraker, Terry - COLORADO STATE UNIVERSITY Gidleweski, Thomas - APHIS-VS, FORT COLLINS Greenlee, Justin Keane, Delwyn - WISCONSIN DIAGNOSTIC LAB Hamir, Amirali O`rourke, Katherine

Submitted to: American Association of Veterinary Laboratory Diagnosticians Publication Type: Abstract Publication Acceptance Date: July 15, 2007 Publication Date: October 18, 2007 Citation: Spraker, T., Gidleweski, T., Greenlee, J., Keane, D., Hamir, A., Orourke, K. 2007. Detection of PrP**CWD in retinal tissues in white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) with CWD [abstract]. American Association of Veterinary Laboratory Diagnosticians 50th Annual Meeting. p. 47.

Technical Abstract: Introduction. Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging mule deer (Odocoileus hemionus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni). An abnormal isoform of a prion protein (PrP**CWD) that has been associated with CWD has been reported in numerous internal organs other than the brain and lymphoid tissues, including the retina of mule deer. The objective of this project was to investigate the possibility of PrP**CWD in the retina of white-tailed deer and Rocky Mountain elk with CWD. Materials and methods. Eyes from 80 captive white-tailed deer that were killed during a depopulation program were collected and placed in 10% neutral buffered formalin. None of these 80 white-tailed deer showed any clinical signs suggestive of CWD prior to euthanasia (gun shot); however 79% were positive for CWD by using immunohistochemical staining of the brain stem and head lymphoid tissues. Eyes from 7 captive and 2 research elk were collected and placed in Davison¿s fixative. Clinical signs typical of CWD were observed in 5 elk (3 with M/M genotype, 1 M/L and 1 L/L). The other 4 were non-clinical (3 with genotype M/M and 1 L/L), but did have a previous positive rectal biopsy. The globe from each animal was trimmed and embedded in a single paraffin block and sectioned at 5 µm. Tissue sections were stained with H&E and immunostained with Anti-Prion 99 and P4 for the detection of PrP**CWD. Results. Prion was only found in the retina, all other regions of the eye (cornea, lens, ciliary body, iris, choroid, sclera) were free of PrP**CWD in the deer. Examination of the eyes from the white-tailed deer revealed 4 to have detectable PrP**CWD within the retina. PrP**CWD was restricted to the inner and outer plexiform layers of these deer. Sections from all 9 elk had PrP**CWD in 8 of the 10 retinal layers and in the optic nerve. All other regions of the eye were free of PrP**CWD. The most prominent features in the elk were heavy PrP**CWD staining in the inner and outer plexiform layers with minimal intracytoplasmic staining in ganglion cells in the M/M and M/L elk. The 2 L/L elk had minimal PrP**CWD staining in the plexiform layers, but relatively heavy staining in the cytoplasm of ganglion cells and an unusual accumulation of PrP**CWD just inside outer limiting membrane layer. An occasional ganglion cell within the ganglion cell layer contained an intracytoplasmic vacuole in the M/M elk. Discussion/Conclusion. Deer and elk do have an abundance of PrP**CWD in retinal tissues and optic nerve (elk). This accumulation of PrP**CWD may affect vision especially in elk. Genotypes did result in different patterns of PrP**CWD accumulation in elk. The LL genotype at codon 132, which has a prolonged incubation period, had much less PrP**CWD in the retina, especially within the inner and outer plexiform layers. In addition, the LL elk seemed to have more intracytoplasmic staining within ganglion cells as compared to the MM and ML elk.


http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=220211



Research Project: Strain Typing of Chronic Wasting Disease (Cwd) and Scrapie by Intracerebral Inoculation into Transgenic and Inbred Mouse Lines Location: Animal Diseases Research

Project Number: 5348-32000-026-07 Project Type: Specific C/A

Start Date: Sep 28, 2007 End Date: Sep 27, 2012

Objective: To identify and differentiate typical and atypical case samples of CWD and Scrapie by characterizing the biologic phenotype and pathologic profile of these agents when administered to susceptible lines of transgenic and inbred mice.

Approach: Tissue samples from deer, elk, sheep and goats with Transmissible Spongiform Encephalopathy (TSE) will be administered to mice by intracerebral injection. Multiple tissue types will be included, such as samples of brain, lymph node, blood, urine, feces, antler velvet and muscle. Mouse models used as recipient hosts will include both preexisting and recently created transgenic and inbred mouse lines. Recipient mouse phenotype will be evaluated by measuring clinical response, population disease rate, incubation time, and pathologic profile within the central nervous system (CNS). Pathologic profile of CNS lesion foci is assessed by evaluating anatomic localization, spongiform change, astrocytic gliosis, and deposition of protease resistant prion protein. BSL-1; 9-4-06. Documents SCA with U. of WA.

