Thursday, May 20, 2010

South Dakota CWD cases mounting

South Dakota CWD cases mounting

Latest Chronic Wasting Disease Testing REsults

From July 1, 2009 to April 30, 2010 a total of 1,823 samples have been collected for Chronic Wasting Disease surveillance in South Dakota. Breakdown of the sampling is as follows:

402 elk sampled - 6 positive

476 mule deer sampled - 8 positive

989 white-tailed deer - 13 positive

1 Moose sampled - 0 positive

Here is a list of positive test results for the surveillance period.

MD male from Rapid City Limits in Pennington County. (Sick/Surveillance)

WT female from Unit 27B in Fall River County. (Sick/Surveillance)

Elk male from Unit H4A in Custer County. (Hunter Harvest)

Elk male from Custer State Park in Custer County. (Hunter Harvest)

Elk female from Unit H3D in Custer County. (Hunter Harvest)

WT male from Unit BH1-11 in Custer County. (Hunter Harvest)

MD male from Unit 21A-08 in Custer County. (Hunter Harvest)

WT male from Unit 27B-08 Fall River County. (Hunter Harvest)

WT female from Unit 27A-08 in Fall River County. (Hunter Harvest)

MD female from Unit 27A-08 in Fall River County. (Hunter Harvest)

WT male from Unit 27B-08 in Fall River County. (Hunter Harvest)

WT female from Unit 21B-09 in Custer County. (Hunter Harvest)

MD male from Unit 21B-08 in Custer County. (Hunter Harvest)

MD male from Unit 27B-08 in Fall River County. (Hunter Harvest)

WT male from Unit 21A-18 in Custer County. (Hunter Harvest)

MD male from Unit 27B-08 in Fall River County. (Hunter Harvest)

WT female from Unit 27B-08 in Fall River County. (Hunter Harvest)

WT female from Unit 27B-08 in Fall River County. (Hunter Harvest)

WT male from Unit 27A-08 ion Fall River County. (Hunter Harvest)

Elk female from Unit H3D-23 in Custer County. (Hunter Harvest)

MD male from Hot Springs City Limits in Fall River County. (Sick/Surveillance)

WT female from Unit 21B-08 in Custer County. (Hunter Harvest)

WT female from Unit 27B in Fall River County. (Hunter Harvest)

WT female from Unit 27A-09 in Fall River County. (Hunter Harvest)

MD female from Unit 27B-08 in Fall River County. (Hunter Harvest)

Elk male from Unit H3A in Fall River County. (Sick/ Surveillance)

Elk male from Wind Cave National Park in Custer County. (sick/Surveillance)

Hunters may get their animal tested for chronic wasting disease by making their own arrangements directly through the SDSU Diagnostic Lab at 605.688.5171

Friday, May 14, 2010

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Published Online May 13, 2010 Science DOI: 10.1126/science.1187107 Science Express Index

Saturday, May 15, 2010

Epidemiology of Chronic Wasting Disease: PrPres Detection, Shedding, and Environmental Contamination REPORT DATE 1 August 2009


Labels: , , ,

Saturday, May 15, 2010

Epidemiology of Chronic Wasting Disease: PrPres Detection, Shedding, and Environmental Contamination REPORT DATE 1 August 2009

Epidemiology of Chronic Wasting Disease: PrPres Detection, Shedding, and Environmental Contamination

1. REPORT DATE 1 August 2009



Obviously the most important goal is to develop an extremely sensitive assay for the infective prion protein. We have made substantial progress since the start of the grant period but are still short of the goal. In a continuation of the proteomics work initiated last year we identified several candidate proteins that are found in urine of infected deer but not in uninfected controls. For some of these we were able to find antibodies and have confirmed the presence of three of these proteins over time using the samples that have been collected for deer over the infection period. Preliminary results indicate that the levels of the proteins increase during the infection period. We have started optimizing the assays on several of the proteins.

As this is a final report I will not reiterate the details presented in the past five years of the grant. However, I will summarize the results and total the publications etc. We are also in the process of filing a patent disclosure on the biomarker proteins we have identified.


