Prions in the environment

Abstract

Scrapie and CWD are horizontally transmissible, and the environment likely serves as a stable reservoir of infectious prions, facilitating a sustained incidence of CWD in free-ranging cervid populations and complicating efforts to eliminate disease in captive herds. Prions will enter the environment through mortalities and/or shedding from live hosts. Unfortunately, a sensitive detection method to identify prion contamination in environmental samples has not yet been developed. An environmentally-relevant prion model must be used in experimental studies. Changes in PrP^Sc structure upon environmental exposure may be as significant as changes in PrP^Sc quantity, since the structure can directly affect infectivity and disease pathology. Prions strongly bind to soil and remain infectious. Conformational changes upon adsorption, competitive sorption, and potential for desorption and transport all warrant further investigation. Mitigation of contaminated carcasses or soil might be accomplished with enzyme treatments or composting in lieu of incineration.

Introduction

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases impacting a number of mammalian species, including cattle (bovine spongiform encephalopathy, BSE or ‘mad cow’ disease), sheep and goats (scrapie), deer, elk and moose (chronic wasting disease, CWD) and humans (Creutzfeldt-Jakob disease, CJD and others).1 Scrapie and CWD are of particular environmental concern as they are horizontally transmissible and remain infectious after years in the environment.2–5 It is likely that the environment serves as a stable reservoir of infectious CWD and scrapie prions. In addition, the disposal of mortalities during BSE outbreaks, both in the past and potential future disposal events, serves as another environmental source of prions with the potential to infect humans. Therefore, it is clear that prions pose a significant environmental concern.

There has been limited risk assessment of prion exposure through environmental pathways, focusing exclusively on BSE. The risk of BSE from sources including landfills and rendering facilities contaminating drinking water has been evaluated,6,7 as well as the risks to cattle from land-applied fly ash, slag, slaughterhouse sludge and meat and bone meal contaminated with BSE.8–10 Due to the large dilution factors, the calculated risk of transmission was extremely low in all cases. However, CWD risks have not been quantified, and it is uncertain if adequate information is available to make credible assessments of prion transmission via environmental pathways. A recent editorial emphasized the need for more information about prion fate in the environment for the development of quantitative risk assessments.11

Although various aspects of prions in the environment have been reviewed previously, a complete review of literature related to prion occurrence, fate and mitigation in the environment is lacking. The potential role of soil in prion transmission was reviewed in 2006,12 but over 30 pertinent studies have been published during the ensuing two years. Snow and colleagues separately reported the prion environmental literature published in 200613 and 2007.14 Recently, Wiggens examined prion stability and infectivity in the environment.15 In contrast, this article will comprehensively review the pertinent literature addressing prion occurrence and fate in the environment, with a specific focus on prion interactions with soil. Potential avenues for environmental mitigation of prion infectivity will be considered. Experimental challenges in studying environmental prions will also be discussed, and important unanswered questions in the field will be highlighted.

Prion Diseases and the Prion Protein

Strong evidence indicates that the infectious agent of prion diseases is solely comprised of PrP^Sc (the prion), an abnormally-folded isoform of a normal cellular protein, PrP^c.1,16 The misfolded conformation of PrP^Sc conveys distinct biological and physicochemical properties, including resistance to proteolysis and inactivation techniques, increased hydrophobicity and a propensity for aggregation.1,17 The CWD and scrapie agent is shed from living hosts and present in mortalities.18,19 CWD and scrapie are orally transmissible,20,21 and the nasal cavity is also an effective route of infectivity.22 Recent bioassay data suggest that prions bound to soil particles remain infectious through oral consumption.23,24

