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Chronic Wasting Disease CWD TSE Prion and Urine
Prions have also been detected in saliva, feces, blood, velvet, and urine (Angers et al. 2006, Angers et al. 2009, Haley et al. 2011, Mathiason et al. 2006). Most urine sold commercially is collected from captive deer facilities. It may be batched from several locations and can be distributed across the country via retail and internet/catalog sales. As stated above, movement of animals and recent failures in CWD certification programs indicate that captive cervids present a higher risk for CWD. If real cervid urine containing prions is put on the landscape by deer hunters, in a scrape or other area used by cervids, prions may bind to soil and contaminate that location for years or decades. Prions deposited in this manner will have a cumulative effect over time. Plants are capable of binding prions on leaves and taking up prions into their tissues; those prions remain infectious (Pritzkow et al. 2015). Cervids attracted to that location (cervid urine is marketed as a deer or elk attractant) have the potential to then ingest prions and become infected. Alaska, Vermont, Virginia, and several Canadian provinces have already banned natural cervid urine for hunting because of these risks. There is no “safe” dose of prion; exposure to one prion may be enough to cause infection (Fryer and McLean 2011). Once infected, exposed cervids shed prions into the environment where they can be encountered by unexposed deer. Infected cervids are known to shed prions prior to showing signs of disease. There is currently no rapid, cost effective test to determine if collected urine contains prions (John et al. 2013). A complete ban on use of products that contain or purport to contain real or “natural” cervid urine and glandular products would limit sales, possession, and use afield, and keep prions off the landscape via this route.
Action 1.1.2: DEC will amend 6 NYCRR Part 189 to prohibit retail sale, and possession, use, and distribution while afield of the urine, glands, or other excreted substances or products containing the urine or excreted substances from any CWD‐susceptible animal for any purpose.
Advantage: 1. Limits abilities of hunters to use urine or urine based products while afield deer hunting; 2. Prohibits the sale of urine by the deer farm industry in New York to send a clear message about the hazard of this product
Disadvantage: Urine producers will no longer have a market for their products in New Yorkhttp://www.dec.ny.gov/docs/wildlife_pdf/cwdpreventionplan2017draft.pdf
Temporal patterns of chronic wasting disease prion excretion in three cervid species
Authors: Ian H. Plummer1​, Scott D. Wright2​,†​, Chad J. Johnson3​, Joel A. Pedersen3​, Michael D. Samuel4​
*Correspondence: Michael D. Samuel, firstname.lastname@example.org
First Published Online: 15 July 2017, Journal of General Virology doi: 10.1099/jgv.0.000845 Subject: Research Article - TSE Agents Received: 07/12/2016 Accepted: 22/05/2017 Cover date: 15/07/2017
Chronic wasting disease (CWD) is the only naturally occurring transmissible spongiform encephalopathy affecting free-ranging wildlife populations. Transmission of CWD occurs by direct contact or through contaminated environments; however, little is known about the temporal patterns of CWD prion excretion and shedding in wild cervids. We tested the urine and faeces of three species of captive cervids (elk, mule and white-tailed deer) at 6, 12, 18 and 24 months after oral inoculation to evaluate the temporal, species- and genotype-specific factors affecting the excretion of CWD prions. Although none of the animals exhibited clinical signs of CWD during the study, we determined that all three cervid species were excreting CWD prions by 6 months post-inoculation. Faecal samples were consistently positive for CWD prions for all three cervid species (88 %), and were more likely to be positive than urine samples (28 %). Cervids with genotypes encoding for the prion protein (PRNP) that were considered to be more susceptible to CWD were more likely to excrete CWD prions (94 %) than cervids with genotypes considered to be less susceptible (64 %).
All cervids with CWD prions in their urine also had positive faeces (n=5), but the converse was not true.
Our study is the first to demonstrate CWD prion excretion in urine by asymptomatic elk and mule deer.
Our results indicate that the excretion of CWD prions in faeces and, to a lesser extent, urine may provide an important avenue for depositing prions in the environment.
Keyword(s): cervids, prions, PMCA, excretion, shedding, chronic wasting diseasehttp://jgv.microbiologyresearch.org/content/journal/jgv/10.1099/jgv.0.000845
P52 NaPTA/RT-QUIC detection of PrPCWD in saliva and urine of CWD-infected cervids and TgElk mice
Hyun Joo Sohn1, Kyung Je Park1, Gordon Mitchell2, In Soon Roh1, Hyo Jin Kim1, Hae Eun Kang1 1Foreign Animal Disease Division Animal And Plant Quarantine Agency(QIA), Gimcheon, South Korea, 2Canadian Food Inspection Agency, Ottawa, Canada
Aims: Chronic wasting disease(CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea and Norway. CWD agents are shed in saliva, urine and feces which most likely contribute to the horizontal transmission between cervid species.The development of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using NaPTA precipitation and real-time quaking-induced conversion(NaPTA/RT-QUIC), we established a ultrasensitive detection method for PrPCWD in the saliva and urine of CWD affected cervids. Also we performed the longitudinal study to detect PrPCWD from ,in the CWD-infected, sequentially sampled transgenic mice overexpressing elk prion protein(TgElk mice).