Chronic wasting disease (CWD) of deer (Odocoileus spp.) and elk (Cervus elaphus) is unique among the transmissible spongiform encephalopathies (TSEs) in that it occurs in free-ranging as well as captive wild ruminants and environmental contamination appears to play a significant role in maintenance of the disease. The precise modes of transmission of CWD are not known although we have shown that horizontal transmission and environmental contamination associated with excreta and carcasses may occur (Miller et al., 2004). But maternal transmission does not appear to play a significant role (Miller and Williams, 2003) in maintenance of CWD in cervid populations. Our long-term goal is to better understand the epidemiology of CWD and apply that information to development of strategies for management and control. To that end we are investigating the dynamics and modes of CWD agent shedding from infected mule deer, white-tailed deer, and elk. The approach includes experimentally infecting cervids, serial collections of a variety of biological samples, and assay of these materials by various means to attempt to detect protease resistant prion protein (PrPres). In addition, because of the concern about environmental contamination associated with excreta, we will be collecting and assaying a variety of environmental specimens collected from areas of presumed high, moderate, and low contamination in CWD endemic facilities.


Aim 1: Develop analytical tools to detect PrPCWD in excreta, blood, and environmental samples.

Biomarker Discovery for Chronic Wasting Disease

Initial Identification of Biomarkers

We have accomplished an extensive analysis of urine from CWD-positive animals. The analysis has identified 11 potential biomarkers, as represented in Table 1. Urine is an ideal source for biomarkers (Aguzzi A, 2004) and we feel strongly that markers found in the urine will also be present in the serum and other tissues of infected animals and our preliminary results are confirming this. The potential protein markers were identified based on their similarity to known proteins from other mammals, since the deer genome sequence has not been characterized. As such, it is imperative that we accurately identify these proteins. We present them here as CWD-1 thru 11 because we are not completely sure of the proteins identity (except where noted) even though we are seeing antibody crossreactivity as will be demonstrated in the following pages. Additionally, several of the proteins have a number of isoforms and we are unsure of which isoform we have identified that we are now seeing in blood and urine samples. Research contained within this proposal will appropriately identify the proteins.

Table 1: The identified potential biomarkers of Chronic Wasting Disease.

Biomarker Possible Physiological Role Summary

CWD-1 Required for a specialized brain endocytosis responsible for generating the synaptic vesicles that store and then release neurotransmitters. Also implicated in Alzheimer’s and early loss of cognitive ability.

CWD-2 Reported roles in cell function, clotting, memory and necrosis. Implicated in Alzheimer’s and its role in cleaving CWD-1 (see above) and is hypothesized to be partly responsible for early loss of cognitive ability.

CWD-3 Molecular chaperone in the eukaryotic cytosol assisting in protein folding.

CWD-4 A protein truncated in some forms of schizophrenia.

CWD-5 Found in Alexander’s disease, a progressive neurological disorder, associated with the destruction of white matter.

CWD-6 Indicator of multi-drug resistance in lung cancer.

CWD-7 A protein scaffold that is involved throughout the cell cycle.

CWD-8 A serine threonine protein kinase involved in mitosis.

CWD-9 Transmembrane protein that plays a critical role in cell adhesion.

CWD-10 Light chain IgG (Serban A, 2004)

CWD-11 An unknown protein that is visibly increased throughout the progression of the disease. Plans for its identification are underway.

Preliminary screening of samples with biomarker antibodies Initially to determine which of the proteins have merit as biomarkers for CWD, we purchased commercially available antibodies against the human or mouse forms of the protein, when available. This predisposes the interpretations to be overly cautious. However, the fact that those proteins for which we were able to obtain antibodies are showing up-regulation, or higher expression, in response to the diseased state strongly suggests we are on the right track. We have not tested some of the biomarkers as commercially available antibodies do not exist for them and we

currently do not have funding to generate those antibodies. So we have 6 markers (CWD 1,2,3,4, 10 and 11) that we have positive preliminary data on and that merit further validation. Given that we developed these protein biomarkers from urine, our initial screens of available proteins focused on urine from both positive and negative animals. Figure 2 shows the potential that these biomarkers hold. Using an off-the-shelf antibody to another species we obtained positive results. Further, we obtained results that show a tendency of the proteins to be increasingly abundant as the disease progresses. Urine is an ideal source of biomarker material in humans, but may prove less than ideal when trying to test for CWD. However, urine has been identified as an acceptable medium for the development of diagnostic tools (Aguzzi A, 2004).

