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From: TSS ()
Subject: Prions Adhere to Soil Minerals and Remain Infectious
Date: April 14, 2006 at 7:10 am PST

Prions Adhere to Soil Minerals

and Remain Infectious

Christopher J. Johnson1,2, Kristen E. Phillips3, Peter T. Schramm3, Debbie McKenzie2, Judd M. Aiken1,2,

Joel A. Pedersen3,4*

1 Program in Cellular and Molecular Biology, University of Wisconsin Madison, Madison, Wisconsin, United States of America, 2 Department of Animal Health and Biomedical

Sciences, School of Veterinary Medicine, University of Wisconsin Madison, Madison, Wisconsin, United States of America, 3 Molecular and Environmental Toxicology Center,

University of Wisconsin Madison, Madison, Wisconsin, United States of America, 4 Department of Soil Science, University of Wisconsin Madison, Madison, Wisconsin, United

States of America

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 diseaseassociated

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.

Citation: Johnson CJ, Phillips KE, Schramm PT, McKenzie D, Aiken JM, et al. (2006) Prions adhere to soil minerals and remain infectious. PLoS Pathog 2(4): e32. DOI: 10.1371/

journal.ppat.0020032

Introduction

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 b-sheet

and lower a-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-toanimal)

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 [6–8].

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 naı¨ve mule deer could contract CWD

Editor: David Westaway, University of Toronto, Canada

Received December 20, 2005; Accepted March 8, 2006; Published April 14, 2006

DOI: 10.1371/journal.ppat.0020032

Copyright:  2006 Johnson et al. This is an open-access article distributed under

the terms of the Creative Commons Attribution License, which permits unrestricted

use, distribution, and reproduction in any medium, provided the original author

and source are credited.

Abbreviations: BH, brain homogenate; BSE, bovine spongiform encephalopathy;

CWD, chronic wasting disease; dpi, days postinoculation; Kte, kaolinite; Mte,

montmorillonite; PK, proteinase K; PrPC, normal cellular isoform of the prion

protein; PrPSc, disease-associated prion protein; TSE, transmissible spongiform

encephalopathy

* To whom correspondence should be addressed. E-mail: joelpedersen@wisc.edu

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0001

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.

Results

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] ¼ 125–

250 lm), representing quartz concentrated in the sand and

silt fractions of soils, and microparticles (dh ¼ 1–5 lm),

representing quartz present in the coarse clay fraction [22].

Purified PrPSc (;0.2 lg) 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).

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 lg) 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

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 ¼ 125–250 lm). PrPSc desorbed from Mte was of lower

molecular mass than the starting material. Adsorption to quartz was

observed when quartz microparticles (dh¼1–5 lm) were employed and

surface area was matched to Mte.

(B) Immunoblotting sensitivity was determined by dilution of Mteadsorbed

PrPSc to the limit of detection. Protein was desorbed from Mte

in 50 ll of SDS-PAGE sample buffer at 100 8C and serially diluted.

Immunoblots used monoclonal antibody (mAb) 3F4. Pel, PrPSc associated

with pelleted mineral particles; Sup, unbound PrPSc in supernatant.

DOI: 10.1371/journal.ppat.0020032.g001

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0002

Sorption of Prions to Soil

Synopsis

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.

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 lgprotein mgMte1).

The adsorption capacity of the quartz microparticles was

nearly 10-fold less (;15.6 lgprotein mgmicroparticle1), and that

of Kte was nearly 100-fold less than Mte (;2 lgprotein mgKte1).

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).

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).

Table 1. PrPSc Adsorption Capacities for the Minerals Examineda

Mineral Binding Capacity

(Sorbent Mass Basis)

(lgprotein mgmineral1)

Binding Capacity

(Sorbent Surface Area Basis)

(mgprotein mmineral2)

Mte 87–174 2.8–5.7

Kte 1.7–2.6 0.15–0.22

Quartz

microparticles

13.6–27.1 2.7–5.4

aProtein concentration determined by Bradford assay; PrPSc concentration was taken as

87% of total protein [45]. Reported adsorption capacities represent upper estimates, as

the fraction of PrPSc in clarified preparations may have been lower.

