• Loading metrics

Prions Adhere to Soil Minerals and Remain Infectious

  • Christopher J Johnson,

    Affiliations Program in Cellular and Molecular Biology, University of Wisconsin Madison, Madison, Wisconsin, United States of America , Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, Wisconsin, United States of America

  • Kristen E Phillips,

    Affiliation Molecular and Environmental Toxicology Center, University of Wisconsin Madison, Madison, Wisconsin, United States of America

  • Peter T Schramm,

    Affiliation Molecular and Environmental Toxicology Center, University of Wisconsin Madison, Madison, Wisconsin, United States of America

  • Debbie McKenzie,

    Affiliation Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, Wisconsin, United States of America

  • Judd M Aiken,

    Affiliations Program in Cellular and Molecular Biology, University of Wisconsin Madison, Madison, Wisconsin, United States of America , Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, Wisconsin, United States of America

  • Joel A Pedersen

    To whom correspondence should be addressed. E-mail:

    Affiliations Molecular and Environmental Toxicology Center, University of Wisconsin Madison, Madison, Wisconsin, United States of America , Department of Soil Science, University of Wisconsin Madison, Madison, Wisconsin, United States of America

Prions Adhere to Soil Minerals and Remain Infectious

  • Christopher J Johnson, 
  • Kristen E Phillips, 
  • Peter T Schramm, 
  • Debbie McKenzie, 
  • Judd M Aiken, 
  • Joel A Pedersen


An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrPSc) with common soil minerals. In this study, we demonstrated substantial PrPSc adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrPSc-binding capacities of each mineral. Furthermore, we observed that PrPSc desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrPSc and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrPSc bound to Mte remained infectious. Results from our study suggest that PrPSc released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.


Transmissible spongiform encephalopathies (TSEs) are a group of incurable diseases likely caused by a misfolded form of the prion protein (PrPSc). TSEs include scrapie in sheep, bovine spongiform encephalopathy (“mad cow” disease) in cattle, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease in humans. Scrapie and CWD are unique among TSEs because they can be transmitted between animals, and the disease agents appear to persist in environments previously inhabited by infected animals. Soil has been hypothesized to act as a reservoir of infectivity, because PrPSc likely enters soil environments through urinary or alimentary shedding and decomposition of infected animals. In this manuscript, the authors test the potential for soil to serve as a reservoir for PrPSc and TSE infectivity. They demonstrate that PrPSc binds to a variety of soil minerals and to whole soils. They also quantitate the levels of protein binding to three common soil minerals and show that the interaction of PrPSc with montmorillonite, a common clay mineral, is remarkably strong. PrPSc bound to Mte remained infectious to laboratory animals, suggesting that soil can serve as a reservoir of TSE infectivity.


Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases that affect a variety of mammalian species and include bovine spongiform encephalopathy (BSE, “mad cow” disease), chronic wasting disease (CWD) of deer and elk, sheep scrapie, and Creutzfeldt-Jakob disease in humans [1]. The agricultural, economic, and social impacts of prion diseases have been intensified by evidence suggesting transmissibility of BSE to humans [2]. The putative infectious agent in these diseases, designated PrPSc, is a misfolded isoform of the normal cellular prion protein (PrPC). The amino acid sequences of PrPSc and PrPC are identical [3]; normal and abnormal forms of the protein differ only in conformation. No differences in posttranslational covalent modification have been demonstrated [3]. Circular dichroism and infrared spectroscopy indicate that the disease-specific isoform has a higher β-sheet and lower α-helix content than PrPC [4]. The normal isoform is soluble and primarily monomeric in solution, whereas PrPSc forms insoluble aggregates.

Sheep scrapie and cervid CWD are unique among TSEs, because epizootics can be sustained by horizontal (animal-to-animal) transmission [5,6]. Routes of natural transmission remain to be clarified, but available evidence indicates that an environmental reservoir of infectivity contributes to the maintenance of these diseases in affected populations [68]. The expanding range of CWD (several regions of North America and Korea) increasingly brings domestic livestock, companion animals, and wildlife species into contact with infected animals and carcasses, and shedded TSE agent, raising the possibility of cross-species transmission. This was demonstrated by the recent detection in Colorado, USA, of a free-ranging, CWD-infected moose, a species not previously known to be affected by the disease in the wild [9].

Although other modes of environmental transmission of scrapie and CWD have been proposed (e.g., flesh flies [10], hay mites [11]), several lines of evidence point to soil as a reservoir for TSE infectivity. TSE infectivity exhibits remarkable resistance to inactivation by most chemical agents, radiation, and heat [12] and has been shown to persist after burial in soil for at least 3 y [13]. Anecdotal observations of healthy sheep contracting scrapie after occupying fields previously containing diseased animals have been reported [7,8]. Although these older studies did not account for the genetic susceptibility of the sheep under study, they suggest that scrapie agent can persist in the environment for years. Recent controlled field experiments provide more compelling evidence of the environmental persistence of prions. Miller et al. [14] demonstrated that naïve mule deer could contract CWD when housed in paddocks previously inhabited by infected animals or containing decomposed infected carcasses.