While human prion diseases are very rare, CWD incidence can be over 15% in free-ranging deer and over 90% in captive deer herds.25 CWD has been identified in cervid populations in 16 US states and two Canadian provinces.26 Scrapie occurs in all sheep-farming countries except Australia and New Zealand.27 The economic impacts of CWD on the affected states may be high due to lost revenues in the agriculture, hunting and tourism industries.28,29 Cross-species transmission of prion diseases can occur, as demonstrated by the transmission of BSE to humans, highlighting the public health risk of animal prion diseases.30 A recent report of a single incident where 81 people were exposed to or consumed CWD-infected venison emphasizes the opportunity for CWD transmission to humans.31 There is growing evidence that the potential for CWD transmission to humans is low, but the risk is still not completely known.30,32–35 Natural transmission of CWD to livestock (especially cattle) has not been demonstrated, but remains an unknown risk. Although transmission of CWD to humans and livestock has not been shown, it might occur if a new strain emerges, either through serial passage in cervids or through environmental pressures. Host range is known to vary between prion strains.36 No distinct CWD strains have yet been identified, but evidence suggests they may exist.36–38

Distinct strains have been identified for other prion diseases, including scrapie and BSE. Prion strains are defined by varied clinical symptoms, incubation times and distinct pathological characteristics.39 The conformation of PrP^Sc (the disease-causing prion protein) is strain dependent.40–44 Because no nucleic acid content has been associated with the prion agent,16,45 it is likely that differences in PrP^Sc conformation are the sole reason for strain differences.46 Thus, the structure of PrP^Sc can affect the nature of the disease and, therefore, we propose that changes in PrP^Sc conformation due to environmental conditions could change prion strain properties.

Routes of Entry, Occurrence and Detection in the Environment

It is now established that scrapie and CWD are horizontally transmissible and can remain infectious after years in the environment.2–4 In one study, the scrapie agent remained infectious after burial in garden soil for three years.47 In another report, a previously scrapie-infected sheephouse and pasture were ‘decontaminated’ and left uninhabited; sheep introduced 16 years later subsequently contracted scrapie suggesting that scrapie remained infectious after 16 years.48 A controlled lab study indicated that the CWD agent remained infectious for at least two years in a pasture.5 Epidemiological modeling suggests that indirect, environmental routes of transmission were responsible for two CWD outbreaks in captive mule deer.49

The existence and nature of environmental reservoirs of prion infectivity have yet to be determined. Recent experiments with prions and soil suggest soil and soil minerals may act as significant reservoirs, but the possibility of environmental transmission sustained by mites or flies cannot be eliminated.50,51 It is also unclear if predators or scavengers (e.g., cougars, vultures) play a significant role in CWD spread in free-ranging cervids.

Prions may enter the environment through a number of routes. First, prions may enter through shedding from live, infected hosts. It has been shown that scrapie and CWD prions can be shed in urine,52,53 feces,54,55 saliva and blood.18 Scrapie can also be shed in birthing matter.56 A second route of entry is through animal mortalities, including farmed sheep, goats and cervids as well as free-ranging cervids. Scrapie, BSE and CWD mortalities contain high levels of infectivity in the central nervous system (CNS), with lower levels of infectivity in extraneural tissues.19 A future outbreak of BSE, scrapie or CWD in captive herds may require the culling of large numbers of animals. While it would be desirable to incinerate these mortalities, biosecurity concerns or other constraints may limit the transport of carcasses over long distances. Thus, other options like on-site burial or composting may be employed. During the early years of the BSE outbreak in the United Kingdom (1988–1991), it is estimated that 6,000 carcasses that were suspected of having BSE were disposed of in 59 landfill sites.7 Another potential route of entry is via solid or liquid waste from rendering plants and slaughterhouses unknowingly processing infected carcasses.

There has been much speculation and interest in environmental locations of concentrated prion infectivity (“hot spots”). Locations of concentrated prion infectivity could be formed at areas of communal activity where shedding of prions in saliva, urine, feces or birthing matter occurs. A recent study of elk wallows suggests that they are used too infrequently to be significant sources of CWD transmission in the wild,57 but mineral licks might be important ‘hot spots’. Animal mortality sites, where highly-infectious CNS matter would enter the environment, could also be hot spots. Hot spots would be important targets for CWD eradiation efforts should a viable mitigation method be developed. These locations would also presumably contain detectable levels of prion contamination, if a sensitive method were successfully developed.