DOI: 10.1371/journal.ppat.0020032.t001

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.

DOI: 10.1371/journal.ppat.0020032.g002

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0003

Sorption of Prions to Soil

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 8C 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.

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 noninfectious

[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).

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 (18) were

followed by secondary extractions (28) extractions with a 10% SDS solution at 1008C. Immunoblots (A–D) employed mAb 3F4. Pel, PrPSc associated with

pelleted mineral particles; Sup, unbound PrPSc in supernatant.

DOI: 10.1371/journal.ppat.0020032.g003

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0004

Sorption of Prions to Soil

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 lg) 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).

Discussion

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 Mtebound

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 8C (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

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.

DOI: 10.1371/journal.ppat.0020032.g004

Table 2. Prions Adsorbed to Montmorillonite Clay Retain

Infectivity

Inoculum Positive Animals/

Total Animals

Onset of Clinical

Symptoms (dpi)a

None 0/8 .200b

Mte (no PrPSc) 0/8 .200b

Mte-PrPSc complex 10/10c 93 6 4d

Mock supernatante (no Mte) 8/8 103 6 0d

Mock pellete (no Mte) 8/8 178 6 21d

aMean dpi 6 SD to the onset of clinical symptoms of TSE infection.

bNone of the animals showed clinical symptoms of TSE infection or had protease-resistant

PrP accumulation at the termination of the experiment at 200 dpi.

cAlthough 12 animals were inoculated, two non-TSE intercurrent deaths occurred at 8 dpi.

dBrains of infected animals were positive for protease-resistant PrP.

eMock supernatant and mock pellet samples were generated by adding clarified PrPSc

(;0.2 lg) to buffer in the absence of soil minerals and processing identically to samples

containing Mte.

DOI: 10.1371/journal.ppat.0020032.t002

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0005

Sorption of Prions to Soil

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 ¼ 0.5–2 lm and saturated with sodium. These reference clay

samples were extensively characterized previously [23,41]. Fine quartz

sand (dh ¼ 125–250 lm) and SiO2 microparticles (dh ¼ 1–5 lm; 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 PKtreated

PrPSc, 20% brain homogenate was treated with 50 lg ml1 of

proteinase K for 30 min at 37 8C. 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 lg) was added to 500 lg of Mte or fine quartz sand, 1,500

lg 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 ll 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 lg 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-ll aliquot of each PrPScsoil

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 8C 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 ll) 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 lg

ml1; 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 lg) was added to

50 lg 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 Naþ-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 CuKa radiation and continuous scanning

from 38 to 158 2h with a step size of 0.028 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 ll

per pellet), 0.1 or 1 M NaCl (25 ll per pellet), or 100 mM sodium

phosphate (pH 2.5 or 11.5; 25 ll per pellet). Primary extractions with

these solutions were followed by secondary extractions with SDSPAGE

sample buffer at 100 8C 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 ll 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 Naþ-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.

Found at DOI: 10.1371/journal.ppat.0020032.sg001 (17 KB PDF).

Table S1. Characteristics of Minerals Used in PrPSc Sorption

Experiments

Found at DOI: 10.1371/journal.ppat.0020032.st001 (25 KB DOC).

PLoS Pathogens | www.plospathogens.org April 2006 | Volume 2 | Issue 4 | e32 0006

Sorption of Prions to Soil

Table S2. Characteristics of Soils Used in PrPSc Sorption Experiments

Found at DOI: 10.1371/journal.ppat.0020032.st002 (26 KB DOC).

Accession Numbers

The GenBank (http://www.ncbi.nlm.nih.gov/) accession number for

PrPSc is M14054.

Acknowledgments

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.

Funding. This work was supported by USEPA grant 4C-R070-

NAEX (JAP) and DOD grant DAMD17–03–1–0369 (JMA).