TSE agents directly enter the environment when carcasses of infected animals decompose [13], through alimentary shedding of the agent from gut-associated lymphoid tissue [15,16], or from urinary excretion from infected, nephritic animals [17]. Furthermore, bovine, sheep, and deer TSE agents have been introduced to soil environments through the burial of diseased carcasses and other infected material [18]. Animals ingest soil both deliberately and incidentally [19]. Cattle, deer, sheep, and other animals can consume hundreds of grams of soil daily [20,21]. Taken together, these data support the notion that PrPSc-contaminated soil may allow intraspecies TSE transmission and enhance the likelihood of spread to other species. As a first step toward understanding the role of soil as a reservoir of TSE infectivity, we investigated the binding of PrPSc to common soil minerals and whole soils and examined the infectivity of mineral-bound prions.


Binding of PrPSc to Soil Minerals

We examined the sorption of purified PrPSc to three common soil minerals (Table S1): quartz, montmorillonite (Mte, an expandable layered silicate clay mineral), and kaolinite (Kte, a nonexpandable phyllosilicate mineral). Quartz of two particle sizes was employed in sorption experiments: fine sand (hydrodynamic diameter [dh] = 125–250 μm), representing quartz concentrated in the sand and silt fractions of soils, and microparticles (dh = 1–5 μm), representing quartz present in the coarse clay fraction [22]. Purified PrPSc (~0.2 μg) was introduced into aqueous suspensions (pH 7.0) of each soil mineral and subjected to 2-h mixing. Unbound PrPSc was separated from bound protein by centrifugation through a 750-mM sucrose cushion. Bound and unbound fractions were analyzed by SDS-PAGE and immunoblotting.

The extent of PrPSc sorption differed among the mineral particles examined. All detectable PrPSc adsorbed to the expandable clay mineral Mte (Figure 1A). X-ray diffraction analysis provided no evidence that PrPSc entered Mte interlayer spaces (Mte d001 spacings were 1.22 nm and 1.47 nm before and after PrPSc adsorption, respectively); prion protein appeared to adsorb to only external clay surfaces. PrPSc did not associate with an equal mass of fine quartz sand at levels detectable by immunoblotting (Figure 1A). A large degree of PrPSc binding to the nonexpandable clay mineral Kte was observed when the surface area was matched to that of external Mte surfaces (Figure 1A). The limited association of PrPSc with fine quartz sand was at least in part attributable to the much smaller specific surface area of these particles as compared to kaolinite and external Mte surfaces (Table S1). When quartz surface area was matched to that of external Mte surfaces, all detectable PrPSc adsorbed to quartz (Figure 1A).

Figure 1. PrPSc Adsorption to Clay Minerals and Quartz Microparticles Substantially Exceeded That to Fine Quartz Sand

(A) Detectable amounts of PrPSc adsorbed to Mte and Kte but not to fine quartz sand (dh = 125–250 μm). PrPSc desorbed from Mte was of lower molecular mass than the starting material. Adsorption to quartz was observed when quartz microparticles (dh = 1–5 μm) were employed and surface area was matched to Mte.

(B) Immunoblotting sensitivity was determined by dilution of Mte-adsorbed PrPSc to the limit of detection. Protein was desorbed from Mte in 50 μl of SDS-PAGE sample buffer at 100 °C and serially diluted. Immunoblots used monoclonal antibody (mAb) 3F4. Pel, PrPSc associated with pelleted mineral particles; Sup, unbound PrPSc in supernatant.

Adsorption Capacities of Soil Minerals for PrPSc

The amount of PrPSc adsorbed to Mte was semiquantitatively assessed by serial dilution of samples to the limit of immunoblotting detection. The dilution at which no detectable immunoreactivity remained provided a basis for comparison with samples lacking immunoreactivity before dilution. PrPSc desorbed from Mte still exhibited immunoreactivity after 100-fold dilution, indicating that the amount of prion protein adsorbed to Mte exceeded that in samples without immunoreactivity (e.g., unbound PrPSc in experiments with Mte) by at least two orders of magnitude (Figure 1B). Furthermore, this result suggests that fine quartz sand was saturated by at least 100-fold less PrPSc (≤ 0.002 μg) than used for sorption experiments (Figure 1A).

To assess the PrPSc-binding capacity of the other soil minerals, increasing quantities of PrPSc were added to each mineral. Protein desorbed from mineral particles was serially diluted and subjected to SDS-PAGE and immunoblotting to semiquantitate the amount of sorbed protein. The binding capacity of a mineral was attained when subsequent PrPSc additions did not further increase the dilution factor required to reach the limit of immunoblotting detection (Table 1). Of the minerals examined, Mte exhibited the highest PrPSc adsorption capacity (~100 μgprotein mgMte−1). The adsorption capacity of the quartz microparticles was nearly 10-fold less (~15.6 μgprotein mgmicroparticle−1), and that of Kte was nearly 100-fold less than Mte (~2 μgprotein mgKte−1). When expressed on a surface-area basis (Table 1), the adsorption capacities of Mte and quartz microparticles were indistinguishable by our measurement method; that of Kte was 25 times less. These data demonstrate that mineral surface properties contribute to differences in the amount of PrPSc bound.