Project Team

Knowles, Donald - Don Schneider, David O`rourke, Katherine

Project Annual Reports

FY 2007

Related National Programs

Animal Health (103)


http://genes.pp.ksu.edu/research/projects/projects.htm?ACCN_NO=411895



1: Vet Res. 2008 Apr 3;39(4):41

A prion disease of cervids: Chronic wasting disease

Sigurdson CJ.

The recent discovery of chronic wasting disease in cervids (CWD) beyond the borders of Colorado and Wyoming, as far east as New York and including two Canadian Provinces, has led to the emergence of CWD as a prion disease of domestic and international importance. The apparent ease of horizontal transmission, potentially via environmental contamination or by prion-containing saliva, creates enormous challenges for disease management. Ongoing studies of CWD interspecies transmission by exposure of domestic and non-domestic species directly or using transgenic mice have shed light on species barriers. Transgenic mice expressing cervid PrP have also proven useful for assessing the genetic influences of Prnp polymorphisms on CWD susceptibility. Accumulating evidence of CWD pathogenesis indicates that the misfolded prion protein, PrPSc, seems to be widely disseminated in many nonneural organs, and CWD infectivity has been recently detected in blood. This review highlights recent research findings in this disease of free-ranging wildlife.

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3. CWD prion spread and target organs

Collectively, CWD pathogenesis studies have revealed extensive deposition of PrPSc in the central nervous system (CNS) and extraneural tissues (Fig. 1). The only other natural prion diseases that even approach this degree of systemic involvement are variant Creutzfeldt-Jakob disease (vCJD) in humans, sheep scrapie, and transmissible mink encephalopathy [22, 23, 30, 61, 62]. In mule deer, PrPSc is detectable in the retropharyngeal lymph node within only 6 weeks following an oral exposure [76]. In a further study of the kinetics of prion

1 http://www.aphis .usda.gov/vs/nahps/cwd/cwd-distribution.html

5

infection in mule deer, Fox et al. showed that PrPSc is widely distributed in lymphoid tissues by 3 months post-oral exposure when it is first detected in brain [17]. By 9 months, PrPSc was detected in the myenteric and submucosal plexi throughout the gastrointestinal tract and in the vagus nerve, and by 16 months, PrPSc deposits were detectable throughout the brain and spinal cord. The Prnp genotype seemed to impact the infection kinetics in that mule deer that were SF heterozygous at codon 225 showed a delay in PrPSc spread; PrPSc was not

detectable in the brain until 16 months post-inoculation which was 13 months later than the 225SS deer. Perhaps the 225F allele confers a dominant negative effect on the kinetics of this CWD strain, as has been described in sheep, where the 171R allele has been shown to have a dominant negative effect on prion susceptibility [20, 33]. CWD pathogenesis seems to vary between deer and elk: PrPSc levels have been found to be lower in lymphoid tissues of elk compared to deer [66]. In a report of 226 CWD-infected elk, 28 had no PrPSc in lymphoid tissues despite having PrPSc in brain [81]. In addition to lymphoid tissues, PrPSc or infectivity has been detected in other non-CNS tissues, including pancreas [17, 77], adrenal gland [17, 77], and skeletal muscle [2]. Recently PrPSc was described in cardiac muscle from 7 of 16 (44%) white-tailed deer and from 12 of 17 (71%) elk [35]. This is the first report of PrPSc in cardiac muscle in any TSE. The cellular and molecular mechanisms of systemic prion spread are under investigation in many laboratories. A recent report showed that blood from CWD-infected deer contained infectivity and could transmit prion disease via a blood transfusion [50]. This finding recapitulates indirect findings of blood infectivity in vCJD affected humans [61] and experimental transfusion studies of scrapie sick sheep [32], and indicates that prion transport

throughout the body may include the blood as a potential vehicle.

snip...