Densitometry on the western blot of CWD-1 tested in urine shows a significant trend for the biomarker to be quantifiably higher as the disease progresses (Figure 3A). Figure 3B illustrates that there are still potential biomarkers to be discovered. Although it is beyond the scope of this grant to identify this potential marker, their existence helps to define the potential that protein biomarkers have in diagnosing CWD. All western blot results shown were performed on 10X concentrated urine. Recent analysis indicates that we can indeed detect the markers in unconcentrated urine (Figure 4). Further, the biomarkers can be observed and demonstrate a quantifiable difference throughout the disease state.

Testing in feces was undertaken as another means by which to indicate disease. Some preliminary successes were accomplished (data not shown). However, fecal samples have proved very difficult to analyze. Given that CWD is the only one of the TSE diseases that lends itself to being monitored through feces, we have not chosen to continue this line of research. Serum and urine are far more useful when applied to human TSE’s and the TSE’s that are known to affect humans, which CWD does not.

Further diversification of the medium of detection to serum broadens the capability of the biomarkers. We have met with limited success in this endeavor largely due to the non specific nature of the antibodies. One clear success on this front is CWD-4 (which according to literature should be represented by a 100kDa and 75kDa band), which is visible at appropriate molecular weights (Figure 5) in the infected animal and clearly less prevalent in the non-infected animals.

We are basing our premise that these will be good biomarkers for the disease on the fact that even with the imperfect antibodies and conditions, we are seeing the protein(s) in the infected animals at well above background levels as the disease progresses in the urine, serum and feces. Perhaps most importantly, we see signal above background very early in the infection.

Preliminary Results Summary

Results obtained thus far are very promising but underscore the need to develop species specific antibodies. The different and complex mediums in which we are testing require specific antibodies if these biomarkers are ever to be used to develop a quick, ante mortem test.

The different TSE diseases lend themselves to detection via biomarkers in different mediums. It is not very efficient to collect urine from wild animals within wild populations such is the case in CWD. As well, blood and serum work well for BSE in the feedlot or slaughter house but urine would seem to be the easiest medium of detection in CJD or vCJD. In both of these mediums we have had success in detecting the markers. The limiting factor is non-specificity to the species. Having multiple biomarkers would allow a testing format that would not rely on a single marker, thus reducing the possibility of getting false positive or negative results. A multiple marker format would also alleviate the argument raised against the use of ESM as an indicator of TSE disease, which is that different individuals have varying levels of transcript (Glock B, 2003) As with all biomarkers there is the potential that the markers may be abundant in other states than the disease of interest. However, a multiple marker format would alleviate that concern. In our proposed system only having one marker indicate positive would not be a positive result. It would require more than one of the markers to indicate the presence of the disease with certainty. Further, our proposed method of utilizing the known light chain IgG (CWD-10) as a fail-safe control alleviates that concern that one marker is insufficient to diagnose the diseased state. With specific antibodies we can determine not only which of the biomarkers are amenable to detection but if they are preferentially detected in one medium and not another.

Given that our laboratory has an extensive library of CWD infected tissues in addition to the facilities and equipment required, we are proposing to develop the biomarkers further using CWD as our TSE of choice. We do expect to be able to test the relevance of our biomarkers in other TSE diseases, but that is beyond the scope of this grant. It is our goal to establish which of the biomarkers, when specific antibodies are used for detection, are useful for confirming the disease. As well, we will establish which biomarkers are useful when applied to urine or serum. With that information we can then develop a test format that will quickly and accurately diagnose the presence of CWD.


Determined that high sensitivity detection of the prion protein cannot be accomplished with out sacrificing both false negative and positive results. Confirmed difficulties reported by others with all of the amplification methods, particularly the false positives, which obviate their standard use for detection. Identified several proteins that can serve as biomarkers for detection of CWD in live animals from both urine and serum. Aim 2. Evaluate multiple biological samples collected from experimentally infected mule deer, white-tailed deer, and elk throughout the CWD incubation period.


• CWD infections established, confirmed, and monitored to terminus in mule deer and white-tailed deer and elk. • Serial samples of excreta collected from throughout the disease course from both mule deer and white-tailed deer and elk are available for analysis of prion shedding patterns. • Genetic influences on disease course in infected white-tailed deer and elk demonstrated, affording opportunities to evaluate the influence of genotype on agent shedding. • Archived materials shared with other laboratories to advance overall progress on developing sensitive assays for prion detection in blood and excreta, investigating potential routes of prion shedding in deer and elk, and exploring patterns of prion shedding during the disease course. Aim 3. The goal of this Aim is to determine if PrPres can be detected in samples collected from facilities contaminated with the CWD agent.


