Transport of the Pathogenic PrionProtein through Landfill MaterialsK U R T H . J A C O B S O N , † S E U N G H A K L E E , †

D E B B I E M C K E N Z I E , ‡ , |

C R A I G H . B E N S O N , † , ⊥ A N DJ O E L A . P E D E R S E N * , † , §

Department of Civil and Environmental Engineering,Department of Comparative Biosciences, and Departmentof Soil Science, University of Wisconsin, Madison,Wisconsin 53706

Received September 16, 2008. Revised manuscript receivedJanuary 8, 2009. Accepted January 13, 2009.

Transmissible spongiform encephalopathies (TSEs, priondiseases) are a class of fatal neurodegenerative diseasesaffecting a variety of mammalian species including humans. Amisfolded form of the prion protein (PrPTSE) is the major, ifnot sole, component of the infectious agent. Recent TSE outbreaksin domesticated and wild animal populations have createdthe need for safe and effective disposal of large quantities ofpotentially infected materials. Here, we report results of a studyto evaluate the potential for transport of PrPTSE derived fromcarcassesandassociatedwastes inmunicipalsolidwaste (MSW)landfills. Column experiments were conducted to evaluatePrPTSE transport in quartz sand, two fine-textured burial soilscurrently used in landfill practice, a green waste residual material(a potential burial material), and fresh and aged MSW.PrPTSE was retained by quartz sand and the fine-texturedburial soils, with no detectable PrPTSE eluted over more than40 pore volumes. In contrast, PrPTSE was more mobile in MSWand green waste residual. Transport parameters wereestimated from the experimental data and used to modelPrPTSE migration in a MSW landfill. To the extent that the PrPTSE

used mimics that released from decomposing carcasses andthe column experiments adequately simulate prion transportthrough burial soils, burial of CWD-infected materials at MSWlandfills could provide secure containment of PrPTSE providedreasonable burial strategies (e.g., encasement in fine-grainedsoil) are used.

IntroductionTransmissible spongiform encephalopathies (TSEs, priondiseases) are fatal neurodegenerative diseases affecting avariety of mammalian species and include bovine spongiformencephalopathy (BSE or “mad cow” disease); chronic wastingdisease (CWD) of cervids (deer, elk, and moose); scrapie of

sheep and goats; and Creutzfeldt-Jakob disease (CJD), fatalfamilial insomnia, and Gerstmann-Straussler-Scheinker dis-ease in humans. The significance of TSEs in North Americahas become increasingly apparent. Sixteen cases of BSE havebeen reported in North America in recent years (www.oie.int/eng/info/en_esbmonde.htm), and the known geographicrange of CWD has expanded dramatically in the past 8 years.

The infectious agent in these diseases is the prion, apathogen apparently lacking nucleic acids and composedprimarily, if not solely, of a misfolded isoform of the normalcellular prion protein (designated PrPTSE) (1). Spongiformdegeneration of the brain occurs as abnormal prion proteinsaccumulate, resulting in personality and memory changes,loss of coordination, and inevitably death (1). No cure existsfor these diseases. Interspecies transmission of some priondiseases has been documented (e.g., refs 2 and 3), includinghumans contracting new variant CJD from consumption ofBSE-infected beef (4). Thus, the presence of TSEs in wild orfarmed animal populations represents a potential risk to bothhuman and domestic animal health, and governments areacting to safeguard human and livestock populations.

Countermeasures to minimize the risk of TSE transmissionoften involve depopulation of infected herds and extensivepostmortem screening. For example, hundreds of cattle wereslaughtered, and 2000 Mg (metric tons) of potentially infectedbeef products were discarded when the first U.S. BSE casewas discovered in 2003 (5). In Wisconsin and other states,CWD management efforts include depopulation of infectedherds to limit intra- and potential interspecies transmission.Large volumes of infected waste are generated by theseresponses to TSE threats, and a significant need exists forsafe and effective disposal of infected carcasses and othermaterials. Because of the large volume of material, under-ground burial and landfilling are attractive disposal options.However, the risks associated with these options remainsunknown. Prions are highly resistant to degradation (e.g.,refs 6-8) and therefore may persist in environments thatmay lead to human and animal exposure. In addition,virtually nothing is known about the transport and fate ofprions in porous media (e.g., soils, landfills, subsurfaceenvironments) (9).