PrPSc Desorbed from Mte Surfaces Is Cleaved

Unexpectedly, PrPSc desorbed from Mte surfaces exhibited a lower molecular mass (~27–31 kDa) than the starting material (~33–35 kDa) (Figure 1A). Neither contaminant proteases nor metal oxide coatings on Mte particles appeared responsible for PrPSc cleavage, as treatments to counteract each did not prevent cleavage (unpublished data). Prior to sorption experiments, Mte was boiled in a solution of 10 mM NaCl for 10 min to denature contaminant proteases, or binding experiments were performed in the presence of a cocktail of protease inhibitors to inactivate them. Neither treatment prevented PrPSc cleavage. Amorphous metal oxide coatings on clay mineral particles can alter their surface reactivities and could potentially be responsible for PrPSc cleavage. The size-fractionated Mte used in this study has been reported to not contain such impurities at levels detectable by X-ray diffraction analysis [23], and precautionary pretreatment of the clay with a buffered neutral citrate-bicarbonate-dithionate solution to remove metal oxide coatings [24] failed to prevent cleavage.

Prion protein desorbed from Kte and quartz did not exhibit a change in molecular mass (Figure 1A), suggesting that surface properties specific to Mte were responsible for the cleavage. Previous studies on protein interaction with Mte have not noted reductions in molecular mass upon desorption [25,26]. We incubated PrPSc with Mte for short time periods (1–15 min) to qualitatively investigate initial adsorption and cleavage kinetics. Adsorption of PrPSc to Mte was apparent within 1 min, and reduction in protein molecular mass was discernable (Figure 2A). Prion protein cleavage consistently occurred early within the first 15 min of contact with Mte and appeared maximal by 60 min. Cleavage of PrPSc caused by sorption to or desorption from Mte seemed to be a phenomenon specific to this protein. We examined sorption and desorption of scrapie-infected hamster brain homogenate (BH) to Mte. Desorption of brain proteins from Mte produced no changes in the overall molecular mass distribution as visualized by Coomassie blue staining (unpublished data). Subunit C2 of the 20S proteasome (~29 kDa), an unrelated protein similar in size to PrP likewise did not appear cleaved upon desorption from Mte (Figure 2B). In contrast, PrPSc in BH was cleaved (Figure 2C).

Figure 2. PrPSc Desorbed from Mte Is Cleaved

(A) PrPSc cleavage occurs after short contact times with Mte surfaces.

(B) The molecular mass protein C2 of the 20S proteasome subunit from BH was unaltered following desorption from Mte.

(C) Cleavage of PrPSc present in infected BH was apparent after desorption from Mte.

(D) PrPSc desorbed from Mte lost immunoreactivity against an antibody recognizing the N-terminal portion of the mature protein.

(E) PrPSc pretreated with PK bound to Mte and did not exhibit further reduction in molecular mass when desorbed.

Immunoblots (A, B, and E) used mAb 3F4. Immunoblots (C and D) employed anti-C2 and R20 polyclonal antibodies, respectively. Pel, PrPSc associated with pelleted mineral particles; Sup, unbound PrPSc in supernatant.

Cleavage of PrPSc involved loss of the N-terminal portion of the protein, which is not necessary for infectivity [3]. Prion protein desorbed from Mte lost immunoreactivity with an antibody directed against amino acids 23–37 on the protein N terminus, indicating that all or part of the epitope of this antibody was missing from the desorbed protein (Figure 2D). In contrast, probing identical samples with a polyclonal antibody against full-length PrP demonstrated that PrPSc was desorbed from the Mte. Although the precise cleavage site was not determined, these data suggest that the N terminus of PrPSc was removed; the fate of the cleaved amino acid residues is not known, as they may have remained bound to the clay or may have been extracted but not detected. When the N-terminal ~70 amino acids were removed from PrPSc by pretreatment with proteinase K (PK) prior to adsorption to Mte, we observed sorption to the Mte, but no further reduction in molecular mass upon desorption, evidence that other regions of the protein remain intact when associated with Mte (Figure 2E). These results also indicate that the N terminus of PrPSc is not necessary for adsorption to Mte.