Highly-sensitive and accurate detection of prion infectivity in the environment is not currently possible. Standard methods such as western blotting fail to detect significant levels of infectivity,58–60 and the most sensitive method of prion detection, animal bioassay, would be impractical for use on large numbers of environmental samples. Protein misfolding cyclic amplification (PMCA),61 developed by Soto and colleagues for detecting small amounts of PrP^Sc, has generated much interest for use as an environmental detection method. PMCA has been used successfully with CWD and with hamster PrP^Sc extracted from soil with SDS.24,62 The recently developed QUIC (quake-induced conversion) method,63,64 which uses recPrP as a substrate instead of uninfected brain homogenate, might be a viable alternative to PMCA as an environmental diagnostic tool. Quantitative tandem mass spectrometric techniques65 may also be developed as a sensitive environmental detection and quantification method for PrP.

Infectivity, Fate and Transport of Prions in the Environment

Understanding the significant route(s) of prion entry into the environment will lead to a better understand of prion fate and transport once in the environment. The matrix in which prions enter soil or water environments (e.g., decomposing tissue, feces, urine, saliva) could greatly affect survivability and transmissibility. Once prions enter the environment, they exist in an exceedingly complex system, detailed in Figure 1, involving a large number of constituents and a wide variety of biological, chemical and physical mechanisms that can influence prion infectivity and fate. Progress towards an understanding of the most significant mechanisms has been made, but more work is needed to determine the behavior of PrP^Sc upon entering the heterogeneous soil-water environment.

Infectivity of adsorbed prions.

One of the most important tasks in the study of CWD and scrapie transmission is identifying the significant route or routes of natural inoculation so that disease mitigation efforts can be effectively designed. The potential for CWD or scrapie transmission by exposure to contaminated soil is possible since cervids and ruminants are known to ingest and inhale large amounts of soil.66,67 Detection of prion infectivity after exposure to the soil environment was originally demonstrated by Brown and Gajdusek in 1991.47 Scrapie-infected hamster brain homogenate mixed with soil and stored outdoors in buried flowerpots for three years remained infectious. Seidel and colleagues recently conducted a similar experiment, demonstrating high levels of infectivity (by repeated oral inoculation) in prion-contaminated soil after 26 months burial.24

Johnson and colleagues have shown that prions bound to soil minerals remain infectious,23,68 and they report prions bound to soil are more infectious than unbound prions.23 Increased infectivity could be due to preferable conformational changes in PrP^Sc (including changes in aggregation) upon binding. Uptake of PrP^Sc upon ingestion or inhalation could also be enhanced by binding to soil, either by increasing PrP^Sc residence time in the animal or improving PrP^Sc accessibility to uptake mechanisms. Further studies are needed to determine how prion infectivity is altered upon interaction with soil.

The infectivity of soil-bound prions has also been confirmed by a cell culture model.69 Unfortunately, the cell line is only susceptible to select mouse prion strains, but this technique might be useful in future soil-prion infectivity studies if new cell lines are developed, as it is less expensive and faster than animal bioassay. Nonetheless, animal bioassay will continue to be an important tool in studying not only the infectivity of soil-bound prions but also changes in disease pathology and strain properties due to environmental exposure and binding to soil.

Environmentally-relevant forms of PrP.

In experimental studies, the PrP source and medium are important to accurately simulate the route of entry, subsequent environmental interactions, and the potential transmissibility of prions in the environment. Studies performed to date have used a wide variety of prion materials, including recombinant PrP (recPrP),70–78 purified hamster PrP^Sc,23,68,79 infectious rodent brain homogenate,23,24,47,68,69,80–84 infectious cervid or ovine brain homogenate,80,82,83 and intact infectious ovine tissue85 (see Table 1, discussed in detail below). As outlined in Figure 2, these prion models vary greatly both in their ease of experimental use and in their applicability to the study of prions in the environment. Recombinant PrP is only useful as a surrogate of PrP^c. Purified PrP^Sc is infectious but artificially aggregated,86 and its production requires a large amount of infectious tissue. Both PrP^c and PrP^Sc exist in vivo as a highly heterogeneous population with both full-length and N-terminal truncated forms as well as glycosylated and unglycosylated forms.87–91 These forms are a small proportion of the overall organic content which will enter the environment simultaneously with prions. This heterogeneity is best modeled using homogenized or intact infectious tissue.

