Friday, September 27, 2013

Uptake of Prions into Plants

Presentation Abstract  


Title: Uptake of Prions into Plants 


Session Title: Current Science of Chronic Wasting Disease: What Have We Learned in the Last 5 Years? 


Session Number: 27 


Session Time: Monday, Oct 07, 2013, 8:30 AM -12:20 PM 


Presentation Time: Monday, Oct 07, 2013, 11:00 AM -11:20 AM 


Presentation Number: 8 


Author(s): Christopher Johnson, U.S. Geological Survey, Madison, WI, Contact: 


Abstract Body:


Chronic wasting disease (CWD) and scrapie-infected animals shed infectious prions during both the preclinical and clinical phases of disease. Contamination of environments with prions released from animals or from infected carcasses appears to contribute to the transmission of these diseases. Previous work has suggested that soil may serve as an environmental disease reservoir. Vegetation is ubiquitous in CWD-contaminated environments and plants are known to absorb a variety of substances from soil, ranging from nutrients to contaminants. The uptake of proteins from soil into plants has been documented for many years and we have been investigating the uptake of prions into plants in vitro. Using laser scanning confocal microscopy, we observed root uptake of fluorescently-tagged, abnormal prion protein in the model plant Arabidopsis thaliana, as well as the crop plants alfalfa (Medicago sativa), barley (Hordeum vulgare) and tomato (Solanum lycopersicum). Using serial protein misfolding cyclic amplification, a sensitive biochemical prion detection method, we have found evidence of prions in aerial tissues from these species, as well as maize (Zea mays). Both stems and leaves of A. thaliana grown in culture media containing prions are infectious when injected into mice and oral bioassays are underway for A. thaliana and other plants. Our results suggest that prions are taken up by plants and that contaminated plants may represent a previously unrecognized risk of human, domestic species and wildlife exposure to CWD and scrapie agents.


Friday, August 09, 2013

CWD TSE prion, plants, vegetables, and the potential for environmental contamination

AD.82: Prion-contaminated plants can transmit prion disease

Sandra J. Pritzkow, Rodrigo Morales, Fabio Moda and Claudio Soto

University of Texas Medical School at Houston; Houston. TX USA

Chronic Wasting Disease (CWD) is a prion disorder affecting deer and elk. The efficient propagation of this disease in captive and free-ranging animals suggest that it may involve horizontal transmission through contaminated environment. It has been shown, that infectious prions can enter the environment through saliva, feces, urine, blood or placenta tissue from infected animals, as well as by carcasses from diseased animals. Various studies have demonstrated that infectious prions bind tightly to soil and remain infectious after years in this material.

We hypothesize that plants, which get in contact with infectious prions, can also play a role on the horizontal transmission of prion diseases. To study whether plants can interact with prions, we analyzed wheat grass roots and leaves incubated with 263K-infected brain homogenate in vitro using the PMCA technique and in vivo in Syrian hamsters. For in vitro analyses, the plant tissue was incubated in serial dilutions of 263K-brain homogenate, washed thoroughly and analyzed for the presence of Prpsc by PMCA. The results show that even highly diluted Prpsc can bind to roots and leaves and sustain the conversion of normal prion protein. Similar experiments are currently ongoing using CWD infected material. In vivo, hamsters were orally infected with leaves or roots incubated in 10% 263K-infected brain homogenate, which were thoroughly washed as well. Hamsters, inoculated with 263K-contaminated roots or leaves, developed typical signs of prion disease, whereas control animals inoculated with non-contaminated plants did not. Prion disease was confirmed by immunohistological and biochemical analyses.

These findings suggest that plants (leaves and roots) can efficiently bind infectious prions and act as carrier of infectivity and may play an important role in horizontal transmission by oral intake of the prion agent.


AD.83: Are plants a potential transmission route for infectious prions?

