Progress 10/01/08 to 09/30/12
Outputs
OUTPUTS: Prions are the causative agents of Transmissible Spongiform Encephalopathies. Prions are composed primarily if not solely of a misfolded form of the cellular prion protein, denoted as PrPTSE. PrPTSE displays remarkable resistance to degradation. Mortality composting may serve as an effective means of TSE waste management and disposal. The diverse microorganisms, elevated temperatures, and alkaline pH may be capable of significant inactivation of PrPTSE. PrPTSE is remarkably resistant to conventional means of pathogen inactivation, and the extent to which composting affects PrPTSE remains unclear. In this project, we sought to determine whether compost microorganisms produce enzymes capable of degrading PrPTSE. Our initial experiments focused on composting prion-infected brain tissue. Carefully conducted control experiments revealed that recovery from compost was incomplete and that compost matrix components co-extracted with PrPTSE severely impacted our ability to measure the protein. Efforts to ameliorate the effect of co-extracted compost matrix components proved unsuccessful. We therefore modified our approach to focus on the degradation of PrPTSE by microorganisms and enzymes isolated from thermophilic compost. We constructed a laboratory-scale composting reactor that allowed us to attain temperatures consistent with thermophilic composting. Compost contains a large variety of microorganisms. We isolated a number of actinomyces species and other protease-producing bacteria from dairy manure + corn grit sawdust compost and dairy manure + feather meal compost. The reason for including feather meal was that it is rich in the insoluble protein, keratin which may encourage growth of protease-producing microorganisms capable of degrading PrPTSE. We developed methods to recover proteases from compost. The most effective protease extractant was used to extract proteases from compost that had passed through different stages of the composting process. The proteolytic activities in these samples were determined. We investigated the protease extracts for their ability to degrade PrPTSE. By use of specific protease inhibitors, we were able to evaluate the classes of proteases responsible for the PrPTSE-degrading ability of the extracts. Our next steps are to identify the protease(s) responsible for degrading PrPTSE. The most significant dissemination activities have been presentations at scientific conferences. An additional manuscript is in revision, and we expect at least one further manuscript to result from this work. PARTICIPANTS: Joel A. Pedersen (PI) - project oversight, experimental design, data interpretation, manuscript preparation. Clarissa Booth (graduate student) - designed and conducted experiments, analyzed data, drafted manuscripts. TARGET AUDIENCES: Academic community, veterinary community, wildlife managers, livestock production community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Controlled lab-scale composting of dairy manure/sawdust led to the effective decomposition of organic material as evidenced by a visible alteration in matrix structure and composition. Temperature progression mimicked that seen in successful compost operations, and appropriate moisture levels were maintained. Composted bovine manure yielded high microbial activity and enzyme production. Protease production in compost increased steadily over time, consistently peaked between days 7-15, and declined to negligible values by day 30. The interference of compost matrix components with prion detection methods highlights the need for caution in interpreting results from experiments intending to examine the efficacy of composting prions. Enzymes extracted from active compost retained proteolytic activity in solution, and autoclaving extracts at 121 C for 15 min eliminated detectable proteolytic activity. Exposure of infected brain homogenate (source of PrPTSE) to 7- and 15-day compost extracts for 2 hours at 28 C resulted in significant reduction of immunodetectable PrPTSE. This reduction was enhanced at higher temperatures (55 C) and at any temperature examined (28-55 C) for longer exposure periods (24 hours). Autoclaved compost extracts did not degrade PrPTSE under identical exposure conditions. The addition of one or more serine protease inhibitors (PMSF or Pefabloc SC) to active compost extracts resulted in substantially less PrPTSE degradation than active extracts in the absence of protease inhibitors. This suggests that one or more serine protease present in our compost extracts is able to degrade PrPTSE. Our next efforts will focus on identifying the responsible enzymes. Although we were able to successfully isolate a variety of actinomyces and other protease-producing bacteria from compost, neither the cultures nor extracellular enzymes produced by them exhibited significant PrPTSE degrading abilities.