Methods: Five saliva and two urine samples from CWD-infected cervids at the terminal stage of disease, and 28 urine samples from sequentially sampled CWD-infected TgElk mice (TgElk CWD) were stored at -80℃. 100uL of each sample was mixed with 10uL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37℃ with shaking at 1,350 rpm. Samples were centrifuged for 30min at 16,100 g. The pellet was resuspended in 10uL of 0.1% SDS/PBS for 30min at 55℃. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98uL RT-QUIC buffer [final concentrations of 1XPBS, 1mM EDTA, 10uM Thioflavin, 300mM NaCl buffer and 0.1mg/ml recombinant hamster recombinant protein(23-231) and 2uL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 55℃ for 60hr with 1min shaking at 700rpm followed by 1min incubation.
Results: NaPTA/RT-QUIC was applied to measure PrPCWD in urine samples collected on every 15days from 30dpi to 120dpi when CWD infected TgElk mice reached terminal stage. . and dpi typicallyCWD in PrPCWD in the urine in TgElk CWD was detectable in early stages(30 and 45dpi), disappeared during the intermediate stages of infection(60 and 75dpi) and reached the highest levels at 90dpi. PrPCWD was also detectable in late and terminal stages(120dpi). In addition, PrPCWD was detected in terminal urine samples from two sika deer(experimental cases) and terminal saliva samples from five cervids were also observed to consistently yield positive results by the NaPTA/RT-QUIC assay.
Conclusions: We demonstrate that CWD prions can be detected by NaPTA/RT-QUIC in the saliva and urine of TgElk mice, red deer and sika deer at the early and terminal stages of disease. Our method appears to be a very useful technique for both diagnosis and surveillance of CWD
P.141: Abundant prion shedding in CWD-infected deer revealed by Realtime conversion
Edward A Hoover,1 Davin M Henderson,1 Nathaniel D Denkers,1 Candace K Mathiason,1 Matteo Manca,2,3 and Byron Caughey2 1Prion Research Center, Colorado State University; Fort Collins, CO USA; 2Laboratory of Persistent Viral Diseases, NI AID; Hamilton, MT USA; 3Department of Biomedical Sciences, University of Cagliari; Monserrato, Italy
Background/Introduction. Chronic wasting disease (CWD) is unique among prion diseases in its efficient lateral transmission in nature. While the presence of infectious prions in body fluids and excreta of infected cervids has been demonstrated by bioassay, the dynamics, magnitude, and consequences of prion shedding remain unknown. The present studies were undertaken to determine the kinetics, duration, and magnitude of prion shedding in infected white-tailed deer.
Materials and Methods. Longitudinal samples were collected from white-tailed deer over a 2-year span after either oral (n=11)] aerosol (n = 6) CWD exposure. The assay protocol employed phosphotungstic acid precipitation of either whole saliva or the pelleted fraction of urine to seed recombinant Syrian hamster prion PrP substrate in RT-QuIC reactions. Prion seeding activity was assayed in 8 replicates of each sample employing thioflavin T detection in a 96-well plate-based fluorometer. Prion seeding reaction rate was determined by taking the inverse of the time at which samples exceeded a threshold of 5 standard deviations above the mean fluorescence of negative controls (1/time to threshold). Seeding activity was quantitated by comparing the realtime conversion reaction rate to a standard curve derived from a reference bioassayed brain pool homogenate from deer with terminal CWD.
Results. We analyzed >200 longitudinally collected, blinded, then randomized saliva and urine samples from 17 CWDinfected and 3 uninfected white-tailed deer. We detected prion shedding as early as 3 months post exposure and sustained thereafter throughout the disease course in both aerosol and orally exposed deer. The incidence of non-specific false positive results from > 500 saliva and urine samples from negative control deer was 0.8%. By comparing real-time reaction rates for these body fluids to a bioassayed serially diluted brain control, we estimated that ≤1 ml of saliva or urine from pre-symptomatic infected deer constitutes a lethal infectious prion dose.