Jay D. Rasmussen,1,3 Brandon H. Gilroyed,2 Tim Reuter,4 Sandor Dudas,5 Catherine Graham,5 Norman F. Neumann.6 Aru Balachandran,7 Stefanie Czub,5 Nat N. Kav1 and Tim A. McAllister3

'Department of Agricultural; Food and Nutritional Sciences; University of Alberta; Edmonton, AB Canada; 2School of Environmental Sciences; University of Guelph Ridgetown Campus; Ridgetown, ON Canada; 3Agriculture and Aqri-Food Canada; Lethbridge Research Centre; Lethbridge, AB Canada; 4Alberta Agriculture and Rural Development; Agriculture Centre; Lethbridge. AB Canada; 5National and OIE Reference Laboratories of BSE; National Centres for Animal Disease Lethbridge Laboratory; Canadian Food Inspection Agency; Lethbridge. AB Canada; School of Public Health; University of Alberta; Edmonton, AB Canada; 'National and OIE Reference Laboratory for scrapie and CWD; Canadian Food Inspection Agency; Ottawa ON Canada

Plants are capable of absorbing large organic materials such as proteins and microorganisms through their roots. This phenomenon introduces the potential for the uptake of infectious prions from the environment and is a possible route for the distribution of prion diseases in natural habitats. Wheat (Triticum aestivum), a major agricultural crop, was used as a model in our experiments to examine prion uptake by plants. In preliminary experiments, model proteins of similar size (Q prions were used (fluorescently-tagged ovalbumin, FT-OV; recombinant cellular PrP, recPrPC). Plants were grown in sterile media (Murashige and Skoog) for 30-45 d before roots were exposed to a model protein solution for 24 h. Foreign target proteins were detected by fluorescent microscopy (FT-OV) and western blotting (FT-OV and recPrPC). FT-OV was found to enter the root system and translocate to the stem. For recPrPc, no detectable uptake or translocation was found, but instead, a strong binding of recPrPc to the outer root surface was observed. These results suggest that uptake by wheat, although possible, might not be universal for all proteins. The consideration of how different plants may respond and how natural root damage may affect protein transport is important. The model described above was used to determine how infectious prions interact with wheat plants. Wheat roots were exposed for 24 h to Chronic Wasting Disease positive and negative elk brain homogenates that were either digested with proteinase K (PK) or left undigested. Plant extracts were analyzed by western blotting to determine the presence of prion proteins, Bands corresponding to PK-sensitive prions were detected in root extracts, but not in other regions of the plant. These results suggest that, similar to model work with recPrPc, PrPc may bind to the outside of the root, without translocation to other areas of the plant. Current work is investigating the implications of exposure of wheat roots to purified PrPCWD on uptake. Future studies will consider the impact of soil on absorption of PrPCWD by roots. Binding of PrPCWD to the surface of wheat roots as shown for PrPc, would open a new discussion on the distribution of infectious prions in the environment.


AD.81: Detection of prion protein associated with cervid chronic wasting disease in environmental samples

Chad J. Johnson, Christen B. Smith, Michael D. Samuel and Joel A. Pedersen University of Wisconsin; Madison. WI USA

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) or prion disease affecting North American members of the deer family (cervids). The disease agent may enter the environment through decomposition of carcasses and shedding in feces, saliva, and urine. Once in the environment disease associated prion protein (PrPTSE) can bind to soil components and remain bioavailable for extended time periods. Assessment of the environmental load of the disease agent is difficult because relevant levels are below the detection limits of immunochemical methods and bioassay is prohibitively expensive to use as a surveillance technique. Here, we report that a combination of detergent extraction and protein misfolding cyclic amplification with beads (PMCAb) substantially improves the sensitivity of PrPTSE detection in environmental samples. Using this technique we are able to achieve detection limits substantially lower than animal bioassay. Working with amended soils we are able to extract and amplify PrPTSE to detectable levels. We have investigated factors contributing to PMCAb inhibition and methods to circumvent those inhibitions. This technique holds promise for helping to clarify the relative importance of direct and indirect transmission of CWD, assess the effectiveness of environmental remediation, and determine environmental loads of infectious agent.