Competing interests. The authors have declared that no competing

interests exist. &

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Sorption of Prions to Soil

http://pathogens.plosjournals.org/archive/1553-7374/2/4/pdf/10.1371_journal.ppat.0020032-S.pdf

http://pathogens.plosjournals.org/perlserv/?request=get-pdf&file=10.1371_journal.ppat.0020032-L.pdf

Epidemiology Update March 23, 2006
As of today, 13 locations and 32 movements of cattle have been examined with
27 of those being substantially completed. Additional investigations of
locations and herds will continue. In addition, state and federal officials
have confirmed that a black bull calf was born in 2005 to the index animal
(the red cow). The calf was taken by the owner to a local stockyard in July
2005 where the calf died. The calf was appropriately disposed of in a local
landfill and did not enter the human or animal food chain.

http://www.aphis.usda.gov/newsroom/hot_issues/bse/bse_al_epi-update.shtml

> The calf was appropriately disposed of in a local
> landfill and did not enter the human or animal food chain.


well, back at the ranch with larry, curly and mo heading up the USDA et al,
what would you expect, nothing less than shoot, shovel and shut the hell up.
no mad cow in USA, feed ban working, no civil war in Iraq either.


but what has past history shown us, evidently it has shown the USDA et al
nothing ;


Disposal of meat and bone meal (MBM) derived from specified risk material
(SRM) and over thirty month scheme carcasses by landfill
The Committee was asked to consider a quantitative risk assessment of the
disposal of meat and bone meal derived from specified risk material and over
thirty month scheme carcasses by landfill, prepared in response to a request
from the Committee at its June 1999 meeting.

The Committee was asked whether, in the light of the results of the risk
assessment, it held to its earlier published (June 1999) view that landfill
was an acceptable outlet for MBM of any origin, although it retained a
preference for incineration. The Committee reiterated that it had a strong
preference for incineration as the favoured route for the disposal of MBM
and were uneasy about the use of landfill for the disposal of this material.
If there were cases where incineration was not practical the Committee felt
it would be preferable for any material going to landfill to be
pressure-cooked first or possibly stored above ground prior to incineration.

http://www.seac.gov.uk/summaries/summ_0700.htm


Disposal of BSE suspect carcases
It is the Department's policy to dispose of BSE suspects by incineration
wherever feasible. No BSE suspect carcases have been landfilled since 1991.

http://www.defra.gov.uk/animalh/bse/publichealth/notification.html#disp


OPINION ON

THE USE OF BURIAL FOR DEALING WITH ANIMAL

CARCASSES AND OTHER ANIMAL MATERIALS THAT

MIGHT CONTAIN BSE/TSE

ADOPTED BY THE

SCIENTIFIC STEERING COMMITTEE

MEETING OF 16-17 JANUARY 2003

The details of the SSC’s evaluation are provided in the attached report. The
SSC

concludes as follows:

(1) The term “burial” includes a diversity of disposal conditions. Although
burial is

widely used for disposal of waste the degradation process essential for
BSE/TSE

infectivity reduction is very difficult to control. The extent to which such
an

infectivity reduction can occur as a consequence of burial is poorly
characterised.

It would appear to be a slow process in various circumstances.

(2) A number of concerns have been identified including potential for
groundwater

contamination, dispersal/transmission by birds/animals/insects, accidental

uncovering by man.

(3) In the absence of any new data the SSC confirms its previous opinion
that animal

material which could possibly be contaminated with BSE/TSEs, burial poses a

risk except under highly controlled conditions (e.g., controlled landfill).

SNIP...

4. CONCLUSION

In the absence of new evidence the opinion of the SSC “Opinion on Fallen
Stock”

(SSC 25th June 1999) must be endorsed strongly that land burial of all
animals and

material derived from them for which there is a possibility that they could

incorporate BSE/TSEs poses a significant risk. Only in exceptional
circumstances

where there could be a considerable delay in implementing a safe means of
disposal

should burial of such materials be considered. Guidelines should be made
available

to aid on burial site selection.

4 PAGES;

http://europa.eu.int/comm/food/fs/sc/ssc/out309_en.pdf


During the 2001 outbreak of FMD in the UK, the

Department of Health prepared a rapid qualitative

assessment of the potential risks to human health

associated with various methods of carcass disposal

(UK Department of Health, 2001c). The most

relevant hazards to human health resulting from

burial were identified as bacteria pathogenic to

humans, water-borne protozoa, and BSE. The main

potential route identified was contaminated water

supplies, and the report generally concluded that an

engineered licensed landfill would always be

preferable to unlined burial. In general terms, the

findings of the qualitative assessment relative to

biological agents are summarized in Table 13.