• CWD infections established and confirmed in mule deer and white-tailed deer. • PrPCWD demonstrated in tonsil and rectal mucosa biopsies from infected mule deer and white-tailed deer.

• Clinical CWD demonstrated in experimentally infected mule deer and white-tailed deer. • Archived materials shared with other laboratories to advance overall progress on developing sensitive assays for prion detection in blood.


(2007) Chang, B., X. Cheng, S. Yin, T. Pan, H. Zhang, P. Wong, S.-C. Kang, F. Xiao, H. Yan, C. Li, L. L. Wolfe, M. W. Miller, T. Wisniewski, M. I. Greene, and M.-S. Sy.. Test for detection of disease-associated prion aggregate in the blood of infected but asymptomatic animals. Clinical and Vaccine Immunology 14:36-43.

(2007) Wolfe, L. L., T. R. Spraker, L. González, M. P. Dagleish, T. M. Sirochman, J. C. Brown, M. Jeffrey, & M. W. Miller. PrPCWD in rectal lymphoid tissue of deer (Odocoileus spp.). Journal of General Virology 88: 2078-2082.

(2008) Benjamin D. Brooks, Amy E. Albertson, Justin A. Jones, Jonathan O. Speare, Randolph V. Lewis, Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target, Analytical Biochemistry 382: 60-62.

(2009) Brooks, Benjamin and Lewis, Randolph V. Identification of Problems Developing an Ultrasensitive Immunoassay for the Ante Mortem Detection of the Infectious Isoform of the CWD-Associated Prion Protein, Journal of Immunoassay and Immunochemistry, 30: 135– 139.


Surplus samples collected in the course of investigations supported by this grant have been shared with at least three other collaborating institutions (Rocky Mountain Laboratories, NIHNIAID; Case Western Reserve University; Institute for Neurodegenerative Diseases, University of California, San Francisco) in the hopes of advancing scientific understanding of CWD in particular and prion diseases in general. Other similar collaborative endeavors will be supported as feasible using materials arising from our work.

Friday, May 14, 2010

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Published Online May 13, 2010 Science DOI: 10.1126/science.1187107 Science Express Index


Labels: , , , , ,

Friday, May 14, 2010

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Published Online May 13, 2010 Science DOI: 10.1126/science.1187107 Science Express Index


Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Rachel C. Angers,1,* Hae-Eun Kang,2 Dana Napier,2 Shawn Browning,1, Tanya Seward,2 Candace Mathiason,4 Aru Balachandran,5 Debbie McKenzie,6 Joaquín Castilla,7 Claudio Soto,8 Jean Jewell,9 Catherine Graham,10 Edward A. Hoover,4 Glenn C. Telling1,2,3,

Prions are infectious proteins composed of PrPSc, which induces conformational conversion of host-encoded PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties, but comprised of PrPSc with indistinguishable biochemical characteristics. While CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk PrP differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.


The identification and characterization here of distinct CWD strains with similar conformations, and the influence of PrP primary structure on their stabilities, is of importance when considering the potential for transmission to species outside the family cervidae. While CWD prions have reassuringly failed to induce disease in transgenic mice expressing human PrP (10, 30), because of the risk of prion exposure from contaminated venison (13) and other infected materials (14), systematically addressing the tissue distributions of CWD1 and CWD2 and their zoonotic potentials would appear to be high priorities.

(10) Transmission of Elk and Deer Prions to Transgenic Mice

(30) Neurobiology of Disease Chronic Wasting Disease of Elk: Transmissibility to Humans Examined by Transgenic Mouse Models

(13) Prions in Skeletal Muscles of Deer with Chronic Wasting Disease;311/5764/1117

(14) Volume 15, Number 5–May 2009 Research Chronic Wasting Disease Prions in Elk Antler Velvet

1 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, Lexington, KY 40536, USA. 2 Sanders Brown Center on Aging, University of Kentucky Medical Center, Lexington, KY 40536, USA. 3 Department of Neurology, University of Kentucky Medical Center, Lexington, KY 40536, USA. 4 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5 Canadian Food Inspection Agency, Ottawa, Ontario, K2H 8P9, Canada. 6 Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada. 7 CIC bioGUNE & IKERBASQUE, Basque Foundation for Science, 48992 Derio & 48011 Bilbao, Bizkaia, Spain. 8 University of Texas Medical School at Houston, Houston, TX 77030, USA. 9 Department of Veterinary Sciences, University of Wyoming, Laramie, WY, USA. 10 Canadian Food Inspection Agency, Lethbridge, Alberta, T1J 3Z4, Canada. * Present address: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Present address: Department of Infectology, Scripps Research Institute, Jupiter, FL, USA.