AD.80: Kinetics of chronic wasting disease prion shedding in cervid saliva and urine

Nicholas J. Haley, Davin Henderson, Glenn C. Telling and Edward A. Hoover

Colorado State University; Fort Collins. CO USA

Efficient horizontal transmission is a unique hallmark of chronic wasting disease (CWD) of deer, elk, and moose. Saliva trans- fer, for example via grazing or mutual grooming, is thought to be the primary mechanism of horizontal transmission, although urine and feces are also thought ro play an important role. It is not known how shortly after exposure an animal may begin shedding PrPCWD, though it has been reported that both clinical and pre-clinical animals may successfully transmit CWD to naive deer. We hypothesized that transmission would occur primarily in end-stage disease, though the purpose of this study was to identify earlier time points during the course of CWD infection in which saliva and urine may carry infectivity. Using both transgenic mouse bioassay and real-rime quaking-induced conversion (RT-QuIC), we evaluated saliva and urine from two experimentally infected white tail deer for which samples were available from multiple time points post-inoculation (p.i.) (e.g., 3, 6 and 12 mo p.i., as well as immediately prior to euthanasia at 24-27 mos). We found that while saliva collected during clinical disease was infectious in mouse bioassay, saliva collected 12 mo p.i., prior to the onset of clinical signs was also variably infectious. Saliva from time points earlier than 12 mo p.i. failed to transmit infection, while urine collected from clinically affected deer had very low potential to transmit infection, as has been reported previously. These findings extend our understanding of CWD shedding in the natural host, and may improve control of CWD transmission in captive and free-ranging settings.


56. Members considered that there is no evidence that crops grown on the land which received composted excreta from BSE-challenged animals pose a TSE risk to humans or animals. One member suggested that, as some of these animals are orally challenged with high doses of BSE-infected materials, and the distribution of infectivity in the digestive system is not completely understood, it might be premature to conclude that there is no infective agent in the manure.

Furthermore, an unpublished study had indicated low level absorption of PrP from soil by tomato plants although it should be noted that this study had not been repeated. Details of this work would be sent to the SEAC Secretary. Dr Matthews explained that most of the manure from animals challenged with high doses of BSE had already been composted and used for coppicing. Members agreed that the risks from disposal of residual manure from experimental animals would be much less than historic risks of on farm contamination from naturally infected animals at the height of the BSE epidemic. ...SNIP...END

SRM are certain cattle tissues capable of transmitting BSE. There is no human health risk assessment to indicate the absence of human health concerns associated with use of composted SRM domestically. To date, scientific evidence has not been able to demonstrate that composting destroys prions. Although domestic use would pose a negligible risk to livestock, there is a potential risk to humans via direct ingestion of the compost or of compost particles adhered to skin or plant material (e.g. carrots). Another potential route of exposure is by ingestion of prions that have been taken up by plants. It has been proven that bacteria are readily taken up by some plants (e.g. E. coli in lettuce) thus the uptake of prions by plants cannot be precluded or dismissed at this time. As a science-based regulator, the CFIA cannot change the policy on this issue without a risk assessment demonstrating that the use of composted SRM poses an acceptable risk to humans.

The BSE Inquiry / Statement No 19B (supplementary) Dr Alan Colchester Issued 06/08/1999 (not scheduled to give oral evidence) SECOND STATEMENT TO THE BSE INQUIRY Dr A Colchester BA BM BCh PhD FRCP Reader in Neurosciences & Computing, University of Kent at Canterbury; Consultant Neurologist, Guy’s Hospital London and William Harvey Hospital Ashford April 1999


88. Natural decay: Infectivity persists for a long time in the environment. A study by Palsson in 1979 showed how scrapie was contracted by healthy sheep, after they had grazed on land which had previously been grazed by scrapie-infected sheep, even though the land had lain fallow for three years before the healthy sheep were introduced. Brown also quoted an early experiment of his own (1991), where he had buried scrapie-infected hamster brain and found that he could still detect substantial infectivity three years later near where the material had been placed. 89. Potential environmental routes of infection: Brown discusses the various possible scenarios, including surface or subsurface deposits of TSE-contaminated material, which would lead to a build-up of long-lasting infectivity. Birds feeding on animal remains (such as gulls visiting landfill sites) could disperse infectivity. Other animals could become vectors if they later grazed on contaminated land. "A further question concerns the risk of contamination of the surrounding water table or even surface water channels, by effluents and discarded solid wastes from treatment plants. A reasonable conclusion is that there is a potential for human infection to result from environmental contamination by BSE-infected tissue residues. The potential cannot be quantified because of the huge numbers of uncertainties and assumptions that attend each stage of the disposal process". These comments, from a long established authority on TSEs, closely echo my own statements which were based on a recent examination of all the evidence. 90. Susceptibility: It is likely that transmissibility of the disease to humans in vivo is probably low, because sheep that die from scrapie and cattle that die from BSE are probably a small fraction of the exposed population. However, no definitive data are available.