Strength of PrPSc Binding to Mte

PrPSc attachment to Mte was avid, and sorbed PrPSc was stable. Washing Mte-PrPSc with the background solution used in sorption experiments did not induce detachment of detectable amounts of PrPSc from Mte (unpublished data). Contact of PrPSc with Mte for up to 1 wk did not result in additional degradation, indicating that the protein was not rendered more susceptible to cleavage by further structural rearrangements on the clay surface (Figure 3A). The strength of PrPSc attachment to Mte was surprising, even in light of reports of protein sorption-desorption hysteresis on mineral surfaces [26]. Conditions previously employed to desorb other proteins from soil minerals were largely ineffective in detaching PrPSc from Mte surfaces [26,27]. In our experiments, described above, a solution containing 10% SDS at 100 °C was used to remove the PrPSc from mineral surfaces. Changes in pH often alter interactions between clay surfaces and sorbed proteins [27,28]. Incubation of Mte-bound PrPSc in 100 mM phosphate buffer at pH 2.5 or 11.5, proton activities substantially higher and lower than the reported isoelectric points for PrPSc [29], failed to release the protein (Figure 3B). Likewise, increases in ionic strength (0.1 M or 1 M NaCl) failed to remove detectable PrPSc from Mte (Figure 3C). Strong chaotropic agents can be effective in desorbing proteins from soil minerals by disrupting hydrogen bonds [26]; however, neither 8 M urea nor 8 M guanidine released detectable amounts of PrPSc from Mte (Figure 3D). Our data indicate the interaction between PrPSc and Mte is strong and of high affinity.

Figure 3. PrPSc Adsorbed to Mte Avidly and Remained Stable

(A) PrPSc was stable when adsorbed to Mte for at least 7 d. (B) Extremes in pH (100 mM phosphate at pH 2.5 or 11.5), (C) sodium chloride (100 mM or 1 M), and (D) chaotropic agents (8 M urea or 8 M guanidine [Gdn]) did not desorb detectable amounts of PrPSc from Mte. Primary extractions (1°) were followed by secondary extractions (2°) extractions with a 10% SDS solution at 100°C. Immunoblots (A–D) employed mAb 3F4. Pel, PrPSc associated with pelleted mineral particles; Sup, unbound PrPSc in supernatant.

PrPSc Bound to Mte Remains Infectious

Sorption of proteins to soil particles often results in structural rearrangements that cause loss or diminution of function [25,27,30]. If binding to Mte surfaces results in (partial) unfolding of PrPSc, a reduction or loss of infectivity would be expected, as denaturation renders the protein non-infectious [31]. We therefore tested whether PrPSc adsorbed to Mte remained infectious by intracerebrally inoculating hamsters with Mte-PrPSc complexes (Table 2). The time to onset of clinical symptoms after inoculation provides a measure of infectivity [32]. Hamsters inoculated with Mte-PrPSc exhibited clinical symptoms of scrapie 93 dpi. To control for any unbound prion protein that may have cosedimented with Mte particles, mineral-free PrPSc suspensions were processed in the same manner as in sorption experiments. The sedimented fraction of these control samples (mock pellets) showed substantially less infectivity than Mte-PrPSc pellets with a mean incubation period of 178 d, 105 d longer than Mte-PrPSc pellets. Hamsters inoculated with supernatants from these control samples (mock supernatants) showed clinical symptoms 103 dpi. Animals intracerebrally inoculated with Mte alone and uninoculated animals did not exhibit TSE symptoms during the course of the experiment (200 d).

Whole Soils Bind PrPSc

To examine the extent of prion protein binding by whole soils, we conducted PrPSc sorption experiments with four soils differing in texture and mineralogy (Table S2). When equal masses of soil (0.5 μg) were used, all soils bound PrPSc to a similar extent (Figure 4); no detectable PrPSc remained in the supernatant at the level of protein used in this experiment. Prion protein desorbed from the soils did not appear cleaved. Several nonmutually exclusive factors may have contributed to this finding, including (1) relatively small amounts of Mte in some samples, (2) occlusion of Mte cleavage sites by metal oxide and/or natural organic matter coatings, and (3) competition among the various sorption domains (both inorganic and organic) for PrPSc, limiting interaction with Mte. The amount of immunoreactive PrPSc recovered from each soil differed slightly; for example, the immunoreactive protein desorbed from the Elliot soil was less than that from the Boardman soil. This may have been due to stronger interaction of PrPSc with the Elliot soil than with the Boardman soil, leading to incomplete extraction, consistent with the larger fraction of clay-sized particles in the Elliot soil (Table S2).

Figure 4. Whole Soils Bind PrPSc

Elliot, Dodge, Bluestem, and Boardman soils bound PrPSc (pelleted soils). No immunoreactivity (i.e., no unbound PrPSc) was detected in the supernatants. Immunoblot employed mAb 3F4.


Environmental transmission of prion diseases has been noted for decades [7,8,14]. In this study, we provide evidence indicating that soil and soil minerals serve as a reservoir of TSE infectivity. While extrapolation of in vitro studies to the environment must be made with caution, our findings suggest that PrPSc released from diseased animals may be sequestered near the soil surface, maintaining the TSE agent in an environmental medium with which livestock and wildlife come in contact. Our experiments demonstrate that Mte-bound PrPSc remains infectious and suggest that soil may harbor more TSE agent than previously assumed on the basis of water extraction of prions from garden soil [13].

