Source: UNIV OF WISCONSIN submitted to
ENVIRONMENTAL CONTAMINATION OF PLANTS WITH PRIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0230855
Grant No.
(N/A)
Project No.
WIS01657
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Pedersen, J. A.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Soil Science
Non Technical Summary
Prion diseases in animals pose a direct threat to food safety and public health. The bovine spongiform encephalopathy (BSE) epidemic of the 1980s and 1990s, for example, infected an estimated one million cattle in the UK, with an cumulative gross budgetary cost of approximately $5 billion between 1996 and 2000. Zoonosis of BSE caused a new variant of Creutzfeldt-Jakob (vCJD) disease in humans following consumption of BSE-contaminated food products and demonstrated that animal prion diseases are capable of crossing the species barrier to infect humans. As of August 2011, 221 cases of vCJD had been reported. Exclusion of prions from products intended for human consumption is required to safeguard public health and retain confidence in the food supply. As chronic wasting disease (CWD) continues to increase in both distribution and prevalence, humans will increasingly be exposed to CWD prions. Human prion disease has not yet been linked to consumption of CWD-infected animal products, but CWD agent has converted human PrPC to an abnormal form in vitro, suggesting the possibility for human infection by CWD exists. The risk that CWD poses to agriculture and the food supply is currently unknown due to incomplete understanding of disease transmission, lack of knowledge regarding the susceptibility of humans to CWD, and a paucity of information on environmental persistence of prions. The discoveries that CWD prions (1) are released into the environment via infectious saliva, blood, urine, feces, and animal mortalities, and (2) can be horizontally transmitted via environmental reservoirs of infectivity after prolonged periods of time (years to decades), suggests prions pose a significant environmental contamination problem now and in the future. The uptake of prions from the environment and into plants may pose a previously unrecognized food safety risk. CWD-infected free-ranging or captive cervids have been found in 19 states, including Wisconsin where the disease negatively impacts not only cervid farming operations, but also the $1.4 billion per year hunting industry. The increasing prevalence and geographical distribution of CWD across North America, coupled with the infectious agent's extraordinary stability and accumulation in the environment, indicates an urgent need to better understand environmental prion disese transmission. The proposed project will provide information on prion uptake in plants and the potential for plant-associated prions to cause disease in mammalian hosts, providing research basic to the problems of agriculture in its broadest aspects. This information will be useful in evaluating the risks posed to crop and livestock production by environmental contamination by prions, thereby facilitating the effective treatment of, and, where possible, prevent animal diseases in both domesticated and wild animals which, if not controlled, would be disastrous to the US livestock industries and endanger the Nation's food supply and ultimately to protect human health through control of animal diseases transmissible to humans.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3110110110120%
3110899110120%
3113899110120%
7110110110120%
7110899110110%
7113899110110%
Knowledge Area
311 - Animal Diseases; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
0110 - Soil; 3899 - Other animals, general; 0899 - Wildlife and natural fisheries, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
Prions deposited into the environment by infected deer or carcasses may contaminate plants and a primary goal of this proposal is to assess the uptake of prions into plants as a possible risk to food safety and human health. The objectives of this project are to (1) Assess uptake of prion protein and prion infectivity in plants; and (2) determine the mechanism(s) of prion uptake into plants and plant cells.
Project Methods
We will assess prion uptake in a diversity of plant species and measure prion replication and infectivity in plant tissues. To accomplish this we will test prion uptake by confocal microscopy using fluorescently tagged prion protein, protein misfolding cyclical amplification, and animal bioassay. We will determine the mechanism(s) of prion uptake into plants and plant cells using common plant cell lines or plant-derived cells and treatments that block specific pathways for biomolecule uptake by cells.

Progress 10/01/12 to 09/30/15

Outputs
Target Audience:The target audience for this work includes the prion research community, wildlife managers, the U.S. Environmental Protection Agency, state environmental agencies, state departments of public health, U.S. Department of Agriculture, andcrop industries in areas impacted by cervid chronic wasting disease and potentially bovine spongiform encephalopathy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Annabel Austin, a DVM student at the University of Pennsylvania, completed a Merial Veterinary Summer Scholarship project focused on evaluating the oral transmissibility prion-contaminated plants. Ms. Austin carried out the PMCA analyses on rodent tissues to detect clinically and subclinically infected hosts resulting from oral consumption of prion-contaminated plants. C. Carlson, and A. Austin presented research findings from this project at national and international conferences. How have the results been disseminated to communities of interest?1.Carlson, C.M. Environmental prion transmission: Identifying novel disease dissemination routes. Invited presentation at the University of Wisconsin Women in Science Seminar Series. University of Wisconsin - River Falls, River Falls, WI (Feb 2015) 2. Johnson, C.J., C.M. Carlson, M.W. Keating, N.M. Gibbs, H. Chang, J.K. Wiepz, and J.A. Pedersen. Uptake of prions into plants.PRION 2015 Conference, Ft. Collins, CO (May 2015). 3. Carlson, C.M. Identifying novel environmental transmission routes for chronic wasting disease.Association of Fish and Wildlife Agencies & USGS Ecosystems Fish and Wildlife Disease Seminar Series. Online Webinar (July 2015). 4. Austin, A.G., C.M. Carlson, and C.J. Johnson. Evaluation of the transmissibility of prions in plants.Merial-NIH National Veterinary Scholars Symposium. University of California - Davis, Davis, CA (August 2015). 5. Pedersen, J.A. Uptake of prions into plants and inactivation of prions. Huazhong Agricultural University, Wuhan, China, (December 2015). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The amount of infectious prion protein taken up by the model plantArabidopsis thalianawas determined taking into account inhibitory effects of plant tissue on protein misfolding cyclic amplification (PMCA), the assay used to amplify, detect and quantify prion protein in plant samples. PMCA analyses of brain and peripheral tissues from rodents orally challenged with prion contaminated plants were used to confirm prion disease in clinical animals and to identify sub-clinically infected animals. Additional procedural controls were performed on our hydroponic, agar, and soil growth systems. We also initiated the study of the uptake of the fluorescent proteinmCherry viaArabidopsis thaliana roots to further examine mechanisms of intact protein uptake by plants. Lastly, one manuscript is being finalized further.