***Conclusion. CWD prions are shed in saliva and urine of infected deer as early as 3 months post infection and throughout the subsequent >1.5 year course of infection. In current work we are examining the relationship of prionemia to excretion and the impact of excreted prion binding to surfaces and particulates in the environment.
Acknowledgments. Support: NIH-RO1-NS-061902; Morris Animal Foundation D12ZO-045
P.154: Urinary shedding of prions in Chronic Wasting Disease infected white-tailed deer
Nathaniel D Denkers,1 Davin M Henderson, 1 Candace K Mathiason,1 and Edward A Hoover1 1Prion Research Center, Department of Microbiology, Immunology, and Pathology, Colorado State University; Fort Collins, CO USA
Background/Introduction. Chronic wasting disease (CWD) is unique among prion diseases in its efficient lateral transmission in nature, yet the dynamics and magnitude of shedding and its immediate and long term consequences remain unknown. The present study was designed to determine the frequency and time span in which CWD prions are shed in urine from infected white-tailed deer using adapted real-time quaking-induced conversion (RT-QuIC) methodology.
Materials and Methods. Longitudinal urine samples were collected by free catch or catheterization over a 2-year period from oral-route infected [CWD+ (n = 11)] and aerosol-route-infected [CWD+ (n = 6); CWD- (n = 3)] white-tailed deer. High speed centrifugation pelleted material from 500 µl of urine was treated with sodium phosphotungstic acid (Na-PTA), resuspended in 0.05% SDS buffer, and used as seed in RT-QuIC assays employing recombinant Syrian hamster prion PrP substrate. Eight (8) replicates of each sample were run and prion seeding activity was recorded as thioflavin T binding fluorescence (480 nm emission) using a fluorimeter-shaker. Samples were considered positive if they crossed an established threshold (5 standard deviations above the negative mean fluorescence).
***Results. In our oral-route inoculation studies, prion seeding activity has been demonstrated in urine collected at 6 months post-inoculation in 6 of 10 deer (11 of 80 replicates; 14%), and intermittently at later time points in all 11 CWD+ exposed deer. Our aerosol-route inoculation studies also showed prion seeding activity in urine collected at 6 months post-inoculation in 1 of 2 deer (3 of 16 replicates; 19%), and intermittently at later time points in 4 of 6 CWD+ exposed deer. Urine from sham-inoculated control deer and all baseline samples yielded 3 false-positive prion seeding activities (3 of 352 replicates; 0.8%).
***Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC) are shed in urine of infected deer as early as 6 months post inoculation and throughout the subsequent disease course. Further studies are in progress refining the real-time urinary prion assay sensitivity and we are examining more closely the excretion time frame, magnitude, and sample variables in relationship to inoculation route and prionemia in naturally and experimentally CWD-infected cervids.
Acknowledgments. Support: NIH: RO1-NS-061902 and Morris Animal Foundation: D12ZO-045
P.178: Longitudinal quantitative analysis of CWD prions shed in saliva of deer
Davin M Henderson, Nina Garbino, Nathaniel D Denkers, Amy V Nalls, Candace K Mathiason, and Edward A Hoover Prion Research Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University; Fort Collins, CO USA
Background/Introduction. Chronic Wasting Disease (CWD) is an emergent rapidly spreading fatal prion disease of cervids (deer, elk and moose). CWD has now been identified in 22 States (including two new states within the last year), 2 Canadian provinces, and South Korea. Shedding of infectious prions in excreta (saliva, urine, feces) may be an important factor in CWD transmission. Here we apply an adapted version of a rapid in vitro assay [real-time quaking-induced conversion (RT-QuIC)] to determine the time of onset, length, pattern, and magnitude of prion shedding in saliva of infected deer.
Materials and Methods. The RT-QuIC assay was performed as previously described in Henderson et al. PLoS-One (2013). Saliva samples were quantitated by comparison to a RT-QuIC reaction rate standard curve of a bioassayed obex sample from a terminally ill cervid.
Results. To better understand the onset and length of CWD prion shedding we analyzed >150 longitudinally collected, blinded, then randomized saliva samples from 17 CWD-infected and 3 uninfected white-tailed deer. We observed prion shedding, as detected by the RT-QuIC assay, as early as 3 months from inoculation and sustained shedding throughout the disease course in both aerosol and orally exposed deer. We estimated the infectious lethal dose of prions shed in saliva from infected deer by comparing real-time reaction rates of saliva samples to a bioassayed serially diluted brain control. Our results indicate that as little as 1 ml of saliva from pre-symptomatic infected deer constitutes a lethal CWD prion dose.