To whom correspondence should be addressed. E-mail:


Received for publication 14 January 2010. Accepted for publication 1 April 2010.

Supporting Online Material for

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Rachel C. Angers, Hae-Eun Kang, Dana Napier, Shawn Browning, Tanya Seward, Candace Mathiason, Aru Balachandran, Debbie McKenzie, Joaquín Castilla, Claudio Soto, Jean Jewell, Catherine Graham, Edward A. Hoover, Glenn C. Telling*

*To whom correspondence should be addressed. E-mail:

Published 13 May 2010 on Science Express DOI: 10.1126/science.1187107

This PDF file includes: Materials and Methods Table S1 References


Supporting Online Material

Materials and Methods

Transgenic mice and inocula Transgenic mice expressing deer or elk PrP coding sequences, referred to as Tg(CerPrP)1536+/- and Tg(CerPrP-E226)5037+/- respectively, have been described previously (S1, S2). All transmitted isolates in this study originated from deer and elk expressing wild type PRNP coding sequences. The 03W1755 elk used in PMCA studies was heterozygous (M/L) at codon 132. Ten % (w/v) homogenates, in phosphate buffered saline (PBS) lacking calcium and magnesium ions, of cervid and mouse brains were prepared by repeated extrusion through an 18 gauge followed by a 21 gauge syringe needle.

Determination of Incubation Periods Groups of anesthetized mice were inoculated intracerebrally with 30 µl of 1 % (w/v) brain extracts prepared and diluted in PBS, or 1 % v/v of the final PMCA product diluted in PBS. Groups of mice were monitored thrice weekly for the development of prion disease. Following a relatively non-specific prodromal phase, early definitive and progressive clinical signs included stimulation-induced hyperexcitability, and flattened posture, culminating in profound ataxia toward the endpoint of disease. CWD-affected mice were rarely kyphotic and maintained a deep pain reflex at end stage. Inoculated mice were diagnosed with prion disease following the progressive development of at least three clinical signs, the time from inoculation to the onset of definitive and subsequently progressive clinical signs being referred to as the incubation time.


Analysis of PrP Animals whose death was obviously imminent were euthanized and their brains taken for biochemical and histopathological studies. For PrP analysis in brain extracts, total protein content from 10 % brain homogenates prepared in PBS was determined by bicinchoninic acid assay (Pierce Biotechnology Inc., Rockford, IL). Brain extracts were either untreated or treated with 40 µg/ml PK for one hour at 37oC in the presence of 2 % sarkosyl and the reaction was terminated with 4 mM phenyl methyl sulfonyl fluoride. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrophoretically transferred to PVDF-FL membranes (Millipore, Billerica, MA), which were probed with anti-PrP mAbs followed by horse radish peroxidaseconjugated sheep anti-mouse IgG, developed using ECL-plus detection (Amersham), and analyzed using an FLA-5000 scanner (Fuji). To determine the relative values of CerPrPSc glycoforms, band intensities were analyzed by densitometry of Western blots using the FLA-5000 scanner.

For histoblot analysis, mice exhibiting neurological dysfunction were humanely killed and their brains immediately frozen on dry ice. Ten µm thick cryostat sections were transferred to nitrocellulose as previously described (S3). Histoblots were immunostained with mAb 6H4 followed by alkaline phosphataseconjugated sheep anti-mouse secondary antibody. Images were captured with a Nikon SMZ1000 microscope with Photometrics Coolsnap CF digital imager and processed with MetaMorph software.