Publications


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: Prion research community, state government officials. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The M.S. student involved in the project defended his thesis titled "Assessment of Prion Uptake by Plants using Protein Misfolding Cyclic Amplification" in partial fulfillment of the requirements for his degree in Civil and Environmental Engineering. Both the M.S. and Ph.D. students involved in the project trained laboratory staff to conduct the novel microplate-based PMCA technique. How have the results been disseminated to communities of interest? A collaborating scientist (C. Johnson) on the project presented results from this study as an invited speaker at an international prion conference. The project director communicated results to state governmental officials. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we intend to publish our findings on prion uptake and quantitation in various species of plants and from various types of media (hydroponic, agar, and soil) as well as those indicating oral transmissibility of prions taken up by plants to rodent hosts. We plan to conduct further experiments to elucidate the mechanisms of prion uptake by plants.

    Impacts
    What was accomplished under these goals? The amount of infectious prion protein taken up by the model plantArabidopsis thalianaand several crop species (alfalfa, barley, maize) grown on hydroponic, agar, and soil media was quantified using protein misfolding cyclic amplification (PMCA) technology. This was accomplished by adaptation and optimization of a new, high throughput and more efficient PMCA method over previous methods used, termed microplate-based PMCA (Moudjou et al. 2013. MBio. 5(1):e00829-13), to the detection of prion protein taken up by plants in our studies. Knowledge of the quantity of infectious prion protein per weight of plant tissue allows us to make initial assessments of risk of infection to mammalian hosts posed by consumption of prion contaminated plants. Rodent bioassays in which animals were orally inoculated with prion contaminated plants and assessed for disease transmission were completed during this reporting period.

    Publications

    • Type: Theses/Dissertations Status: Submitted Year Published: 2014 Citation: Keating, M.W. Assessment of Prion Uptake by Plants using Protein Misfolding Cyclic Amplification. M.S. dissertation, University of Wisconsin, Madison, USA, 2014


    Progress 01/01/13 to 09/30/13

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The Ph.D. student involved in the project participated in an international prion conference where she won an award for the best poster. The Ph.D. student involved in the project trained a M.S. student to conduct analyses for prions. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we intend to publish our initial findings, conduct further experiments to assess prion uptake from soil to plants, and begin examining mechanisms of uptake.

    Impacts
    What was accomplished under these goals? To determine plant uptake and transmission of infectious prion protein, we exposed the model plant Arabidopsis thaliana and several crop species (alfalfa, barley, maize, tomato) to prion-contaminated hydroponic, agar, and soil media and tested for the presence of prion protein and infectivity by confocal laser scanning microscopy (CLSM), protein misfolding cyclic amplification with beads (PMCAb) and rodent bioassay. CLSM microscopy was used to assess uptake of fluoresecently labeled pathogenic prion protein into roots. PMCAb and rodent bioassays (intracerebral and oral routes of inoculation) were used to assess the translocation of prion infectivity to aerial portions of plants (i.e., leaves, stems).

    Publications

    • Type: Theses/Dissertations Status: Submitted Year Published: 2013 Citation: Carlson, C.M. Environmental prion transmission: Identifying novel prion dissemination routes. Ph.D. dissertation, University of Wisconsin, Madison, USA, 2013


    Progress 01/01/12 to 12/31/12

    Outputs
    OUTPUTS: This project began in October. We have set up plant exposures to prions. We have adapted our detection method method for prion protein in plant extracts. PARTICIPANTS: Joel A. Pedersen (principal investigator) - overall supervision of project; Clarissa Booth (graduate research assistant) - trained prospective (rotating) graduate student on techniques relevant for project; Christina Carlson (graduate research assistant) - initiated plant exposures, optimized detection method; Christopher J. Johnson (collaborator, USGS National Wildlife Health Center) - supervision of activities at his facility related to project; Haeyoon Chang (biological science technician, USGS National Wildlife Health Center) - provided laboratory assistance to Carlson. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    Conditions have been identified to allow successful detection of pathogenic prion protein in plant extracts.

    Publications

    • No publications reported this period