The objective of this study was to assess the potential forPrPTSE transport through materials present in conventionalmunicipal solid waste (MSW) landfills. A series of laboratory-scale column experiments was conducted to quantify migra-tion of PrPTSE through landfill burial materials and solid waste,and mathematical modeling was used to estimate theconcentration of PrPTSE in landfill leachate for a typicaldisposal scenario. All experiments were conducted using thepathogenic form of the prion protein (PrPTSE) as a biomarkerfor infectivity.

Materials and MethodsPorous Media. Six porous media were selected for use inprion transport experiments: quartz sand, two natural soils,green waste residual material (GWR), and fresh and agedMSW. Quartz sand was selected as a material with lowpotential for binding of PrPTSE (10, 11), whereas the naturalsoils, GWR, and MSW were intended to represent more typicalburial materials that might be encountered in landfill practice.Properties of these porous media are presented in Table 1.

The quartz sand (Iota 6, Unimen, New Canaan, CT) wasuniformly graded, consisting of particles with a nominal sizebetween 0.18 and 0.25 mm. Metal and organic contaminantswere removed from the fractionated sand by 24-h soakingin 12 N HCl, rinsing with distilled deionized water (ddH2O;

* Corresponding author phone: (608)263-4971; fax: (608)265-2595;e-mail: japedersen@soils.wisc.edu. Corresponding author address:Department of Soil Science, University of Wisconsin, 1525 Observa-tory Drive, Madison, WI 53706-1299.

† Department of Civil and Environmental Engineering.| Present address: Alberta Centre for Prions and Protein Folding

Diseases, 2-04 Environmental Engineering Building, University ofAlberta, Edmonton, Alberta T6G 2M8, Canada.

‡ Department of Comparative Biosciences.⊥ Present address: Department of Civil & Environmental Engi-

neering, University of Washington, Seattle, WA.§ Department of Soil Science.

Environ. Sci. Technol. 2009, 43, 2022–2028

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18 MΩ-cm resistivity), and baking overnight at 800 °C (12).Prior to use, the sand was rehydrated by 1-h boiling in ddH2Owater. Attachment of PrPTSE to this quartz sand in batchexperiments was described previously (11).

Two soils used as burial materials at MSW landfills wereselected for study: Boardman silt and Bluestem clay soils.These soils represent a range of PrPTSE binding potentialsthat might be encountered in practice (silt: lower bindingpotential, clay: higher binding potential). Sorption of PrPTSE

to these soils is described by Johnson et al. (10). The soilswere dried for 24 h at 110 °C and then moistened togravimetric water contents typical of field conditions priorto being compacted in the columns (15% Boardman, 11%Bluestem, ref 13).

Fresh MSW was obtained from the Wake County TransferStation in Wake County, North Carolina and was composedof 70.7% (w/w) paper, 14.6% plastic, 3.9% metal, 3.5% glass,0.6% wood, and 6.7% miscellaneous materials (e.g., fabric,food debris, cigarette butts, rubber, excrement). Aged MSW(∼10 y old) was obtained from a bioreactor landfill inKentucky and was composed of 50.3% (w/w) soil, 24.0% wood,10.3% paper, 4.1% plastic, 2.2% glass, 0.5% metal, and 8.5%miscellaneous materials. Both types of MSW were shreddedto a maximum particle size of 10 mm prior to packing intothe columns. GWR was obtained from a composting opera-tion at a MSW landfill.