Our results demonstrate that all soil mineral surfaces examined bound PrPSc and that Mte and quartz have larger specific binding capacities for PrPSc than does Kte (Figure 1). Although not relevant to TSE transmission, nonglycosylated, recombinant PrPC has been shown to bind to Mte [33]. Interestingly, the N terminus of PrPSc desorbed from Mte was truncated (Figures 1A and 2). While Mte is known to catalyze several reactions, including the deamination of free glutamine and aspartic acid [34] and the polymerization of RNA into oligomers [35], protease activity has not been noted previously. The interaction between Mte and PrPSc is remarkably avid, as the only extractant used in this study that effected desorption was a solution containing 10% SDS at 100 °C (Figure 3B–3D). Prion protein appears unlikely to readily desorb from Mte in the environment. The propensity for PrPSc to tenaciously bind to Mte could be exploited in landfills to isolate prion-infected materials and prevent migration of the infectious agent.

The observation that prions remained infectious when bound to Mte is intriguing in light of the results of the desorption experiments; PrPSc adsorbed to Mte was extremely difficult to remove. Current mechanistic models for conversion of PrPC to the pathological form require direct PrPC–PrPSc interaction [36]. The brain is unlikely to possess microenvironments capable of extracting significant amounts of PrPSc from clay surfaces. The 10-d increase in incubation period for Mte-adsorbed PrPSc relative to clay-free controls (mock supernatant) was statistically significant (p < 0.05) and would correspond to approximately a 1-log increase in infectivity [32]. This result suggests that PrPSc-Mte complexes are inherently more infectious than the unbound protein and/or adsorption to Mte reduces clearance from the brain. We consider it likely that PrPSc adsorbed to Mte surfaces was available to convert PrPC in the brain to the pathological isoform. Our findings are reminiscent of reports in which metal wires exposed to scrapie agent harbored significant infectious agent despite attempts to remove attached PrPSc [37,38].

The infectivity of soil- and soil mineral-sorbed PrPSc following oral exposure warrants investigation. The binding of PrPSc to soil particles could reduce oral bioavailability such that soil serves as a sink rather than a reservoir for infectivity. Conversely, association with mineral particles may protect the agent from degradation in the gastrointestinal tract, possibly enhancing transmission [39]. For example, bovine rotaviruses and coronaviruses retain infectivity via the oral route when bound to clay minerals [40]. While desorption of the protein from soil particles is more likely to occur in the gut than in the brain, removal of PrPSc from mineral particles may not be necessary to initiate infection.

In conclusion, soil and soil minerals have the potential to bind PrPSc and maintain infectivity. These findings will serve as the basis for further study on the interaction of PrPSc with other soil components (humic substances, quartz, and other minerals), the stability of soil-bound PrPSc under typical environmental conditions (UV light, freeze-thaw cycles) and the effect of soil microorganisms and extracellular enzymes on protein integrity. Our current results suggest that sorption of PrPSc to clay minerals may limit its migration through the soil column. Maintenance of prion infectivity at the soil surface may contribute to the propagation of CWD and scrapie epizootics and enhance the likelihood of interspecies transmission of these diseases.

Materials and Methods

Preparation of soil minerals and soils.

Montmorillonite (SWy-2) and kaolinite (KGa-1b) clays, obtained from the Clay Minerals Society Source Clays Repository (West Lafayette, Indiana, United States), were size-fractionated by wet sedimentation to obtain particles with dh = 0.5–2 μm and saturated with sodium. These reference clay samples were extensively characterized previously [23,41]. Fine quartz sand (dh = 125–250 μm) and SiO2 microparticles (dh = 1–5 μm; 99% purity) were obtained from Sigma (St. Louis, Missouri, United States). The fine quartz sand was soaked for 24 h in 12 N HCl to remove impurities. X-ray diffraction analysis and infrared photoacoustic spectroscopy indicated that the SiO2 microparticles were composed of quartz.

We examined PrPSc sorption to four soils (Table S2). The Elliot soil was a silty clay loam purchased from the International Humic Substances Society (St. Paul, Minnesota, United States). Organically amended Dodge soil (sandy clay loam) was obtained from a glaciated upland area in Madison, Wisconsin. The Bluestem soil was a sandy clay loam collected from a fluvial deposit in Cedar Rapids, Iowa. The Boardman soil was a silt loam taken from an eolian deposit in Boardman, Oregon. Characteristics of these soils are presented in Table S2.

Source of PrPSc.

Syrian hamsters (cared for according to all institutional animal care and handling protocols of the University of Wisconsin, Madison) were experimentally infected with the Hyper strain of hamster-adapted transmissible mink encephalopathy agent. PrPSc was purified to a P4 pellet from brains of infected hamsters by a modification of the procedure described by Bolton et al. [42,43]. The P4 pellet prepared from four brains was resuspended in 1 ml of 10 mM Tris (pH 7.4) with 130 mM NaCl. For experiments employing PK-treated PrPSc, 20% brain homogenate was treated with 50 μg ml−1 of proteinase K for 30 min at 37 °C. After blocking PK activity with 5 mM phenylmethylsulfonyl fluoride, purification was performed as above.

Batch sorption experiments.