91. Recommendations for disposal procedures: Brown recommends that material which is actually or potentially contaminated by BSE should be: 1) exposed to caustic soda; 2) thoroughly incinerated under carefully inspected conditions; and 3) that any residue should be buried in landfill, to a depth which would minimise any subsequent animal or human exposure, in areas that would not intersect with any potable water-table source.

92. This review and recommendations from Brown have particular importance. Brown is one of the world's foremost authorities on TSEs and is a senior researcher in the US National Institutes of Health (NIH). It is notable that such a respected authority is forthright in acknowledging the existence of potential risks, and in identifying the appropriate measures necessary to safeguard public health. Paper by SM Cousens, L Linsell, PG Smith, Dr M Chandrakumar, JW Wilesmith, RSG Knight, M Zeidler, G Stewart, RG Will, "Geographical distribution of variant CJD in the UK (excluding Northern Ireland)". Lancet 353:18-21, 2 nd January 1999 93. The above paper {Appendix 41 (02/01/99)} (J/L/353/18) examined the possibility that patients with vCJD (variant CJD) might live closer to rendering factories than would be expected by chance. All 26 cases of vCJD in the UK with onset up to 31 st August 1998 were studied. The incubation period of vCJD is not known but by analogy with other human TSEs could lie within the range 5-25 years. If vCJD had arisen by exposure to rendering products, such exposure might plausibly have occurred 8-10 years before the onset of symptoms. The authors were able to obtain the addresses of all rendering plants in the UK which were in production in 1988. For each case of vCJD, the distance from the place of residence on 1st January 1998 to the nearest rendering plant was calculated


Friday, February 08, 2013

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

Saturday, March 10, 2012

CWD, GAME FARMS, urine, feces, soil, lichens, and banned mad cow protein feed CUSTOM MADE for deer and elk




Friday, August 09, 2013

***CWD TSE prion, plants, vegetables, and the potential for environmental contamination







Sunday, August 25, 2013


HD.13: CWD infection in the spleen of humanized transgenic mice


Liuting Qing and Qingzhong Kong


Case Western Reserve University; Cleveland, OH USA


Chronic wasting disease (CWD) is a widespread prion disease in free-ranging and captive cervid species in North America, and there is evidence suggesting the existence of multiple CWD strains. The susceptibility of human CNS and peripheral organs to the various CWD prion strains remains largely unclear. Current literature suggests that the classical CWD strain is unlikely to infect human brain, but the potential for peripheral infection by CWD in humans is unknown. We detected protease-resistant PrpSc in the spleens of a few humanized transgenic mice that were intracerebrally inoculated with natural CWD isolates, but PrpSc was not detected in the brains of any of the CWD-inoculated mice. Our ongoing bioassays in humanized Tg mice indicate that intracerebral challenge with such PrpSc-positive humanized mouse spleen already led to prion disease in most animals. These results indicate that the CWD prion may have the potential to infect human peripheral lymphoid tissues.