6. CWD strains among deer and elk

Prion strains, such as those seen in sheep scrapie, show distinct incubation periods in differentially susceptible inbred mice and lesions target discrete brain regions [11, 18, 19]. CWD in deer and elk has been considered a single disease entity, and western blot glycoform patterns of PrPSc are similar among deer and elk [67]. However, some new data indicate otherwise, suggesting that conformational variants, or strains, may exist. In a study by Raymond et al., Syrian golden hamsters were infected with mule deer or elk CWD, but with an incomplete attack rate; only 2 of 7 and 0 of 7 hamsters developed terminal disease, respectively. Indeed, second and third passage of the mule deer derived strain resulted in a short incubation period of only 85-89 days, whereas the elk-derived strain led to an incubation period of 408-544 days. Surprisingly, when mule deer CWD was first passaged in hamster PrP expressing transgenic mice and then into hamsters, a slowly replicating strain

with distinct clinical disease and PrPSc deposition patterns in brain ensued. Therefore two different strains could be passaged from a single mule deer CWD isolate, a rapid and a slowly replicating strain with differing disease phenotypes [70]. Alternatively, these strains could have been generated upon interspecies transmission [6].

We have also observed two strains arising from a single CWD-infected mule deer upon

passage in transgenic mice overexpressing murine PrP. Here, mice developed different

PrPSc aggregate morphologies in brain, either dense, congophilic plaques or fine, diffuse aggregates which could be selectively passaged [78]. LaFauci et al. have reported that elk PrP expressing transgenic mice developed phenotypically divergent diseases when inoculated with either mule deer or elk CWD, which was also suggestive of different strains [44]. In each of these studies, it is not clear whether mule deer and elk possess heterogeneous PrP aggregates (strain mixture), or whether the strains may have developed in the new host. However, Safar et al. have reported differing conformational characteristics for PrPSc from CWD-infected white-tailed deer and elk directly, using a conformation dependent immunoassay (CDI) [71], which supports the existence of CWD strains. The possible existence of CWD strains is perhaps not entirely surprising, considering that there are genetic Prnp differences among deer and elk that could influence PrPSc conformation [34, 36, 58].

7

7. Interspecies CWD transmission

Wild predators and scavengers are presumably feeding on CWD-infected carcasses.

Skeletal muscle has been shown to harbor CWD prion infectivity [2], underscoring that other species will almost certainly be exposed to CWD through feeding. However, CWD has not been successfully transmitted by oral inoculation to species outside of the cervid family, suggestive of a strong species barrier for heterologous PrP conversion. Ferrets (family Mustelidae) can be infected with deer CWD after intracerebral (ic) but not oral exposure [5, 80]. Raccoons resisted even ic infection for up to 2 years thus far [24]. Mountain lion (Puma concolor) susceptibility to experimental feeding of CWD prions is currently under investigation (M. Miller and L. Wolfe, personal communication).

Could wild rodents colonizing CWD- or scrapie-infected pastures serve as an environmental reservoir of prion infectivity? Interestingly, bank voles (Clethrionomys glareolus), are readily infected with CWD and sheep scrapie by intracerebral inoculation ([64]; U. Agrimi, unpublished data) and are considered as a potential reservoir for sheep scrapie [64]. Many vole species occur in North America [65, 83] and further research may determine whether voles enhance CWD or scrapie spread through environmental contamination.

Given that environmental contamination with CWD prions likely occurs [55], domestic

ruminants may be exposed to CWD through common grazing areas. However, sheep and

cattle appear to be poorly susceptible to mule deer CWD: ic inoculation with mule deer CWD succeeded to infect only 2 of 8 sheep [28]; likewise cattle have not been infected after cograzing with CWD-infected mule deer, or after a direct oral exposure (over 6 years) (M. Miller, personal communication). Even direct ic inoculation led to CWD infection in only 5 of 13 cattle (38%) after 2-5 years [26]. In contrast, cattle are highly susceptible to white-tailed deer CWD with 12 of 14 animals developing neurologic disease and PrPSc by only 22 months post-ic inoculation (+/-0.5 months) [29]. Further studies are planned to determine whether

cattle are susceptible to white-tailed deer prions after an oral exposure (J. Richt, personal communication). The differential susceptibility of cattle to CWD from mule deer versus whitetailed deer suggests that CWD strains exist, and that CWD may differentially cross species barriers depending on the strain. Nevertheless, to date, natural CWD infections have been detected only in cervids.

Is the converse true, are cervids susceptible to sheep scrapie? Only one study has been performed on cervid susceptibility to sheep scrapie by the ic route, and showed that 3 of 6 elk developed neurologic signs, spongiform encephalopathy and PrPSc in brain [25]. Further experiments to address this question may be interesting since sheep scrapie is considered a possible source for CWD in North America [89, 91].