Full-length PrP^Sc might be rare in the brain matter of animal carcasses greater than 1 week old.83 Thus, a large population of PrP will enter and exist in the environment without the N-terminal domain, and the environmental behavior of PrP^Sc lacking the N-terminal region could be markedly different from full-length PrP^Sc. The N-domain has been shown to affect PrP aggregation,92 and evidence suggests the N-domain might play a role in sorption to soils.68,70,73,74,76 Therefore, both full-length and truncated prions should be used to study environmental fate.

Rodent-adapted prions may not simulate prion fate in the environment. In one study, PrP from rodent (HY TME) brain homogenate significantly degraded upon incubation at room temperature while CWD-elk PrP did not.83 A transgenic (Tg) CWD model mimicked the natural elk PrP. Differences between rodent PrP degradation and natural (sheep scrapie, BSE) PrP degradation were also seen in two other degradation studies.80,82 Thus, caution should be taken when extrapolating rodent results to a natural prion system. Recently developed Tg mouse models expressing the elk, ovine, bovine or human prion protein (PrP^c) may be valuable research tools in studying the infectivity of environmental samples.37,93,94 In addition, because Tg tissue is in many cases easier to procure than natural CWD or scrapie tissue, transgenic tissue might be used as PrP source material for environmental fate studies.

Because the structure of PrP is likely the sole determinant of infectivity and strain properties,16,41–46,95 changes in PrP structure due to environmental mechanisms could directly impact transmissibility and pathogenesis. This is analogous to other environmental contaminants such as hormones, pesticides or other organic compounds, where metabolites can have differential toxicity from the parent compound. However, the prion-environment system is more complex due to the initial heterogeneity of PrP^Sc (in vivo) and the three-dimensional and yet undefined structure of PrP^Sc.

Prion sorption to soil.

Prion sorption (physical or chemical binding) to soil could play an important role in prion transport or immobilization, provide protection from or enhance in situ proteolysis, and induce conformational changes that enhance or decrease infectivity, all of which could affect the transmissibility of CWD, scrapie and BSE in the environment. The fate of proteins in soil environments has relevance not only to the study of prion fate and transmission but also the fate and activity of viruses and bacteria as well as extracellular enzymes such as Bacillus thuringiensis (Bt) toxins released from transgenic crops.96 Answers to basic questions such as the role protein sorption to soil minerals plays in soil ecology are currently lacking. Recent studies, detailed in Table 1 and summarized below, have started to explore the complex interactions between prions and soil (Fig. 1).

Protein sorption has generally been observed to be strong, fast and irreversible for a wide range of surfaces.97 Prions are no exception and both PrP^c and PrP^Sc appear to have an affinity for quartz sands and soils and a particularly strong affinity for clay minerals.68,73,74,77,79 Prion sorption is strongly irreversible and resistant to detergent and chaotropic treatments.71,81 Studies using recPrP and purified PrP^Sc hours;68,71,78 have observed maximal sorption in less than two however, PrP sorption kinetics might be significantly longer in the complex, competitive soil environment.

As with other proteins, prion sorption is most likely a function of electrostatic attractions and repulsions and hydrophobic interactions. RecPrP studies have identified electrostatic attraction between positively-charged peptides and negatively-charged mineral surfaces as the most significant adsorption mechanism.70,71 However, since the three-dimensional structure of PrP^Sc remains unknown, it is a challenge to model the specific mechanisms that are significant in PrP^Sc adsorption. It is known that PrP^Sc is highly insoluble and aggregated, and hydrophobic interactions could therefore play a larger role in PrP^Sc sorption to amorphous clay minerals. PrP^Sc adsorption to quartz sand was shown to be maximal around the isoelectric point (IEP, at pH ≈ 4 for prions), corresponding to maximal PrP^Sc aggregation.79 Maximum adsorption near the IEP has been shown for many other proteins and surfaces.98

Because the N-terminal domain is known to be flexibly-disordered and contains a high number of positively-charged amino acids, it may play a significant role in electrostatic attraction to negatively-charged mineral surfaces.70 The N-terminal domain is lost upon desorption of PrP^Sc from clay,68 and PK-digestion is required to desorb maximal PrP^Sc from clay and sandy soils.74 More full-length recPrP adsorbs to montmorillonite than N-terminal truncated recPrP.73 However, the N-domain is not needed for prion adsorption.68,74,76