Oral.15: Molecular barriers to zoonotic prion transmission: Comparison of the ability of sheep, cattle and deer prion disease isolates to convert normal human prion protein to its pathological isoform in a cell-free system


Marcelo A.Barria,1 Aru Balachandran,2 Masanori Morita,3 Tetsuyuki Kitamoto,4 Rona Barron,5 Jean Manson,5 Richard Kniqht,1 James W. lronside1 and Mark W. Head1


1National CJD Research and Surveillance Unit; Centre for Clinical Brain Sciences; School of Clinical Sciences; The University of Edinburgh; Edinburgh, UK; 2National and OIE Reference Laboratory for Scrapie and CWD; Canadian Food Inspection Agency; Ottawa Laboratory; Fallowfield. ON Canada; 3Infectious Pathogen Research Section; Central Research Laboratory; Japan Blood Products Organization; Kobe, Japan; 4Department of Neurological Science; Tohoku University Graduate School of Medicine; Sendai. Japan; 5Neurobiology Division; The Roslin Institute and R(D)SVS; University of Edinburgh; Easter Bush; Midlothian; Edinburgh, UK


Background. Bovine spongiform encephalopathy (BSE) is a known zoonotic prion disease, resulting in variant Creurzfeldt- Jakob disease (vCJD) in humans. In contrast, classical scrapie in sheep is thought to offer little or no danger to human health. However, a widening range of prion diseases have been recognized in cattle, sheep and deer. The risks posed by individual animal prion diseases to human health cannot be determined a priori and are difficult to assess empirically. The fundamemal event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein (PrPC) to its pathological isoform (PrPSc). Here we report the use of a rapid molecular conversion assay to test whether brain specimens from different animal prion diseases are capable of seeding the conversion of human PrPC ro PrPSc.


Material and Methods. Classical BSE (C-type BSE), H-type BSE, L-type BSE, classical scrapie, atypical scrapie, chronic wasting disease and vCJD brain homogenates were tested for their ability to seed conversion of human PrPC to PrPSc in protein misfolding cyclic amplification (PMCA) reactions. Newly formed human PrPSc was detected by protease digestion and western blotting using the antibody 3F4.


Results. C-type BSE and vCJD were found to efficiently convert PrPC to PrPSc. Scrapie failed to convert human PrPC to PrPSc. Of the other animal prion diseases tested only chronic wasting disease appeared to have the capability ro convert human PrPC to PrPSc. The results were consistent whether the human PrPC came from human brain, humanised transgenic mouse brain or from cultured human cells and the effect was more pronounced for PrPC with methionine at codon 129 compared with that with valine.


Conclusion. Our results show that none of the tested animal prion disease isolates are as efficient as C-type BSE and vCJD in converting human prion protein in this in vitro assay. However, they also show that there is no absolute barrier ro conversion of human prion protein in the case of chronic wasting disease.







Sunday, August 25, 2013


***Chronic Wasting Disease CWD risk factors, humans, domestic cats, blood, and mother to offspring transmission




Sunday, July 21, 2013


*** As Chronic Wasting Disease CWD rises in deer herd, what about risk for humans?




> sCJDMM1-2 should be considered as a separate entity at this time.


> All of the Heidenhain variants were of the methionine/ methionine type 1 molecular subtype.












Thursday, August 08, 2013


Characterization of the first case of naturally occurring chronic wasting disease in a captive red deer (Cervus elaphus) in North America




Sunday, September 01, 2013


hunting over gut piles and CWD TSE prion disease




Wednesday, September 04, 2013


cwd - cervid captive livestock escapes, loose and on the run in the wild...




Tuesday, September 10, 2013


Review and Updates of the USDA-APHIS Veterinary Services (VS) National Chronice Wasting Disease (CWD) Program 2012-2013





Sunday, August 11, 2013


Creutzfeldt-Jakob Disease CJD cases rising North America updated report August 2013


Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing an extreme increase of 48% between 2008 and 2010





Friday, August 16, 2013


*** Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K. and Contaminated blood products induce a highly atypical prion disease devoid of PrPres in primates





Sunday, September 08, 2013


Iatrogenic Creutzfeldt-Jakob disease via surgical instruments and decontamination possibilities for the TSE prion





Tuesday, September 17, 2013


USAHA 116TH ANNUAL MEETING October 18 – 24, 2012 CWD, Scrapie, BSE, TSE prion (September 17, 2013)





*** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, and there may be asymptomatic individuals infected with the CWD equivalent. These circumstances represent a potential threat to blood, blood products, and plasma supplies.





Wednesday, September 25, 2013


Wisconsin Hunters will again be able to have adult deer tested for chronic wasting disease CWD




kind regards, terry


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