8. Human susceptibility to CWD

Millions of North Americans hunt deer and elk (U.S. Department of the Interior, Census Bureau), and there is no doubt that people have been exposed to CWD through venison consumption, particularly in light of recent data showing CWD prions in muscle [2]. Human susceptibility to CWD or to other newly emerging animal TSE [9, 14] is still unclear, although we can be somewhat reassured in that there have been no large scale outbreaks of human TSE cases in Colorado and Wyoming, where CWD has existed for decades [51]. Up until approximately 10 years ago, autopsies were not performed on suspect human TSE cases in many states due to biosafety concerns, therefore the diagnosis of potential new TSE strains has been hampered. This indicates that clinical TSE diagnoses in humans were not confirmed, nor was any strain typing done to look for the appearance of potentially subtle or unusual pathological or biochemical phenotypes of a new TSE strain. Fortunately, the

autopsy rate for suspect cases is improving. At the National Prion Disease Pathology

Surveillance Center at Case Western Reserve University (Cleveland, Ohio), Creutzfeldt-Jakob disease (CJD) suspect cases are studied and classified by CJD subtype. Thus far,

8

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

Surveillance Center***,

however there have been no unusual or novel prion subtypes that might indicate the appearance of a new prion strain [7, 41]. Other indirect studies of human susceptibility to CWD also suggest that the risk is low. In biochemical conversion studies, Raymond et al. [68] showed that the efficiency of CWD to

convert recombinant human PrP into amyloid fibrils was low, but similar to that of both BSE and scrapie fibrils to do the same. These results suggest that there is a molecular incompatibility in the conversion of human PrPC by CWD, sheep scrapie, or BSE, and that cross species infections in humans may be rare events.

To determine whether common PrPSc strain features may link CWD and CJD, histopathology and the PrPSc biochemical characteristics from deer and elk were compared with that of humans with sporadic CJD (sCJD) cases that are methionine homozygous at codon 129 of the Prnp gene by Xie et al. [96], although strain features including histologic profile, target organs, and glycoform patterns will not necessarily remain the same upon crossing species barriers [6, 5, 8, 57]. The PrPSc form is cleaved by proteinase-K (PK) at different sites depending on the conformation of the protein and may aid determination of whether the PrPSc conformation is similar. By western blot (SDS-PAGE) of elk CWD, the unglycosylated

PK-resistant PrPSc migrated at 21 kDa, similar to sCJD (MM1 subtype) and the PK cleavage site was the same, occurring at residues 78 and 82 as assessed by N-terminal sequencing. Conformational stability was evaluated by measuring the PrPSc stability under partially denaturing conditions and also showed no significant difference between elk CWD and sCJD MM1 PrPSc. However, elk CWD and human sCJD MM1 strains exhibited distinct glycoform patterns by two dimensional gel electrophoresis, suggesting that the strains differed. Future studies may utilize luminescent conjugated polymers, which were recently shown to distinguish naturally- and experimentally-derived prion strains [79].

To study elk-human prion species barriers, Kong et al. inoculated elk CWD into transgenic mice expressing either human PrP or elk PrP. Whereas the elk PrP expressing mice developed disease 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 than 756 days [41].

In accordance with these results, Tamgüney et al. also reported that human PrP

overexpressing mice were not susceptible to 9 CWD isolates from mule deer, white-tailed deer, and elk [84]. However, mice have a limited lifespan and further passages may be necessary to detect low levels of prion infectivity that may be present subclinically. Although indirect evidence is accumulating that there may be a robust species barrier for CWD transmission to humans, one report indicates nonhuman primate susceptibility to CWD. Intracerebral inoculation of squirrel monkeys (Saimiri sciureus) demonstrated a positive CWD transmission [49]. Among non-human primates, however, the Prnp sequence of the new world monkeys are the most distant from humans [72], and therefore may not indicate that human prion conversion would occur by CWD.

snip...

11. Disease control challenges posed by CWD

Evidence is building that indicates efficient horizontal transmission occurs in CWD, indeed a complicating aspect in disease control [91]. Potential transmission mechanisms range from spread via direct contact among animals to environmental exposure through grazing in areas contaminated by prion-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]. An additional experiment by Miller and colleagues showed that CWD-infected carcasses allowed to decay naturally in confined pastures can lead to CWD infections in captive deer, demonstrating the potential for

environmental contamination to spread infection [55]. Modelling studies have provided further

10

support that environmental contamination is likely playing a significant role in transmitting CWD [56, 53]. Additionally, infectious prions have been demonstrated to bind soil particles and remain infectious to animals by both intracerebral and oral exposure routes [38, 37]. Prion infectivity has been recovered from soil more than two years after experimental exposure to prions, suggesting the soil may serve as a reservoir for CWD prions [75]. Taken together, these results indicate that there may even be multiple sources for CWD exposure, perhaps through direct contact and environmental routes.