Publications
- Booth, C.J., Johnson, C.J., and Pedersen, J.A. (2013) Microbial and enzymatic inactivation of prions in soil environments. Soil Biol. Biochem. (accepted)
- Booth, C.J., Sibley, S.D., Kumar, K., and Pedersen, J.A. 2012. Inactivation of soil-bound prion protein by proteolytic enzymes from compost. Eurosoil 2012, Bari, Italy, July 2012; S11.08-P-8.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Prions are the causative agent of Transmissible Spongiform Encephalopathies. Prions are composed of a misfolded form of cellular prion protein, denoted as PrPTSE. PrPTSE displays remarkable resistance to degradation. Mortality composting may serve as an effective means of TSE waste management and disposal. The diverse microorganisms, elevated temperatures, and alkaline pH may be capable of significant inactivation of PrPTSE. PrPTSE is remarkably resistant to conventional means of pathogen inactivation, and the extent to which composting affects PrPTSE remains unclear. The outputs during the third project year are as follows: 1. Controlled composting carried out in laboratory-scale compost reactor. We constructed a lab-scale reactor (~50 mL) housed at 55 C for controlled, aerobic composting of dairy manure. We monitored temperature, moisture, and microbial activity of the compost to ensure efficient decomposition of organic matter. 2. Isolation of enzymes from active compost. We extracted thermophilic enzymes from compost using an ionic buffer. We monitored proteolytic activity in the extracts using a colorimetric, non-specific protease assay. Analysis of proteolytic activity over time allowed us to estimate microbial activity and enzyme production in the compost matrix over the course of composting time. 3. Exposure of prions to proteolytically active compost extracts. Infectious brain homogenate containing hamster-adapted prions was incubated with enzyme extracts from compost. Control samples in which enzyme extracts were autoclaved to denature enzymes were also analyzed. Prion protein was detected via immunoblot. Dissemination of findings to scientific community. Pertinent results of this project have been shared with the scientific community through poster presentations at scientific meetings both nationally and internationally. PARTICIPANTS: Joel A. Pedersen (PI) - project oversight, experimental design, data interpretation, manuscript preparation Clarissa Booth (graduate student) - designed and conducted experiments, analyzed data, drafted manuscripts, presentations at international meetings. TARGET AUDIENCES: Academic community, veterinary community, wildlife managers, livestock production community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. Controlled lab-scale composting of dairy manure led to the effective decomposition of organic material as evidenced by a visible alteration in matrix structure and composition. Temperature progression mimicked that seen in successful compost operations, and appropriate moisture levels were maintained. 2. Composted bovine manure yielded high microbial activity and enzyme production. Protease production in compost increased steadily over time, peaked at day 7, and declined to negligible values by day 30. Enzymes extracted from active compost retained proteolytic activity in solution. 3. A 2-hour incubation of PrPTSE with day 7 compost extracts at 37 C and 55 C caused a loss of detectable protein. Autoclaving the compost extracts eliminates proteolytic activity. Incubation of PrPTSE with autoclaved compost extracts results in no loss of protein signal, suggesting that loss of PrP immunoreactivity is due to degradation of PrPTSE by enzymes in compost. Compost extracts incubated with the serine protease inhibitor, Pefabloc SC, exhibited a significant loss of proteolytic activity. Incubation of PrPTSE with compost extracts containing Pefabloc SC result in no apparent loss of PrP immunoreactivity, suggesting that a serine protease may be at least partially involved in PrPTSE degradation by compost enzymes. The outcomes to date of this project, including presentation of results to the scientific community would not have been possible without the aid of Hatch funding. The budget allowed us to purchase supplies, reagents, and equipment necessary for conducting lab-scale composting, enzyme extractions, microbial isolations, and experiments involving pathogenic prion protein.
Publications
- Smith, C.B., Booth, C.M., and Pedersen, J.A. 2011. Fate of prions in soils: A review. J. Environ. Qual. 40: 449-461.
- Booth, C.J., and Pedersen, J.A. 2011. [Abstract 702] Degradation of pathogenic prion protein by enzymes extracted from manure and compost. Ecology of Soil Microorganisms, Prague, Czech Republic, 04/27/11-05/01/11.
- Booth, C.J., and Pedersen, J.A. 2011. [Abstract 326] Degradation of pathogenic prion protein by enzymes extracted from manure and compost. Enzymes in the Environment: Activity, Ecology and Applications, Bad Nauheim, Germany, 07/17/11-07/21/11.