Leachate. Landfill leachate was obtained from a MSWlandfill in southern Wisconsin operating with leachaterecirculation. Leachate was collected from a lift station withpoly(tetrafluoroethylene) (PTFE) bailers and stored at 4 °Cunder inert gas until used. Major cations and anions, pH,redox potential, and alkalinity of the leachate are describedin Table S2 in the Supporting Information (SI).

Prion Source. Prion-enriched fractions were preparedfrom brain tissue of hamsters clinically affected with the HYagent using a modification of the Bolton et al. (14) proceduredescribed by McKenzie et al. (15). Such prion enrichmentscontain∼109 infectious units (IU50) ·mL-1 (11). Hydrodynamicradii of PrPTSE aggregates present in these prion enrichmentsrange from 250 to 500 nm under the pH and ionic strengthconditions relevant for the present study (11).

PrPTSE Transport Experiments. Column tests to evaluatePrPTSEtransport were conducted with quartz sand, Boardmansilt, Bluestem clay, GWR, and MSW. All column tests wereconducted in custom-fabricated columns (10-mm ID, 24-mm height for quartz sand, soil, and GWR experiments; 50-mm ID, 50-mm height for MSW experiments). Different sizedcolumns were employed to allow even packing of eachmaterial and to minimize the amount of PrPTSE used.Poly(tetrafluoroethylene) (PTFE) was used for all columnparts based on preliminary experiments that indicatedminimal PrPTSE binding to PTFE relative to other potentialcolumn materials (viz. glass, polyvinyl chloride, polymethylmethacrylate). A 1-mm thick perforated PTFE frit was placedat the bottom of each column to retain the porous medium.Ferrules were PTFE or ethylene-tetrafluoroethylene, fittingswere made of fluorinated ethylene polypropylene (TeflonFEP), and seals were PTFE-coated Viton.

For quartz sand experiments, sand was placed by sedi-mentation in ddH2O while gently tapping the column. TheBoardman silt and Bluestem clay were compacted to drydensities of 1.62 and 1.65 Mg ·m-3, the GWR was compactedto a dry density of 1.09 Mg ·m-3, and the fresh and aged MSWwere compacted to dry densities of 0.49 and 0.77 Mg ·m-3.These densities represent typical field conditions (16).

End plates were attached to the columns, and the enclosedmedium was saturated by pumping ddH2O (quartz sand) or5 mM CaCl2 (soils, GWR, MSW) through the column for atleast 15 pore volumes (PV). A pore volume was defined asthe total volume of pore space in the specimen as definedby weight-volume computations. This gross pore volumemay neglect micropores within particles. Flow was orienteddownward to be consistent with the landfill setting.

To determine the hydraulic properties of the porousmedia, a tracer test was conducted by pumping 1.2 mM KBrthrough each column. The effluent Br- concentration wasdetermined using a microplate-based colorimetric method(17). The effective porosity and dispersivity (Table 1) weredetermined by fitting the advection-dispersion-reactionequation (ADRE) to the Br- breakthrough curve using themethod in Lee and Benson (18). Tailing was absent in theBr- effluent data, and good agreement was obtained between

TABLE 1. Properties of Packing Materials Used in Column Experimentsa

parameter quartz sand Bluestem clay Boardman silt GWRb fresh MSW aged MSW

specific surface areac (m2 ·g-1) 0.2 10.3 13 11 ND NDfoc

d,e 0 0.0042 0.0047 0.022 0.42 0.18dry density (Mg ·m-3), Fd 1.38 1.62 1.65 1.09 0.49 0.77effective porosity, ne 0.38 0.27 0.34 0.45 0.64 0.52dispersivity (mm) 1.03 2.68 0.60 1.41 32 51residual volumetric water content, θr 0.45 0.068 0.034 0.095 0.11 0.11saturated water content, θs 0.43 0.38 0.46 0.45 0.53 0.53R (m-1) 14.5 0.8 1.6 20 26 26n 2.68 1.09 1.37 1.31 2.22 2.22saturated hydraulic conductivity (m ·d-1),K 7.128 0.048 0.06 0.062 0.086 0.086