Prions enter the environment concurrently with organic matter from the host. In addition, the soil environment contains native organic matter. Thus, it is important to consider the role organic matter plays in prion sorption. recPrP has been shown to have a high affinity for organic matter, equal to or greater than that calculated for mineral surfaces.76–78 In addition, humic acid was found to increase the recPrP sorption capacity of kaolinite about 10-fold.77 It is also important to note that solution ionic strength and the homoionic salt species (e.g., Na⁺, Ca²⁺) can significantly affect the measured sorption capacities for organic matter and clay minerals, including PrP capacity.77,79 While other proteins have been shown to enter the interlayer areas of expandable clays (e.g., smectites),99 potentially decreasing their bioavailability, Johnson et al. found that purified PrP^Sc did not enter the interlayer area of Na⁺-montmorillonite.68 However, choice of homoionic species can affect the height of the interlayer area.

Conformational changes in protein structure upon interaction with soil minerals have been well documented and can range from slight unfolding to significant changes in secondary structure and can also include aggregation or disaggregation.97 Proteins can also reorient over time between side-on and end-on orientations. PrP^Sc is aggregated, and changes in aggregation could occur, potentially affecting infectivity.95 One study did find that recPrP does not form β-sheets or self-aggregate, as seen in vitro, when adsorbed to clay.70 More must be done to determine what conformational changes (if any) occur to PrP^Sc when it binds to minerals and how these changes affect agent survival and infectivity.

Experimental methods for prion sorption have initially focused on using detergents and boiling to desorb prions into solution where they are detected by immunoblotting techniques (Table 1).81 Bound PrP is usually first separated from unsorbed prions by low-speed centrifugation (in some cases by using a sucrose cushion).68,69,81 Unsorbed PrP can be detected by normal immunoblotting, but bound PrP must be desorbed before detection. Unfortunately, detergent and boiling extraction methods typically have very low PrP recoveries, presumably due to the strong and near-irreversible binding of PrP to soil particles. Recoveries reported are 61–67% (wastewater sludge)84 and 5–40% for sandy and clay soils.74,75 An electroelution method developed by Rigou et al. reported similar recoveries (5–40%).73 It is likely that desorption selects for a certain PrP population, such as loosely-bound aggregates.

A direct detection method has been developed by Genovesi and colleagues which bypasses the need to desorb and instead directly detects the PrP bound to soil via immunological methods.69 This method holds promise for use in future studies, including studies of desorption and desiccation behavior. While short-term prion desorption has not been observed except after detergent treatments, longer-term desorption and mobilization could occur. Drying and desiccation, which will undoubtedly take place in many prion-contaminated environments, could significantly alter prion sorption and infectivity. Desiccation can result in protein conformational changes,100 although low-heat treatments of prions do not affect infectivity.101

Abiotic pathways may play a key role in prion transport. Due to their insolubility and high affinity for clays and silts, prions are unlikely to be transported long distances in surface water. Recent studies simulating prion fate in wastewater found that PrP strongly partitioned into the sludge solids.80,84 To date, only one study has directly evaluated the mobility of prions in soil.75 In this study, recPrP was placed in a soil column and unsaturated pore water was sampled for nine months. RecPrP was detected in the original contamination layer throughout the experiment, even in columns with active soil microbial populations. Only slight recPrP migration was observed, indicating that the recPrP was strongly sorbed to soil and underwent limited transport in soil systems. The potential for colloid-facilitated prion transport was not investigated in this study. Colloid-facilitated transport has been shown to be a significant transport process for many strongly-sorbing contaminants.102 In addition, infectious prions can form aggregates of colloidal size95 and might be transported unassociated. Macro-pore colloid-facilitated transport could quickly move prions into groundwater or surface waters and therefore warrants further study.

Almost all prion sorption studies have used recPrP or purified PrP^Sc as the source of infectious material (Table 1). These systems do not take into account the competitive matrix (animal tissue, blood, saliva or excreta) in which prions will enter the environment. Competition for sorption sites and interactions on mineral surfaces between prions and other proteins, carbohydrates, lipids and nucleic acids could all be significant factors. Many studies have found significant effects on sorption capacity and on conformational changes due to competition between plasma proteins exposed to surfaces.103 Therefore, it is uncertain how applicable the previously published results are in simulating the complex nature of prion fate in the environment.