- Booth, C.J.; Johnson, C.J.; Pedersen, J.A. 2011. [Abstract ENVR-339] Proteolytic enzymes isolated from thermophilic compost rapidly degrade pathogenic prion protein. Abstracts Papers Amer. Chem. Soc., 242, ENVR 339, Denver, CO, 08/28/11-09/01/11.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Prions are the causative agent of Transmissible Spongiform Encephalopathies. The prion consists predominantly, if not entirely, of the misfolded form of cellular prion protein, denoted as PrPTSE. PrPTSE displays remarkable resistance to degradation. Mortality composting may serve as an effective means of TSE waste management and disposal. The diverse microorganisms, elevated temperatures, and alkaline pH may be capable of significant inactivation of PrPTSE. PrPTSE is remarkably resistant to conventional means of pathogen inactivation, and the extent to which composting affects PrPTSE remains unclear. The outputs during the second project year are as follows: 1. Optimization of PrPTSE extraction from spent compost. Assessment of biodegradation by compost microorganisms requires efficient protein recovery from the matrix. We expect the fate of PrPTSE during composting to be influenced by association with particulate matter. We employed several extraction methods that have successfully recovered PrPTSE from soils. We used prion-infected hamster brain homogenate (BH) a prion source. Spent compost was spiked with BH, and PrPTSE was extracted with various surfactants. In control experiments, spent compost was first extracted, and BH was added to the extract afterwards. Immunoblotting was used to detect the protein. 2. Isolation of proteases from manure. Manure microorganisms produce a abundance of enzymes, including proteases and are thus of particular interest for potential PrPTSE digestion. Proteases have successfully been extracted from soil and sludge with low ionic strength solutions; we employed similar methods to extract proteases from cattle manure. Enzyme activity in extracts was measured colorimetrically. BH was incubated with protease extracts, and immunoblotting was used to detect remaining protein. Control samples were prepared in which protease extracts were autoclaved to denature enzymes. 3. Isolation of aerobic microorganisms from cattle manure. Animal manure hosts a large variety of microorganisms. We examined the ability of isolated manure microorganisms to degrade PrPTSE. We used enriched and selective media to isolate a microorganisms from manure, exposed BH to the isolates, and used immunoblotting to detect remaining protein. Control samples were autoclaved to destroy microbial activity and denature enzymes. 4. Fabrication of lab-scale compost reactor. The composting process proceeds efficiently when conditions are optimal for microbial growth and activity. Temperature, moisture content, and oxygen level must be controlled to promote biodegradation of compost contents. We prepared a lab-scale setup for composting of prions. Atmospheric air is pumped through a humidification chamber at low flow rate, and enters the composting chamber. Air exits through a small needle at the bottom of the chamber, and a thermocouple monitors internal temperature. The humidification and composting chambers are housed in an incubator to provide initial heat to the system. Compost starting material is dairy cattle manure mixed with small grain sawdust. This arrangement promotes efficient microbial breakdown of compost material. PARTICIPANTS: Joel A. Pedersen (PI) - project oversight, experimental design, data interpretation, manuscript preparation Clarissa Booth (graduate student) - designed and conducted experiments, analyzed data, drafted manuscripts TARGET AUDIENCES: Academic community, veterinary community, wildlife managers, livestock production community PROJECT MODIFICATIONS: One of the primary objectives of this project was to evaluate the potential of compositing to inactivate infectious prion proteins. We had planned to actively compost infectious material and extract any remaining protein using low concentrations of surfactants. We would then detect protein using a variety of methods including immunoblot, protein misfolding cyclic amplification (PMCA), and animal bioassay. In the Project Outcomes section, we describe the difficulty we encountered in extracting prion protein from the organic matter-rich compost matrix. We were unable to detect any prion protein in our control samples. Our data suggests this is due to the interference of natural organic matter (NOM) with immunoblotting. This is a serious concern for our project as we cannot attribute a diminished protein signal on an immunoblot to degradation by composting. We have made efforts to remove NOM from compost extracts, but agents such as polyvinylpolypyrrolidone (PVPP) which retain to humic substances also retain to prion protein, removing it from solution. These detection challenges prompted us to modify our approach. Enzymes, such as proteases, can be extracted from matrices like soil (Ladd, 1972; Nannipieri et al., 1980) and sludge (Gessesse et al., 2003). We are using similar methods to extract active enzymes from animal manure and compost. Pathogenic prion protein (PrPTSE) is then exposed to these enzymes to evaluate their ability to degrade the protein. We are also culturing whole microorganisms from manure and compost for exposure to PrPTSE. Immunoblotting and PMCA are still used as protein detection methods, and we plan to conduct animal bioassay to monitor remaining infectivity. These modifications will us to investigate the degradation of prion protein by compost enzymes or microorganisms without confusing poor recovery from the compost matrix or NOM interference with protein degradation. Discovery of PrPTSE-degrading enzymes or microorganisms from compost would be a novel finding, and one with potential implications for disposal of TSE-infected animals.