Larger prion aggregates were removed from purified PrPSc by collecting supernatants from two sequential 5-min centrifugations at 800 g (clarification step). Clarified PrPSc (~0.2 μg) was added to 500 μg of Mte or fine quartz sand, 1,500 μg of Kte, or 3.2 mg of quartz microparticles in 10 mM NaCl buffered to pH 7.0 with 10 mM 3-N-morpholinopropanesulfonic acid (MOPS) (500 μl final volume). In some cases, Mte experiments were conducted in unbuffered 10 mM NaCl. Sorption experiments with Mte performed in buffered and unbuffered 10 mM NaCl yielded comparable results. Experiments with Mte, Kte, and quartz microparticles each employed equivalent (external) mineral surface areas. In sorption experiments with whole soil samples, ~2 μg of clarified PrPSc was added to 5-ml suspensions of each soil (5 mg) in 5 mM CaCl2. Samples were rotated at ambient temperature for 2 h or an indicated time period. Sorption appeared complete within 2 h, as longer incubation times did not result in changes in levels of bound protein.

Each PrPSc-mineral suspension and a 500-μl aliquot of each PrPSc-soil suspension was placed over a 750 mM sucrose cushion prepared in a solution of the same composition as the background solution in the sorption experiment, and centrifuged at 800 g for 7 min to sediment mineral or soil particles and adsorbed PrPSc. A sucrose cushion was found necessary to prevent a fraction of unbound PrPSc from sedimenting during centrifugation. Clarified PrPSc did not sediment through the sucrose cushion (Figure S1).

Unbound PrPSc remaining in the supernatant was precipitated with four volumes of cold methanol and resuspended in SDS-PAGE sample buffer (100 mM Tris [pH 8.0], 10% SDS, 7.5 mM EDTA, 100 mM dithiothreitol, and 30% glycerol). PrPSc was extracted from pelleted mineral particles with SDS-PAGE sample buffer at 100 °C for 10 min. The same procedure was followed for PrPSc-soil suspensions. To determine mineral adsorption capacities for prion protein, varying volumes of clarified PrPSc preparation were added to a 1:100 dilution of each mineral suspension. All adsorption experiments were repeated at least three times.

For BH sorption experiments, 10% BH was clarified by collecting supernatants from two sequential 5-min centrifugations at 800 g. Aliquots (10 or 30 μl) of clarified BH were rotated with Mte in 10 mM NaCl at ambient temperature for 2 h; complexes of Mte and BH constituents were then sedimented through a sucrose cushion and processed as described in the preceding paragraphs.

All samples prepared for SDS-PAGE were separated on 4%−20% precast gels (BioRad, Hercules, California, United States) under reducing conditions. Proteins were transferred to polyvinyl difluoride membranes and immunoblotted with mAb 3F4 (1:40,000 dilution), R20 N-terminal pAb (1:10,000 dilution), Rab 9 pool 2 full-length PrP pAb (1:10,000 dilution), or anti-20S proteosome subunit C2 pAb (1 μg ml−1; A.G. Scientific, San Diego, California, United States). Detection was achieved with an HRP-conjugated goat anti-mouse immunoglobulin G (IgG) (BioRad) for mAb 3F4 and an HRP-conjugated goat anti-rabbit IgG (BioRad) for all pAbs.

X-ray diffraction analysis.

PrPSc preparation (10 μg) was added to 50 μg of Mte in 10 mM NaCl (final volume of 0.5 ml). Samples were rotated at ambient temperature for 2 h and centrifuged at 16,100 g for 7 min. After centrifugation, the bulk of the supernatant was removed, leaving a small amount of solution above the clay pellet. The clay was resuspended in the remaining supernatant, and the slurry was placed on silica wafer slides and stored in a desiccator for over 12 h. The basal d001 spacings of near homoionic Na+-SWy-2 before and after adsorption of PrPSc were determined by X-ray diffraction on a Scintag PAD V diffractometer (Cupertino, California, United States) using CuKα radiation and continuous scanning from 3° to 15° 2θ with a step size of 0.02° and a dwell time of 2 s.

Extraction experiments.

PrPSc adsorbed to Mte was incubated for 30 min at room temperature in 8 M urea or 8 M guanidine HCl (50 μl per pellet), 0.1 or 1 M NaCl (25 μl per pellet), or 100 mM sodium phosphate (pH 2.5 or 11.5; 25 μl per pellet). Primary extractions with these solutions were followed by secondary extractions with SDS-PAGE sample buffer at 100 °C to assess the efficacy of the primary extraction. Urea and guanidine primary extracts were dialyzed against double distilled water for 2 h (nominal molecular weight cutoff, 12–14 kDa; Fisher Scientific, Pittsburgh, Pennsylvania, United States) prior to SDS-PAGE analysis.

Infectivity bioassay.

PrPSc-Mte pellets prepared as above were resuspended in pH 7.4 PBS (50 μl per pellet) and intracerebrally inoculated into male, weanling Syrian hamsters (Harlan, Indianapolis, Indiana, United States). Equivalent amounts of PrPSc starting material or Mte without PrPSc were inoculated into control animals. Hamsters were monitored every 3 d for the onset of clinical symptoms [32,44]. Brains from clinically positive hamsters and uninfected controls were analyzed for protease-resistant PrP by immunoblotting.