Degradation and Mitigation of Prions in the Environment

Prions are subject to degradation in both natural and engineered settings. Bacterial enzymes which effectively degrade prions have been identified, but they are most effective at high pH (10–12) and high temperature (50–60°C).59,104 Microbiological consortia taken from the rumen and colon of cattle could degrade PrP^Sc to undetectable levels within 20 hours under anaerobic conditions at 37°C, although infectivity remained.60,105 Using an enzyme treatment, it may be possible to lower or eliminate the infectivity at identified or presumed CWD and scrapie ‘hot spots’ in captive and wild settings.

Incineration of prion-contaminated material is considered the most effective method of disposal. Combustion at 1,000°C can destroy prion infectivity, however, low infectivity remains after treatment at 600°C.106 Despite its effectiveness, incineration may not be a practical solution, such as during a large outbreak of BSE, scrapie or CWD requiring a mass culling. Incineration of contaminated soil, vegetation and farm infrastructure (paddocks, fences) to eliminate CWD or scrapie environmental infectivity is also not practical.

Composting mortalities might be an attractive alternative if incineration is not practical. Huang et al. found composting of scrapie-infected tissue reduced or completely eliminated PrP^Sc as detected by western blot.85 Others have demonstrated PrP^Sc reductions in CWD-infected elk and deer brain homogenates incubated at 55°C.107 Infectivity was not determined in either study. The risk of landfilling carcasses, residuals from composting or other prioncontaminated materials has not yet been thoroughly investigated. Thus, the ultimate fate of the PrP^Sc from the 6,000 landfilled carcasses in the UK suspected of being BSE-contaminated remains unclear.

PrP^Sc degradation might occur in the subsurface environment. One study found recPrP was degraded by soil extracted soluble enzymes after eight days incubation at 28°C,72 but the use of recPrP greatly limits the significance of these results. Previous studies found prion infectivity two or more years after burial of infectious brain homogenates.24,47 Therefore, it is highly likely that infectivity will remain many years after burial. Evaluation of the potential for prion transport in unsaturated and saturated zones will help determine the mobility of prions in landfill environments, better quantifying the risk of landfilling infectious material.

The risk of prions in wastewater and biosolids has recently gained some attention in the wastewater industry.11,108,109 Prions are unlikely to enter wastewater except through effluent from slaughterhouses unknowingly rendering prion mortalities or through contaminated effluent from hospital or research facilities. Hinckley and colleagues recently determined that most PrP^Sc and prion infectivity would associate with the activated sludge solids, survive mesophilic anaerobic digestion, and be present in the remaining biosolids.84 Likewise, Kirchmayr et al. found no significant decrease in PrP^Sc after 16 days incubation in mesophilic anaerobic sludge and observed PrP^Sc solids association.80 PrP^Sc degradation was observed in thermophilic anaerobic sludge, although maximum degradation occurred in sterilized samples.80 Others found a large decrease in PrP^Sc within 15 days after incubating BSE brain homogenates in municipal sewage at 20°C.82 Sheep scrapie brain homogenates were somewhat more resistant to degradation. Based on these studies, it can be assumed that most prion infectivity will be conserved during normal wastewater treatment processes, and prions would thus enter the environment, highly diluted, via landfill disposal or landspreading of biosolids.

Conclusion

Determination of the optimum conditions and treatments for prion degradation and immobilization should lead to best management practices for combating CWD and scrapie and for dealing with future outbreaks of prion disease requiring the disposal of a large number of carcasses. To answer practical questions about disease mitigation, it will be necessary to determine (1) the routes of prion entry into the environment, (2) the reservoirs of prions in the environment, and (3) the mechanisms for disease transmission via the environment. The results of fundamental research on prion interactions with soil will undoubtedly begin to answer these questions, and may also lead to a better understanding of basic protein-soil adsorption processes.