particle size distributions

parameter quartz sand Bluestem clay Boardman silt GWRb fresh MSW aged MSW

% sand 100 50 37 26 ND ND% silt 0 23 55 56 ND ND% clay 0 27 8 18 ND ND

a Abbreviations: foc, mass fraction of organic carbon; GWR, green waste residual; MSW, municipal solid waste; R and n,van Genuchten parameters for the water retention function; ND, not determined. b GWR was obtained from the compostingoperation of a landfill in southern Wisconsin where disposal of infected carcasses has been proposed. c Specific surfacearea measured by N2 adsorption (BET method). d Mass fraction of organic carbon in soils determined by the organiccarbon dry combustion method using a Leco CNS-2000 ((0.0001) (St. Joseph, MI). e Mass fraction of organic carbon inMSW calculated from loss on ignition (loi) data using the relationship foc) 0.476 (floi-0.0133) (Mort Barlaz, personalcommunication).

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the data and the fit of the ADRE, indicating that classicaladvection-dispersion theory represented the transport pro-cesses in the experiments. However, in some waste materialswith significant secondary porosity within the organicfraction, other transport processes could be important (e.g.,micropore diffusion).

Columns were flushed for g15 PV with the chosen eluent(10 mM Tris pH 7.0 + 10 mM NaCl for quartz sand to allowcomparison with previous research (11); leachate for soils, MSW,and GWR to represent conditions prevailing in landfill environ-ments) after the tracer test. PrPTSE-enriched preparations werethen applied directly to the top of the column (10 μL or ∼50ng for quartz sand, soil, and GWR; 300 μL or∼1500 ng for MSW).Forty PV of eluent were then pumped downward through eachcolumn at a seepage velocity of ∼0.2 m ·d-1. For the quartzsand, soil, and GWR columns, four samples were collected perPV in Protein Lo-Bind microcentrifuge tubes (Eppendorf AG,Hamburg, Germany). After elution, samples were snap-frozenin liquid nitrogen and stored at -80 °C until analysis. For theMSW columns, two or four samples were collected per PV in50-mL PTFE centrifuge tubes (Nalg Nunc, Rochester, NY) andstored at-80 °C prior to analysis. All column experiments wereconducted at least twice.

Prior to analysis, PrPTSE was precipitated from solution byaddition of ice-cold methanol (4 × sample volume), overnightstorage at-20 °C, and 30-min centrifugation at 0 °C (24,400g).Supernatants were aspirated and discarded. The resultingpellets were dried by vacuum centrifugation (Speed VacSC110, Thermo Savant, Waltham, MA) and resuspended ina 1:1 mixture of water and 5 × sample buffer (100 mM Tris,7.5 mM EDTA, 100 mM DTT, 350 mM SDS pH 8.0) used forSDS-PAGE. All samples were analyzed by immunoblotting(vide infra).

After eluting 40 PV, the columns were frozen for 1 h at-80 °C and sectioned using a razor blade (quartz sand, soiland GWR columns) or overnight at -20 °C and sectionedwith an Oster 2803 reciprocating electric knife (MSWcolumns). PrPTSE was extracted from a portion of each sectionwith 5 × SDS-PAGE sample buffer at 100 °C and analyzed byimmunoblotting (10).

Immunoblot Analysis. Samples containing PrPTSE werefractionated by SDS-PAGE and analyzed by immunoblottingfollowing the method in Johnson et al. (10). The PrP-specificmonoclonal antibody 3F4 (Signet, Dedham, MA) was usedat a 1:40,000 dilution. Detection was achieved with HRP-conjugated goat antimouse immunoglobulin G (Bio-Rad,Hercules, CA) and West Pico peroxidase detection substrate(Pierce, Rockford, IL). Control experiments with leachate andsoil extracts in the absence of PrPTSE demonstrated nononspecific binding of the primary antibody and limitednonspecific binding of the immunoglobulin G to constituentsin the leachate. This nonspecific binding gave signals near50 kD and 37 kD and did not interfere with detection of PrP.Spiking PrPTSE into leachate or column eluent did notsignificantly diminish immunoreactivity of the PrPTSE.