TABLE 13. Potential health hazards and associated pathways of exposure
resulting from landfill or burial of

animal carcasses (adapted from UK Department of Health, 2001c).

PLEASE SEE TABLE AT;

http://www.k-state.edu/projects/fss/research/books/carcassdispfiles/PDF%20Fi
les/CH%201%20-%20Burial.pdf


PART 2

Rendering and fixed-facility incineration were

preferred, but the necessary resources were not

immediately available and UK officials soon learned

that the capacity would only cover a portion of the

disposal needs. Disposal in commercial landfills was

seen as the next best environmental solution, but

legal, commercial, and local community problems

limited landfill use. With these limitations in mind,

pyre burning was the actual initial method used but

was subsequently discontinued following increasing

public, scientific, and political concerns. Mass burial

and on-farm burial were last on the preferred

method list due to the complicating matter of bovine

spongiform encephalopathy (BSE) and the risk posed

to groundwater (Hickman & Hughes, 2002).


http://www.k-state.edu/projects/fss/research/books/carcassdispfiles/PDF%20Fi
les/Introduction%20to%20Part%202%20-%20Cross-Cutting%20&%20Policy%20Issues.p
df


Carcase disposal:

A Major Problem of the

2001 FMD Outbreak

Gordon Hickman and Neil Hughes, Disposal Cell,

FMD Joint Co-ordination Centre, Page Street

snip...


http://www.defra.gov.uk/animalh/svj/fmd/pages27-40.pdf


3. Prof. A. Robertson gave a brief account of BSE. The US approach
was to accord it a _very low profile indeed_. Dr. A Thiermann showed
the picture in the ''Independent'' with cattle being incinerated and thought
this was a fanatical incident to be _avoided_ in the US _at all costs_...

snip...


http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf


PAUL BROWN SCRAPIE SOIL TEST


http://www.bseinquiry.gov.uk/files/sc/seac07/tab03.pdf

Some unofficial information from a source on the inside looking out -

Confidential!!!!

As early as 1992-3 there had been long studies conducted on small
pastures containing scrapie infected sheep at the sheep research station
associated with the Neuropathogenesis Unit in Edinburgh, Scotland.
Whether these are documented...I don't know. But personal recounts both
heard and recorded in a daily journal indicate that leaving the pastures
free and replacing the topsoil completely at least 2 feet of thickness
each year for SEVEN years....and then when very clean (proven scrapie
free) sheep were placed on these small pastures.... the new sheep also
broke out with scrapie and passed it to offspring. I am not sure that TSE
contaminated ground could ever be free of the agent!!
A very frightening revelation!!!

----------

You can take that with however many grains of salt you wish, and
we can debate these issues all day long, but the bottom line,
this is not rocket-science, all one has to do is some
experiments and case studies. But for the life of me,
I don't know what they are waiting on?

Kind regards,

Terry S. Singeltary Sr.
Bacliff, Texas USA

More here:

http://www.bseinquiry.gov.uk/files/ws/s018.pdf


INCINERATION TEMPS

Requirements include:

a. after burning to the range of 800 to 1000*C to eliminate smell;

well heck, this is just typical public relations fear factor control.
do you actually think they would spend the extra costs for fuel,
for such extreme heat, just to eliminate smell, when they spread
manure all over your veg's. i think not. what they really meant were
any _TSE agents_.

b. Gas scrubbing to eliminate smoke -- though steam may be omitted;

c. Stacks to be fitted with grit arreaters;

snip...

1.2 Visual Imact

It is considered that the requirement for any carcase incinerator
disign would be to ensure that the operations relating to the reception,
storage and decepitation of diseased carcasses must not be publicly
visible and that any part of a carcase could not be removed or
interfered with by animals or birds.

full text;


http://www.bseinquiry.gov.uk/files/yb/1989/04/03006001.pdf


http://europa.eu.int/comm/food/fs/sc/ssc/out311_en.pdf


TSS




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