The study of prions in the environment is highly challenging. No other environmental ‘contaminant’ has the complex structure of prions. The fact that this structure is not even known and that many standard analytical methods are not well-developed adds to the challenge. Monitoring PrP^Sc structure during environmental studies is as equally important as quantification measurements, since changes in structure can directly affect infectivity and disease pathology. Although some innovation will be required, applications of analytical techniques such as dynamic light scattering, liquid chromatography-mass spectrometry, and atomic force microscopy hold promise in yielding more quantitative and/or more precise structural measurements of environmental prions compared to current immunological methods, which also require disruptive purification methods. Of critical importance is the ability to study prion quantity and structure in complex systems such as tissue homogenates and intact soils where there are countless interferences and competing factors. In particular, measuring changes in aggregation state as prions are exposed to the environment may be the key to assessing prion mobility, stability, infectivity and ultimately, transmissibility in the environment.

Abbreviations

CWD	=	chronic wasting disease
BSE	=	bovine spongiform encephalopathy
PrP	=	prion protein
PrPc	=	cellular, noninfectious prion protein
PrPSc	=	disease-causing prion protein
recPrP	=	recombinant prion protein

Figures and Tables

Figure 1 The complex nature of prion-soil interactions.

Figure 2 Prion models used to study environmental fate.

Table 1 Review of prion sorption literature

Soil/Mineral	Sorption capacity (µgPrP/mg)	Equilibration time	Prion material used	Method(s) used	Reference
Whole soils	ND	24 h, 7 d, 21d	ME7 (mouse) BH	Sarkosyl extraction, supernatant & pellet WB	74
		2 h–90 d	ovine recPrP	Sarkosyl extraction, supernatant & pellet WB	74
		1–60 d	ovine recPrP	SDS boiling, electroelution, WB & ELISA	73
		1 h	263K (hamster) BH	Detergent extract, low speed sep., supernatant WB	81
		2 h	purified HY (hamster) PrP^Sc	Sucrose sep., SDS boiling extraction, pellet WB	68
		1, 3 & 6 months	ovine recPrP	SDS boiling extraction, WB	75
		1 h	RML (mouse) BH	Direct detection 96-well assay	69
		1–26 months	263K (hamster) BH	SDS boiling extraction, pellet WB	24
Loamy soil (w/high OM)	30.5	2 h	ovine recPrP	Batch & flow through, supernatant UV spec	78
Sandy soil (w/high OM)	15.5	2 h	ovine recPrP	Batch & flow through, supernatant UV spec	78
Organic matter	333 or 1000	2 h	ovine recPrP	Batch & flow through, supernatant UV spec	78
Catechol (synthetic OM)	ND	1 h–72 h	ovine recPrP, C-term recPrP	Detergent Extraction, CD measurements	76
Fine quartz sand	>110 pH 4, >50 pH 7	4 h	purified HY (hamster) PrP^Sc	Sucrose sep., supernatant ELISA	79
Quartz microparticles	13.6–27.1	2 h	purified HY (hamster) PrP^Sc	Sucrose sep., SDS boiling extraction, pellet WB	68
Mica	ND	1 h	ovine recPrP	Radiolabeled recPrP in flow-through setup	71
Kaolinite	1.7–2.6	2 h	purified HY (hamster) PrP^Sc	Sucrose sep., SDS boiling extraction, pellet WB	68
	≈50	10 min	murine recPrP	Batch, high speed centrifug. sep., supernatant UV spec	77
Montmorillonite-Na^+	ND	10 h	ovine recPrP	FTIR-spectra comparisions	70
	87–174	2 h	purified HY (hamster) PrP^Sc	Sucrose sep., SDS boiling extraction, pellet WB	68
	1000	2 h	ovine recPrP, C-term recPrP	SDS boiling, electroelution, WB & ELISA	73
	>1200	10 min	murine recPrP	Batch, high speed centrifug. sep., supernatant UV spec	77
Montmorillonite-Ca^2+	>400	10 min	murine recPrP	Batch, high speed centrifug. sep., supernatant UV spec	77

ND: not determined, OM: organic matter, BH: brain homogenate, WB: western blot, UV spec: ultraviolet spectroscopy.

Acknowledgements

This work was supported by the National Center for Research Resources (C06 RR17417-01).