Significant challenges to CWD eradication exist in free-ranging cervids. Infected deer and elk range over a broad geographic region, and even previously surmised geographic barriers such as the Continental Divide have proven passable by infected animals. Ridding the environment of CWD-contaminated soil or even CWD-infected carcasses is not possible.

Moreover, the available ante-mortem diagnostic tests for surveillance are laborious and impractical for large numbers of free-ranging animals [74, 88, 95]. Therefore for a wildlife manager, this disease is costly to survey and difficult to control.

12. Conclusion

CWD in cervids is efficiently transmitted, likely more than any other TSE in animals or humans. Therefore, it is unlikely that this TSE can be eradicated, but perhaps through an improved understanding of transmission routes, biological factors influencing pathogenesis, and the molecular basis of CWD prion conversion, a targeted strategy for interrupting disease spread may be developed.

Acknowledgements

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

snip...see full text 19 pages ;


http://www.vetres.org/index.php?option=article&access=standard&Itemid=129&url=/a



Elk with a long incubation prion disease phenotype have a unique PrPd profile.

CELLULAR, MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE

Neuroreport. 18(18):1935-1938, December 3, 2007. O'Rourke, Katherine I. a; Spraker, Terry R. c; Zhuang, Dongyue a; Greenlee, Justin J. b; Gidlewski, Thomas E. d; Hamir, Amir N. b Abstract: The transmissible spongiform encephalopathies (TSEs) invariably result in fatal neurodegeneration and accumulation of PrPd, an abnormal form of the host prion protein PrPc, encoded by the PRNP gene. A naturally occurring polymorphism (methionine/valine) at PRNP codon 129 is associated with variation in relative disease susceptibility, incubation time, clinical presentation, neuropathology, and/or PrPd biochemical characteristics in a range of human TSEs. A methionine/leucine polymorphism at the corresponding site in the Rocky Mountain elk PRNP gene is associated with variation in relative susceptibility and incubation time in the cervid TSE chronic wasting disease. We now report that elk lacking the predisposing 132-methionine allele develop chronic wasting disease after a long incubation period and display a novel PrPd folding pattern.

(C) 2007 Lippincott Williams & Wilkins, Inc.


http://www.neuroreport.com/pt/re/neuroreport/abstract.00001756-200712030-00012.htm;jsessionid=L2nZpnpJQkMxcnsp2J2210J46CXBQSl7nPhwGfLyGyn2J5p0bTQR!231517226!181195629!8091!-1



Transmission and Detection of Prions in Feces

Jiri G. Safar,1,2 Pierre Lessard,1 Gültekin Tamgüney,1,2 Yevgeniy Freyman,1 Camille Deering,1 Frederic Letessier,1 Stephen J. DeArmond,1,3 and Stanley B. Prusiner1,2,4

1Institute for Neurodegenerative Diseases, Departments of 2Neurology, 3Pathology, and 4Biochemistry and Biophysics, University of California, San Francisco, San Francisco

In chronic wasting disease (CWD) in cervids and in scrapie in sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as the mode of transmission of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of 140 days and a rate of prion infection of 80%-100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrPSc) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored 60 ng/g PrPSc and prion titers of 106.6 ID50/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Received 9 October 2007; accepted 15 November 2007; electronically published 27 May 2008.

(See the editorial commentary by Bosque and Tyler, on pages 8-9.)

Potential conflicts of interest: none reported.

Financial support: National Institutes of Health (grants AG02132, AG010770, NS22786, and NS14069); G. Harold and Leila Y. Mathers Foundation; Sherman Fairchild Foundation.

Reprints or correspondence: Dr. Stanley B. Prusiner, 513 Parnassus Ave., HSE-774, San Francisco, CA 94143-0518 (mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:stanley@ind.ucsf.edu).


http://www.journals.uchicago.edu/doi/abs/10.1086/588193



LIVE TESTING

Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa Lorenzo González1, Robert Horton, Drew Ramsay, Reet Toomik, Valerie Leathers, Quentin Tonelli, Mark P. Dagleish, Martin Jeffrey and Linda Terry Correspondence: 1Corresponding Author: Lorenzo González, Veterinary Laboratories Agency, Pentlands Science Park, Bush Loan, PENICUIK, Midlothian EH26 0PZ, UK, e-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:l.gonzalez@vla.defra.gsi.gov.uk