PrPSc in brain homogenates of terminally sick mice was also analyzed by conformational stability assay (S4-S7). The relative amounts of bands


representing PK resistant CerPrPSc were analyzed by densitometry of Western blots using the FLA-5000 scanner. The sigmoidal dose-response was plotted using a four-parameter algorithm and non-linear least square fit. The Gdn.HCl concentration required to denature 50% of CerPrPSc is denoted as the (Gdn.HCl)1/2 value. For histopathological stu dies, brains were dissected rapidly after sacrifice of the animal and immersion fixed in 10% buffered formalin. Tissues were embedded in paraffin and 8 µm thick coronal microtome sections were mounted onto positively charged glass slides. Analysis of PrP in the brains of mice by IHC was performed as previously described (S8) using anti-PrP mAb 6H4 as primary antibody, and IgG1 biotinylated goat anti-mouse secondary antibody (Southern Biotech). Following inactivation of endogenous peroxidases by incubation in 3% H2O2 in methanol, peroxidase immunohistochemistry was used to evaluate the extent of reactive astrocytic gliosis using antibodies to glial fibrillary acidic protein. Detection was with Vectastain ABC reagents and slides were developed with diaminobenzidine. Digitized images for figures were obtained by light microscopy using a Nikon Eclipse E600 microscope equipped with a Nikon DMX 1200F digital camera.

Neuropathological Lesion Profiling Paraffin-embedded mouse brains were sectioned coronally to areas corresponding to the five levels of the brain that contained the mouse brain regions of interest. Brain sections were stained with hematoxylin and eosin. Images of the each of the brain regions were captured using a Photometrics Cool Snap digital camera and a Nikon Eclipse E600


microscope. The extent of vacuolar degeneration in the cerebral grey matter was assessed using a semi-quantitative method for discriminating prion strains (S9). The numbers of vacuoles per field were manually counted. Text In addition to intrinsic strain characteristics, time to onset of disease is dependent on prion titers (S10). Both effects were evident in these studies. Preparations containing low CWD prions titers produce longer incubation times in Tg(CerPrP)1536+/- mice than higher titer isolates (S2). Prolonged incubation times resulting from primary transmission of low titer CWD isolates generally shorten on second passage in syngeneic hosts, while strain-related incubation time properties are expected to persist during serial transmission. To begin to distinguish the effects of strain and titer on the variable incubation times observed during primary transmissions, we performed serial transmissions in Tg(CerPrP)1536+/- mice (Table S1). Titer-related reduction in mean incubation time was a feature of many of serially passaged isolates (Table S1). Generally, on second passage, there was less variance of incubation times for both strains, an effect that was also likely to be related to more consistent prion titers. Factors affecting CWD prion titers include the stage of disease in affected deer and elk, the neuroanotomical locations from which prions were isolated, and possible effects of post-mortem interval.

The most extreme effects of titer were observed during transmission of the 012-22012 elk isolate. Despite a 387 d incubation time, CWD1 neuropathology


was registered in the only mouse available for analysis following transmission of elk isolate 012-22012 (Fig. 1A); 3 of 8 inoculated mice in this cohort did not develop disease (Table S1 and Fig. 1A). The protracted time to onset of disease and the less than 100 % attack rate on primary passage suggests that the titer of CWD1 prions in this elk isolate was close to the endpoint of sensitivity of the bioassay. Consistent with this notion, serial passage of 012-22012 prions from the brain of a second diseased mouse with a 380 d incubation time, produced a rapid mean incubation time of 208 ± 4 d in 8 inoculated mice and CWD1 neuropathology in all analyzed mice (n = 5) (Fig. 2A and Table S1).


Table S1: Transmission of CWD prions to Tg(CerPrP)1536+/- mice Incubation time, mean days ± SD (n/n0)

Inoculum Origin First Second


012-22012 Colorado 384 ± 3.3 (5/8) 208 ± 3.5 (8/8) 012-09442 Colorado 208 ± 16.9 (8/8) 307 ± 25.3 (6/6) 02-0306 Saskatchewan 225 ± 8.3 (7/7) 238 ± 38.1 (7/7) 12389 Wyoming 230 ± 24.4 (8/8) 001-44720 Colorado 231 ± 13.7 (7/7) 248 ± 37.5 (8/8) 7378-47 Wyoming 235 ± 5.5 (8/8) 230 ± 28.6 (7/7) 001-403022 Colorado 271 ± 35.9 (8/8) 235 ± 38.1 (8/8) 04-0306 Saskatchewan 281 ± 14.9 (7/7) 211 ± 7.5 (7/7) CWD pool Alberta 293 ± 30.9 (6/6) 01-0306 Saskatchewan 322 ± 25.3 (8/8) 274 ± 29.3 (8/8) 03-0306 Saskatchewan 335 ± 12.6 (7/7) 226 ± 45.9 (9/9)