Impacts
1. Pathogenic prion protein (PrPTSE) extraction from a spent compost matrix. Association with particulate matter is expected to influence the fate and recovery of PrPTSE during composting. We examined a variety of ionic and non-ionic detergents for their ability to extract PrPTSE from an inactive compost matrix. Many of the extractants used were relatively harsh and likely denatured the protein, in principle allowing higher protein recovery than would gentler extractants that preserve the tertiary structure of PrPTSE. However, we were unable to detect recovered protein by immunoblotting. We were also unable to recover detectable PrPTSE in control samples in which infected brain homogenate (BH) was spiked into compost extracts. We determined that natural organic matter (NOM) negatively affects sample detection by immunoblotting. Spent compost extracts released significantly higher amounts of NOM than soil from which PrPTSE has been successfully extracted. Co-extraction of NOM in compost extracts appears to interfere with PrPTSE detection by immunoblotting. This prevents accurate determination of whether reduced protein recovery is due to degradation during composting, inefficient release from compost particles, or NOM interference with immunoblotting. 2. Protease extraction from dairy cattle manure. Animal manure often serves as a compost inoculant because of its high microbial activity. We extracted of active proteolytic enzymes produced by manure microbes based on methods for the extraction of proteases from soil and sludge. Enzyme activity of samples was measured colorimetrically using a non-specific protease assay. Solutions including sodium pyrophosphate, KCl, and McDougall's mineral salt solution were used to extract proteases. Compost extracts obtained with McDougall's mineral salt solution exhibited high proteolytic activity, and were used for subsequent studies. When BH was incubated with active protease extracts overnight at 37C, immunoreactivity was diminished compared to autoclaved extracts. Addition of a serine protease inhibitor limited loss of PrPTSE immunoreactivity, suggesting that a serine protease may be involved in PrPTSE degradation. 3. Isolation of microorganisms from dairy cattle manure. Bacteria and fungi were isolated aerobically at 37C using enriched and selective media. Isolates have not yet been identified, but were separated by colony morphology and transferred to separate tubes of fluid thiogylcollate media. BH was exposed to media cultures overnight at 37C, and five isolates (assigned provisional numerical codes) significantly diminished PrPTSE immunoreactivity compared to inactive cultures.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: An urgent need exists for safe and cost-effective means for disposing of carcasses infected with transmissible spongiform encephalopathies (TSEs). Examples include chronic wasting disease (CWD)-infected carcasses, downer cattle and dead stock potentially infected with bovine spongiform encephalopathy (BSE), and sheep infected with scrapie. Mortality composting (composting of animal carcasses) may represent an effective means for the disposal of TSE-infected carcasses and potentially contaminated animal products. The alkaline pH, elevated temperatures and unique microbial communities present in compost may be capable of significant inactivation of infectious prion proteins. We are investigating the inactivation of pathogenic prion proteins (PrPSc) achieved during simulated mortality composting. During the first project year, we focused on developing and optimizing extraction protocols to recover PrPSc from soils and compost and adapting a novel method (protein misfolding cyclical amplification, PMCA) for the detection of PrPSc in compost extracts. For detection by PMCA (and bioassay), the prion protein must be in its pathogenic conformation. Optimal extraction conditions therefore represent a tradeoff between maximizing prion protein recovery and maintaining the pathogenic conformation. We have designed composting reactors for the project and initiated trial composting experiments. PARTICIPANTS: Joel A. Pedersen (PI), Judd M. Aiken (collaborator, University of Alberta, Edmonton, Alberta, Canada), Clarissa Booth (PhD student) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The information generated by this project will be useful in assessing the suitability of mortality composting for the disposal of TSE-infected carcasses and other prion-contaminated material, and in evaluating the safety of using the resulting compost as a soil amendment. If we demonstrate significant reduction in prion infectivity, a logical extension of this work would be to isolate novel microorganisms and proteases capable of degrading PrPSc. Such organisms or proteases could have utility in decontamination efforts.
Publications
- No publications reported this period
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