Supporting Information

Figure S1. Sucrose Cushion Prevented Sedimentation of Unbound PrPSc under Conditions Necessary to Pellet Soil Minerals

A substantial amount of unbound PrPSc pelleted when centrifuged under conditions required to remove Na+-Mte from suspension, but was prevented from sedimenting by a sucrose cushion. Sucrose cushions were therefore employed in batch sorption experiments to prevent sedimentation of unbound PrPSc. Results from representative mock adsorption experiments are shown. PrPSc was rotated in a solution of 10 mM NaCl in the absence of soil minerals for 2 h and was either placed above a 750 mM sucrose cushion and centrifuged (two right lanes), or centrifuged without a sucrose cushion (two left lanes). Supernatants (Sup) and pellets (Pel) were analyzed by immunoblotting with mAb 3F4.

(17 KB PDF)

Table S1. Characteristics of Minerals Used in PrPSc Sorption Experiments

(25 KB DOC)

Table S2. Characteristics of Soils Used in PrPSc Sorption Experiments

(26 KB DOC)

Accession Numbers

The GenBank ( accession number for PrPSc is M14054.


We thank Richard Rubenstein (SUNY Downstate Medical Center) for the gift of mAb 3F4 and Byron Caughey (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories) for pAb R20. We thank Allen Herbst, Chad Johnson, Mine Ekenler, Juan Gao, and Laura Sullivan for technical assistance. We thank Harry Read and Beatriz Quinchia-Rios for their critical reading of this manuscript. We gratefully acknowledge the constructive comments of three anonymous reviewers.

Author Contributions

CJJ, DM, JMA, and JAP conceived and designed the experiments. CJJ, KEP, and PTS performed the experiments. CJJ, KEP, PTS, DM, JMA, and JAP analyzed the data. JMA and JAP contributed reagents/materials/analysis tools. CJJ, DM, JMA, and JAP wrote the paper.