Mule deer

8481 Wyoming 173 ± 3.8 (7/7) 217 ± 28.8 (7/7) 978-24384 Colorado 211 ± 22.5 (7/7) 229 ± 26.8 (5/5) D10 Colorado 228 ± 28.9 (15/15) 217 ± 28.3 (8/8) D92 Colorado 232 ± 48.8 (15/15) 244 ± 46.6 (7/7) 9179 Wyoming 239 ± 64.6 (7/7) 216 ± 32.0 (7/7) 989-09147 Colorado 250 ± 6.5 (8/8) 325 ± 36.0 (5/5) W97 Colorado 254 ± 27.1 (5/5) 226 ± 43.9 (7/7) 8905 Wyoming 259 ± 63.3 (8/8) 238 ± 28.6 (8/8) Db99 Colorado 259 ± 11.2 (7/7) 246 ± 15.8 (4/4) 7138 Wyoming 260 ± 46.6 (7/7) 216 ± 22.9 (7/7) CWD Pool Colorado 264 ± 9.3 (7/7) 207 ± 6.0 (6/6) 33968 Colorado 278 ± 27.0 (6/6) 239 ± 19.5 (8/8) H92 Colorado 283 ± 20.4 (6/6) 259 ± 44.6 (8/8) 04-22412 Wyoming 284 ± 54.4 (6/6) 001-39647 Colorado 289 ± 7.9 (5/5) 217 ± 47.1 (8/8) V92 Colorado 310 ± 30.5 (7/7) 288 ± 16.0 (8/8)

Whitetail deer

Wisconsin 200 ± 19.2 (6/6)2 206 ± 3.7 (8/8)


Elk 03W1755 Wyoming/Texas3 446 ± 22.6 (5/5)

Deer 04-22412 Wyoming/Texas3 264 ± 73.1 (6/6)

Total CWD14 212 ± 34.1 (n = 64) 206 ± 11.9 (n = 68)

Total CWD24 306 ± 48.5 (n = 78) 286 ± 22.1 (n = 46)

Saline 410 – 597 (0/7)

None 421 – 490 (0/7)

1 The number of mice developing prion disease (n), divided by the number inoculated (n0) is shown in parentheses. Mice dying of causes unrelated to prion disease were excluded.

2 PrPSc in this sample was precipitated with sodium phosphotungstate prior to inoculation.


3 Samples originated from Wyoming elk and deer; PMCA was accomplished in Texas.

4 Mean incubation times for primary transmission of naturally-occurring and PMCAgenerated CWD prions were determined in 64 neuropathologically confirmed mice with the CWD1 pattern, and 78 neuropathologically confirmed mice with the CWD2 pattern. Mean incubation times for secondary transmissions of CWD prions were determined in 68 neuropathologically confirmed mice with the CWD1 pattern, and 46 neuropathologically confirmed mice with the CWD2 pattern. For both primary and secondary passages, incubation times of CWD1 and CWD were different (p



Chad Johnson1, Judd Aiken2,3,4 and Debbie McKenzie4,5 1 Department of Comparative Biosciences, University of Wisconsin, Madison WI, USA 53706 2 Department of Agriculture, Food and Nutritional Sciences, 3 Alberta Veterinary Research Institute, 4.Center for Prions and Protein Folding Diseases, 5 Department of Biological Sciences, University of Alberta, Edmonton AB, Canada T6G 2P5

The identification and characterization of prion strains is increasingly important for the diagnosis and biological definition of these infectious pathogens. Although well-established in scrapie and, more recently, in BSE, comparatively little is known about the possibility of prion strains in chronic wasting disease (CWD), a disease affecting free ranging and captive cervids, primarily in North America. We have identified prion protein variants in the white-tailed deer population and demonstrated that Prnp genotype affects the susceptibility/disease progression of white-tailed deer to CWD agent. The existence of cervid prion protein variants raises the likelihood of distinct CWD strains. Small rodent models are a useful means of identifying prion strains. We intracerebrally inoculated hamsters with brain homogenates and phosphotungstate concentrated preparations from CWD positive hunter-harvested (Wisconsin CWD endemic area) and experimentally infected deer of known Prnp genotypes. These transmission studies resulted in clinical presentation in primary passage of concentrated CWD prions. Subclinical infection was established with the other primary passages based on the detection of PrPCWD in the brains of hamsters and the successful disease transmission upon second passage. Second and third passage data, when compared to transmission studies using different CWD inocula (Raymond et al., 2007) indicate that the CWD agent present in the Wisconsin white-tailed deer population is different than the strain(s) present in elk, mule-deer and white-tailed deer from the western United States endemic region.