In recent publications, it was shown that disease-associated prion protein (PrPd) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrPd can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrPd could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.


http://jvdi.org/cgi/content/abstract/20/2/203



Detection of PrPCWD in postmortem rectal lymphoid tissues in Rocky mountain elk (Cervus elaphus nelsoni) infected with chronic wasting disease Terry R. Spraker1, Thomas L. Gidlewski, Aru Balachandran, Kurt C. VerCauteren, Lynn Creekmore and Randy D. Munger

Correspondence: 1Corresponding Author: Terry R Spraker, Colorado State University Diagnostic Laboratory, Colorado State University, 300 W Drake Rd, Fort Collins, CO 80526, e-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:Terry.Spraker@colostate.edu

Preclinical diagnostic tests for transmissible spongiform encephalopathies have been described for mule deer (Odocoileus hemionus), using biopsy tissues of palatine tonsil, and for sheep, using lymphoid tissues from palatine tonsil, third eyelid, and rectal mucosa. The utility of examining the rectal mucosal lymphoid tissues to detect chronic wasting disease (CWD) was investigated in Rocky Mountain elk (Cervus elaphus nelsoni), a species for which there is not a live-animal diagnostic test. Postmortem rectal mucosal sections were examined from 308 elk from two privately owned herds that were depopulated. The results of the postmortem rectal mucosal sections were compared to immunohistochemical staining of the brainstem, retropharyngeal lymph nodes, and palatine tonsil. Seven elk were found positive using the brainstem (dorsal motor nucleus of the vagus nerve), retropharyngeal lymph nodes, and palatine tonsil. Six of these elk were also found positive using postmortem rectal mucosal sections. The remaining 301 elk in which CWD-associated abnormal isoform of the prion protein (PrPCWD) was not detected in the brainstem and cranial lymphoid tissues were also found to be free of PrPCWD when postmortem rectal mucosal sections were examined. The use of rectal mucosal lymphoid tissues may be suitable for a live-animal diagnostic test as part of an integrated management strategy to limit CWD in elk.


http://jvdi.org/cgi/content/abstract/18/6/553



PrPCWD in rectal lymphoid tissue of deer (Odocoileus spp.) Lisa L. Wolfe1, Terry R. Spraker2, Lorenzo González3, Mark P. Dagleish4, Tracey M. Sirochman1,5, Jeremy C. Brown6, Martin Jeffrey3 and Michael W. Miller1

1 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA 2 Colorado State University Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 80523, USA 3 Veterinary Laboratories Agency – Lasswade, Pentlands Science Park, Penicuik EH26 0PZ, UK 4 Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, UK 5 Department of Molecular Biology, University of Wyoming, 1000 E. University Avenue, Laramie, WY 82071, USA 6 Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070, USA

Correspondence Lisa L. Wolfe mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:lisa.wolfe@state.co.us

The utility of rectal lymphoid tissue sampling for the diagnosis of chronic wasting disease (CWD) infections in mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) was evaluated. CWD-associated prion protein (PrPCWD) deposits were observed in the rectal mucosa from 19 orally inoculated mule deer by 381 days post-inoculation (p.i.); similarly, 45 out of 50 naturally infected mule deer had PrPCWD in their rectal mucosa. In orally inoculated white-tailed deer, the presence of glycine (G) or serine (S) at codon 96 of the native PrP (denoted 96GG, 96GS or 96SS) appeared to influence the temporal patterns of PrPCWD deposition: nine out of 11 infected 96GG individuals had PrPCWD in their rectal mucosa by 342 days p.i., whereas only three out of seven infected 96GS individuals had PrPCWD in their rectal mucosa by 381 days p.i. and none of three 96SS individuals had PrPCWD in their rectal mucosa by 751 days p.i. These findings support further evaluation of rectal mucosa sampling in CWD surveillance.


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Subject: Prions in Skeletal Muscles of Deer with Chronic Wasting Disease [SCIENCE FULL TEXT] Date: January 26, 2006 at 12:23 pm PST

Prions in Skeletal Muscles of Deer with Chronic Wasting Disease

Rachel C. Angers,1* Shawn R. Browning,1*† Tanya S. Seward,2 Christina J. Sigurdson,4‡ Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3§

1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders Brown Center on Aging, 3Department of Neurology, University of Kentucky, Lexington, KY 40536, USA. 4Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526, USA.

*These authors contributed equally to this work.