  1. 1. Prusiner SB (1998) The prion diseases. Brain Pathol 8: 499–513.
  2. 2. Belay ED, Schonberger LB (2005) The public health impact of prion diseases. Annu Rev Public Health 26: 191–212.
  3. 3. Prusiner SB (1998) Prions. Proc Natl Acad Sci U S A 95: 13363–13383.
  4. 4. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, et al. (1991) Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy. Biochemistry 30: 7672–7680.
  5. 5. Hoinville LJ (1996) A review of the epidemiology of scrapie in sheep. Rev Sci Tech 15: 827–852.
  6. 6. Miller MW, Williams ES (2003) Prion disease: Horizontal prion transmission in mule deer. Nature 425: 35–36.
  7. 7. Greig JR (1940) Scrapie: Observations on the transmission of the disease by mediate contact. Vet J 96: 203–206.
  8. 8. Palsson PA (1979) Rida (scrapie) in Iceland and its epidemiology. In: Prusiner SB, Hadlow WJ, editors. Slow transmissible diseases of the nervous system. 1st Ed. New York: Academic Press. pp. 357–366. pp.
  9. 9. Division of Wildlife, Colorado Department of Natural Resources (2005) Hunter harvested moose tests positive for CWD. Available at: Accessed 17 March 06.
  10. 10. Post K, Riesner D, Walldorf V, Mehlhorn H (1999) Fly larvae and pupae as vectors for scrapie. Lancet 354: 1969–1970.
  11. 11. Carp RI, Meeker HC, Rubenstein R, Sigurdarson S, Papini M, et al. (2000) Characteristics of scrapie isolates derived from hay mites. J Neurovirol 6: 137–144.
  12. 12. Taylor DM (2000) Inactivation of transmissible degenerative encephalopathy agents: A review. Vet J 159: 10–17.
  13. 13. Brown P, Gajdusek DC (1991) Survival of scrapie virus after 3 years' interment. Lancet 337: 269–270.
  14. 14. Miller MW, Williams ES, Hobbs NT, Wolfe LL (2004) Environmental sources of prion transmission in mule deer. Emerg Infect Dis 10: 1003–1006.
  15. 15. Hadlow WJ, Kennedy RC, Race RE (1982) Natural infection of Suffolk sheep with scrapie virus. J Infect Dis 146: 657–664.
  16. 16. Sigurdson CJ, Williams ES, Miller MW, Spraker TR, O'Rourke KI, et al. (1999) Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus). J Gen Virol 80: 2757–2764.
  17. 17. Seeger H, Heikenwalder M, Zeller N, Kranich J, Schwarz P, et al. (2005) Coincident scrapie infection and nephritis lead to urinary prion excretion. Science 310: 324–326.
  18. 18. Brown P (1998) BSE: The final resting place. Lancet 351: 1146–1147.
  19. 19. Hui CA (2004) Geophagy and potential contaminant exposure for terrestrial vertebrates. Rev Environ Contam Toxicol 183: 115–134.
  20. 20. Weeks HP, Kirkpatrick CM (1976) Adaptations of white-tailed deer to naturally occurring sodium deficiencies. J Wildl Manage 40: 610–625.
  21. 21. Fries GF (1996) Ingestion of sludge applied organic chemicals by animals. Sci Total Environ 185: 93–108.
  22. 22. Drees LR, Wilding LP, Smeck NE, Senkagi AL (1989) Silica in soils: Quartz and disordered silica polymorphs. In: Dixon JB, Weed SB, Dinauer RC, editors. Minerals in soil environments, Soil Science Society of America book series. 2nd ed. Madison, Wisconsin: Soil Science Society of America. pp. 913–974. pp.
  23. 23. Chipera SJ, Bish DL (2001) Baseline studies of the Clay Minerals Society source clays: Powder X-ray diffraction analyses. Clays Clay Miner 49: 398–409.
  24. 24. Jackson ML (2005) Soil chemical analysis. Revised 2nd Ed. Madison, Wisconsin: Parallel Press.
  25. 25. Vettori C, Calamai L, Yoderc M, Stotzky G, Gallori E (1999) Adsorption and binding of AmpliTaq DNA polymerase on the clay minerals, montmorillonite and kaolinite. Soil Biol Biochem 31: 587–593.
  26. 26. Docoslis A, Rusinski LA, Giese RF, van Oss CJ (2001) Kinetics and interaction constants of protein adsorption onto mineral microparticles—Measurement of the constants at the onset of hysteresis. Colloids Surf B Biointerfaces 22: 267–283.
  27. 27. Morgan HW, Corke CT (1976) Adsorption, desorption, and activity of glucose oxidase on selected clay species. Can J Microbiol 22: 684–693.
  28. 28. Quiquampoix H, Staunton S, Baron MH, Ratcliffe RG (1993) Interpretation of the pH dependence of protein adsorption on clay mineral surfaces and its relavance to the understanding of extracellular enzyme activity in soil. Colloids Surf A Physiochem Eng Aspects 75: 85–93.
  29. 29. Bolton DC, Meyer RK, Prusiner SB (1985) Scrapie PrP 27–30 is a sialoglycoprotein. J Virol 53: 596–606.
  30. 30. Lecomte S, Hilleriteau C, Forgerit JP, Revault M, Baron MH, et al. (2001) Structural changes of cytochrome c(552) from Thermus thermophilus adsorbed on anionic and hydrophobic surfaces probed by FTIR and 2D-FTIR spectroscopy. Chembiochem 2: 180–189.
  31. 31. Caughey B, Raymond GJ, Kocisko DA, Lansbury PT Jr (1997) Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J Virol 71: 4107–4110.
  32. 32. Prusiner SB, Groth DF, Cochran SP, Masiarz FR, McKinley MP, et al. (1980) Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent. Biochemistry 19: 4883–4891.
  33. 33. Revault M, Quiquampoix H, Baron MH, Noinville S (2005) Fate of prions in soil: Trapped conformation of full-length ovine prion protein induced by adsorption on clays. Biochim Biophys Acta 1724: 367–374.
  34. 34. Naidja A, Siffert B (1989) Glutamic acid deamination in the presence of montmorillonite. Clay Miner 25: 27–37.
  35. 35. Ferris JP, Hill AR Jr, Liu R, Orgel LE (1996) Synthesis of long prebiotic oligomers on mineral surfaces. Nature 381: 59–61.
  36. 36. Caughey B (2001) Interactions between prion protein isoforms: The kiss of death? Trends Biochem Sci 26: 235–242.
  37. 37. Zobeley E, Flechsig E, Cozzio A, Enari M, Weissmann C (1999) Infectivity of scrapie prions bound to a stainless steel surface. Mol Med 5: 240–243.
  38. 38. Flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, et al. (2001) Transmission of scrapie by steel-surface-bound prions. Mol Med 7: 679–684.
  39. 39. Martinsen TC, Taylor DM, Johnsen R, Waldum HL (2002) Gastric acidity protects mice against prion infection? Scand J Gastroenterol 37: 497–500.
  40. 40. Clark KJ, Sarr AB, Grant PG, Phillips TD, Woode GN (1998) In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus. Vet Microbiol 63: 137–146.
  41. 41. Fripiat JJ, Van Olphen H (1979) Data handbook for clay minerals and other non-metallic minerals. Oxford, New York: Pergamon Press. 346 p.
  42. 42. Bolton DC, Bendheim PE, Marmorstein AD, Potempska A (1987) Isolation and structural studies of the intact scrapie agent protein. Arch Biochem Biophys 258: 579–590.
  43. 43. McKenzie D, Bartz J, Mirwald J, Olander D, Marsh R, Aiken J (1998) Reversibility of scrapie inactivation is enhanced by copper. J Biol Chem 273: 25545–25547.
  44. 44. Bessen RA, Marsh RF (1992) Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters. J Gen Virol 73: 329–334.
  45. 45. Silveira JR, Raymond GJ, Hughson AG, Race RE, Sim VL, et al. (2005) The most infectious prion protein particles. Nature 437: 257–261.