Sunday, April 12, 2009

CWD UPDATE Infection Studies in Two Species of Non-Human Primates and one Environmental reservoir infectivity study and evidence of two strains

Thursday, April 03, 2008

A prion disease of cervids: Chronic wasting disease

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

A prion disease of cervids: Chronic wasting disease

Sigurdson CJ.


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


full text ;

From: TSS (


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

From: "Belay, Ermias"


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


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


Dear Sir/Madam,

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

That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091).

Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.

Ermias Belay, M.D.

Centers for Disease Control and Prevention

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


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

To:;; ebb8@CDC.GOV



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

SEE also ;

A. Aguzzi - Chronic Wasting Disease (CWD) also needs to be addressed. Most serious because of rapid horizontal spread and higher prevalence than BSE in UK, up to 15% in some populations. Also may be a risk to humans - evidence that it is not dangerous to humans is thin.


Chronic Wasting Disease and Potential Transmission to Humans

Ermias D. Belay,* Ryan A. Maddox,* Elizabeth S. Williams,? Michael W. Miller,? Pierluigi Gambetti,§ and Lawrence B. Schonberger*

*Centers for Disease Control and Prevention, Atlanta, Georgia, USA; ?University of Wyoming, Laramie, Wyoming, USA; ?Colorado Division of Wildlife, Fort Collins, Colorado, USA; and §Case Western Reserve University, Cleveland, Ohio, USA

Suggested citation for this article: Belay ED, Maddox RA, Williams ES, Miller MW, Gambetti P, Schonberger LB. Chronic wasting disease and potential transmission to humans. Emerg Infect Dis [serial on the Internet]. 2004 Jun [date cited]. Available from:


Chronic wasting disease (CWD) of deer and elk is endemic in a tri-corner area of Colorado, Wyoming, and Nebraska, and new foci of CWD have been detected in other parts of the United States. Although detection in some areas may be related to increased surveillance, introduction of CWD due to translocation or natural migration of animals may account for some new foci of infection. Increasing spread of CWD has raised concerns about the potential for increasing human exposure to the CWD agent. The foodborne transmission of bovine spongiform encephalopathy to humans indicates that the species barrier may not completely protect humans from animal prion diseases. Conversion of human prion protein by CWD-associated prions has been demonstrated in an in vitro cell-free experiment, but limited investigations have not identified strong evidence for CWD transmission to humans. More epidemiologic and laboratory studies are needed to monitor the possibility of such transmissions.

snip...full text ;

Volume 12, Number 10-October 2006


Human Prion Disease and Relative Risk Associated with Chronic Wasting Disease

Samantha MaWhinney,* W. John Pape,? Jeri E. Forster,* C. Alan Anderson,?§ Patrick Bosque,?¶ and Michael W. Miller#

*University of Colorado at Denver and Health Sciences Center, Denver, Colorado, USA; ?Colorado Department of Public Health and Environment, Denver, Colorado, USA; ?University of Colorado School of Medicine, Denver, Colorado, USA; §Denver Veteran's Affairs Medical Center, Denver, Colorado, USA; ¶Denver Health Medical Center, Denver, Colorado, USA; and #Colorado Division of Wildlife, Fort Collins, Colorado, USA

Suggested citation for this article

The transmission of the prion disease bovine spongiform encephalopathy (BSE) to humans raises concern about chronic wasting disease (CWD), a prion disease of deer and elk. In 7 Colorado counties with high CWD prevalence, 75% of state hunting licenses are issued locally, which suggests that residents consume most regionally harvested game. We used Colorado death certificate data from 1979 through 2001 to evaluate rates of death from the human prion disease Creutzfeldt-Jakob disease (CJD). The relative risk (RR) of CJD for CWD-endemic county residents was not significantly increased (RR 0.81, 95% confidence interval [CI] 0.40-1.63), and the rate of CJD did not increase over time (5-year RR 0.92, 95% CI 0.73-1.16). In Colorado, human prion disease resulting from CWD exposure is rare or nonexistent. However, given uncertainties about the incubation period, exposure, and clinical presentation, the possibility that the CWD agent might cause human disease cannot be eliminated.

snip... full text ;

full text ;


Labels: , , , ,