†Present address: Department of Infectology, Scripps Research Institute, 5353 Parkside Drive, RF-2, Jupiter, Florida, 33458, USA.

‡Present address: Institute of Neuropathology, University of Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.

§To whom correspondence should be addressed: E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:gtell2@uky.edu

Prions are transmissible proteinaceous agents of mammals that cause fatal neurodegenerative diseases of the central nervous system (CNS). The presence of infectivity in skeletal muscle of experimentally infected mice raised the possibility that dietary exposure to prions might occur through meat consumption (1). Chronic wasting disease (CWD), an enigmatic and contagious prion disease of North American cervids, is of particular concern. The emergence of CWD in an increasingly wide geographic area and the interspecies transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt Jakob disease (vCJD) have raised concerns about zoonotic transmission of CWD.

To test whether skeletal muscle of diseased cervids contained prion infectivity, Tg(CerPrP)1536 mice (2) expressing cervid prion protein (CerPrP), were inoculated intracerebrally with extracts prepared from the semitendinosus/semimembranosus muscle group of CWD-affected mule deer or from CWD-negative deer. The availability of CNS materials also afforded direct comparisons of prion infectivity in skeletal muscle and brain. All skeletal muscle extracts from CWD-affected deer induced progressive neurological dysfunction in Tg(CerPrP)1536 mice with mean incubation times ranging between 360 and ~490 d, whereas the incubation times of prions from the CNS ranged from ~230 to 280 d (Table 1). For each inoculation group, the diagnosis of prion disease was confirmed by the presence of PrPSc in the brains of multiple infected Tg(CerPrP)1536 mice (see supporting online material for examples). In contrast, skeletal muscle and brain material from CWD-negative deer failed to induce disease in Tg(CerPrP)1536 mice (Table 1) and PrPSc was not detected in the brains of sacrificed asymptomatic mice as late as 523 d after inoculation (supporting online material).

Our results show that skeletal muscle as well as CNS tissue of deer with CWD contains infectious prions. Similar analyses of skeletal muscle BSE-affected cattle did not reveal high levels of prion infectivity (3). It will be important to assess the cellular location of PrPSc in muscle. Notably, while PrPSc has been detected in muscles of scrapie-affected sheep (4), previous studies failed to detect PrPSc by immunohistochemical analysis of skeletal muscle from deer with natural or experimental CWD (5, 6). Since the time of disease onset is inversely proportional to prion dose (7), the longer incubation times of prions from skeletal muscle extracts compared to matched brain samples indicated that prion titers were lower in muscle than in CNS where infectivity titers are known to reach high levels. Although possible effects of CWD strains or strain mixtures on these incubation times cannot be excluded, the variable 360 to ~490 d incubation times suggested a range of prion titers in skeletal muscles of CWD-affected deer. Muscle prion titers at the high end of the range produced the fastest incubation times that were ~30% longer than the incubation times of prions from the CNS of the same animal. Since all mice in each inoculation group developed disease, prion titers in muscle samples producing the longest incubation times were higher than the end point of the bioassay, defined as the infectious dose at which half the inoculated mice develop disease. Studies are in progress to accurately assess prion titers.

While the risk of exposure to CWD infectivity following consumption of prions in muscle is mitigated by relatively inefficient prion transmission via the oral route (8), these

results show that semitendinosus/semimembranosus muscle, which is likely to be consumed by humans, is a significant source of prion infectivity. Humans consuming or handling meat from CWD-infected deer are therefore at risk to prion exposure.

References and Notes

1. P. J. Bosque et al., Proc. Natl. Acad. Sci. U.S.A. 99, 3812 (2002).

2. S. R. Browning et al., J. Virol. 78, 13345 (2004).

3. A. Buschmann, M. H. Groschup, J. Infect. Dis. 192, 934 (2005).

4. O. Andreoletti et al., Nat. Med. 10, 591 (2004).

5. T. R. Spraker et al., Vet. Pathol. 39, 110 (2002).

6. A. N. Hamir, J. M. Miller, R. C. Cutlip, Vet. Pathol. 41, 78 (2004).

7. S. B. Prusiner et al., Biochemistry 21, 4883 (1980).

8. M. Prinz et al., Am. J. Pathol. 162, 1103 (2003).

9. This work was supported by grants from the U.S. Public Health Service 2RO1 NS040334-04 from the National Institute of Neurological Disorders and Stroke and N01-AI-25491 from the National Institute of Allergy and Infectious Diseases.

Supporting Online Material



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Materials and Methods

Fig. S1

21 November 2005; accepted 13 January 2006 Published online 26 January 2006; 10.1126/science.1122864 Include this information when citing this paper.