***Conclusions. During the pre-symptomatic stage of CWD infection and throughout the course of disease deer may be shedding multiple LD50 doses per day in their saliva. CWD prion shedding through saliva and excreta may account for the unprecedented spread of this prion disease in nature.
Acknowledgments. Supported by NIH grant RO1-NS-061902 and grant D12ZO-045 from the Morris Animal Foundation.https://www.landesbioscience.com/journal...n%20Animals.pdf
Sunday, September 13, 2015
urine, feces, and chronic wasting disease cwd tse prion risk factors, loading up the environmenthttp://chronic-wasting-disease.blogspot.com/2015/09/urine-feces-and-chronic-wasting-disease.html
snip...see more here;
SUNDAY, JULY 16, 2017
*** Temporal patterns of chronic wasting disease prion excretion in three cervid species ***http://chronic-wasting-disease.blogspot.com/2017/07/temporal-patterns-of-chronic-wasting.html
Using in vitro prion replication for high sensitive detection of prions and prionlike proteins and for understanding mechanisms of transmission.
Claudio Soto Mitchell Center for Alzheimer's diseases and related Brain disorders, Department of Neurology, University of Texas Medical School at Houston.
***Recently, we have been using PMCA to study the role of environmental prion contamination on the horizontal spreading of TSEs. These experiments have focused on the study of the interaction of prions with plants and environmentally relevant surfaces. Our results show that plants (both leaves and roots) bind tightly to prions present in brain extracts and excreta (urine and feces) and retain even small quantities of PrPSc for long periods of time. Strikingly, ingestion of prioncontaminated leaves and roots produced disease with a 100% attack rate and an incubation period not substantially longer than feeding animals directly with scrapie brain homogenate. Furthermore, plants can uptake prions from contaminated soil and transport them to different parts of the plant tissue (stem and leaves). Similarly, prions bind tightly to a variety of environmentally relevant surfaces, including stones, wood, metals, plastic, glass, cement, etc. Prion contaminated surfaces efficiently transmit prion disease when these materials were directly injected into the brain of animals and strikingly when the contaminated surfaces were just placed in the animal cage. These findings demonstrate that environmental materials can efficiently bind infectious prions and act as carriers of infectivity, suggesting that they may play an important role in the horizontal transmission of the disease.
Since its invention 13 years ago, PMCA has helped to answer fundamental questions of prion propagation and has broad applications in research areas including the food industry, blood bank safety and human and veterinary disease diagnosis. https://prion2015.files.wordpress.com/2015/05/programguide1.pdf http://www.tandfonline.com/doi/pdf/10.4161/pri.28467
In conclusion, the results in the current study indicate that removal of furniture that had been in contact with scrapie-infected animals should be recommended, particularly since cleaning and decontamination may not effectively remove scrapie infectivity (31), even though infectivity declines considerably if the pasture and the field furniture have not been in contact with scrapie-infected sheep for several months. As sPMCA failed to detect PrPSc in furniture that was subjected to weathering, even though exposure led to infection in sheep, this method may not always be reliable in predicting the risk of scrapie infection through environmental contamination. These results suggest that the VRQ/VRQ sheep model may be more sensitive than sPMCA for the detection of environmentally associated scrapie, and suggest that extremely low levels of scrapie contamination are able to cause infection in susceptible sheep genotypes.
Keywords: classical scrapie, prion, transmissible spongiform encephalopathy, sheep, field furniture, reservoir, serial protein misfolding cyclic amplification http://journal.frontiersin.org/article/10.3389/fvets.2015.00032/full
FRIDAY, NOVEMBER 03, 2017
TPWD Texas Deer Hunters Set for Saturday’s Opening Day with CWD UPDATE !http://chronic-wasting-disease.blogspot.com/2017/11/tpwd-texas-deer-hunters-set-for.html
WEDNESDAY, OCTOBER 18, 2017
TEXAS Medina County Elk Tests Positive for Chronic Wasting Disease CWD TSE PRION harvested on a high-fenced premiseshttp://chronic-wasting-disease.blogspot.com/2017/10/texas-medina-county-elk-tests-positive.html
WEDNESDAY, SEPTEMBER 27, 2017
TEXAS, TPWD, WIN CWD COURT CASE AGAINST DEER BREEDERS CAUSE NO. D-1-GN-15-004391http://chronic-wasting-disease.blogspot.com/2017/09/texas-tpwd-win-cwd-court-case-against.html
MONDAY, AUGUST 14, 2017
*** Texas Chronic Wasting Disease CWD TSE Prion History ***http://chronic-wasting-disease.blogspot.com/2017/08/texas-chronic-wasting-disease-cwd-tse.htmlhttp://chronic-wasting-disease.blogspot.com/
kind regards, terry