Table 1. Incubation times following inoculation of Tg(CerPrP)1536 mice with prions from skeletal muscle and brain samples of CWD-affected deer.

Inocula Incubation time, mean d ± SEM (n/n0)*

Skeletal muscle Brain

CWD-affected deer

H92 360 ± 2 d (6/6) 283 ± 7 d (6/6)

33968 367 ± 9 d (8/8) 278 ± 11 d (6/6)

5941 427 ± 18 d (7/7)

D10 483 ± 8 d (8/8) 231 ± 17 d (7/7)

D08 492 ± 4 d (7/7)

Averages 426 d 264 d

Non-diseased deer

FPS 6.98 >523 d (0/6)

FPS 9.98 >454 d (0/7) >454 d (0/6)

None >490 d (0/6)

PBS >589 d (0/5)

*The number of mice developing prion disease divided by the original number of inoculated mice is shown in parentheses. Mice dying of intercurrent illnesses were excluded.



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Supporting Online Material for

Prions in Skeletal Muscles of Deer with Chronic Wasting Disease

Rachel C. Angers, Shawn R. Browning, Tanya S. Seward, Christina J. Sigurdson,

Michael W. Miller, Edward A. Hoover, Glenn C. Telling§

§To whom correspondence should be addressed: E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000076/!x-usc:mailto:gtell2@uky.edu

Published 26 January 2006 on Science Express

DOI: 10.1126/science.1122864

This PDF file includes:

Materials and Methods

Fig. S1

Supporting Online Materials

Materials and Methods

Homogenates of semitendinosus/semimembranosus muscle (10% w/v in phosphate

buffered saline) were prepared from five emaciated and somnolent mule deer, naturally

infected with CWD at the Colorado Division of Wildlife, Wildlife Research Center.

These deer were identified as D10, D08, 33968, H92, and 5941. CWD infection was

confirmed in all cases by the presence of histologic lesions in the brain including

spongiform degeneration of the perikaryon, the immunohistochemical detection of

disease-associated PrP in brain and tonsil, or by immunoblotting of protease-resistant,

disease associated PrP (CerPrPSc). Semitendinosus/semimembranosus muscle was also

obtained from two asymptomatic, mock inoculated deer, referred to as FPS 6.68 and 9.98,

that originated from a CWD non-endemic area and which were held indoors at Colorado

State University from ten days of age. These control deer were confirmed negative for

CWD by histopathological and immunohistochemical analysis of brain tissue at autopsy.

The utmost care was taken to avoid inclusion of obvious nervous tissue when muscle

biopsies were prepared and to ensure that contamination of skeletal muscle samples with

CNS tissue did not occur. Fresh, single-use instruments were used to collect each sample

biopsy and a central piece from each sample was prepared with fresh, disposable

instruments to further isolate muscle tissue for inoculum preparation. Brain samples for

transmission were prepared separately from muscle as additional insurance against cross

contamination.

1

Groups of anesthetized Tg(CerPrP)1536 mice were inoculated intracerebrally with 30 µl

of 1 % skeletal muscle or brain extracts prepared in phosphate buffered saline (PBS).

Inoculated Tg(CerPrP) mice were diagnosed with prion disease following the progressive

development of at least three neurologic symptoms including truncal ataxia, ‘plastic’ tail,

loss of extensor reflex, difficultly righting, and slowed movement. The time from

inoculation to the onset of clinical signs is referred to as the incubation time.

For PrP analysis in brain extracts of Tg(CerPrP)1536 mice, 10 % homogenates prepared

in PBS were either untreated (-) or treated (+) with 40 µg/ml proteinase K (PK) for one

hour at 37oC in the presence of 2% sarkosyl. Proteins were separated by sodium dodecyl

sulfate polyacrylamide gel electrophoresis, analyzed by immunoblotting using anti PrP

monoclonal antibody 6H4 (Prionics AG, Switzerland), incubated with appropriate

secondary antibody, developed using ECL-plus detection (Amersham), and analyzed

using a FLA-5000 scanner (Fuji).

2

Fig. S1

PrP in brain extracts from representative Tg(CerPrP)1536 mice receiving muscle or CNS

tissue inocula from CWD-affected or CWD-negative deer. Extracts were either treated

(+) or untreated (-) with proteinase K (PK) as indicated. The positions of protein

molecular weight markers at 21.3, 28.7, 33.5 kDa (from bottom to top) are shown to the

left of the immunoblot.

3



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