Estimation of Transport Parameters. When PrPTSE wasobserved in the effluent (i.e., present above the detectionlimit), transport parameters were obtained by fitting thebreakthrough curve with the one-dimensional ADRE withlinear and instantaneous sorption and first-order attachmentand detachment (19)

∂θC∂t

) ∂

∂z(θD∂C∂z )- ∂qC

∂z-

FdKd

θ∂θC∂t

- θkattC+FdkdetS

θ(1)

where θ is volumetric water content, C is the PrPTSE

concentration in the aqueous phase, t is time, z is distancein vertical direction, v is seepage velocity, D is the dispersioncoefficient, q is the Darcy velocity, Fd is the dry density ofmedium in the column, Kd is the equilibrium distributioncoefficient describing linear, instantaneous, and reversible

sorption, katt is a first-order attachment coefficient, kdet is afirst-order detachment coefficient, and S is the solid-phase(attached) PrPTSE concentration. Various forms of eq 1 havebeen used successfully for modeling the transport of colloidsin porous media (e.g. refs 19-21).

Equation 1 was fitted to the data using the inversealgorithm in the program HYDRUS (v1.02 (22)), which usesthe finite-element method to solve eq 1 and employs aMarquardt-Levenberg optimization algorithm for inversion.The seepage velocity was set at q/ne, where ne is the effectiveporosity, and the dispersion coefficient was set as the productof the dispersivity and seepage velocity using parametersfrom the tracer tests (Table 1). Molecular diffusion wasignored because advection dominated transport in thecolumns (Peclet number. 10). The reaction parameters (Kd,katt, and kdet) were independent variables fitted via theinversion algorithm.

When no PrPTSE was observed in the column effluent (i.e.,below detection limit), the governing equation was simplifiedby neglecting Kd and kdet:

∂C∂t

)D∂

2C

∂z2- v

∂C∂z

- kattC (2)

The solution to eq 2 for instantaneous slug input of mass Mat z ) 0 is (23)

C(z, t)){ M ⁄ A

√4πDtexp[- (z- vt)2

4Dt ]}e-kattt (3)

where A is the cross sectional area of the column. This approachwas based on a parametric evaluation of the potential impactsof Kd, katt, and kdet on PrPTSE transport, which showed that theabsence of PrPTSE in the effluent over at least 40 PV was possibleonly when Kd and kdet were nil for the column testing conditionsthat were employed (see the SI). Equation 3 was solved for katt

assuming that the PrPTSE concentration in the effluent was 80ng ·L-1 (based on detection limit for the immunoblot analysisof the sand and soil column effluent samples) at one PV. Theseepage velocity and dispersion coefficient were set as previ-ously described. Use of eq 3 in this manner provides a lowerbound estimate of katt.

Results and DiscussionTransport of PrPTSE through Quartz Sand and Soils. Effluentfrom the quartz sand and soil columns contained no PrPTSE

detectable by immunoblotting (detection limit ∼25 pg PrPTSE,equivalent to 0.05% of the protein loaded onto columns).Dissection and analysis of sections from the quartz sand,Boardman silt, and Bluestem clay columns revealed intensePrPTSE signals from the topmost section (upper 3 mm) andstrongly attenuated signals from the subsequent two or threesections (Figure 1).

Mass balances for the quartz sand and soil columns couldnot be closed with the analytical methods used (i.e., not allof the PrPTSE loaded onto the columns could be recovered).More than 95% of the mass of protein applied was extractedfrom the sand column, while ∼55% and ∼38% were recoveredfrom the Boardman silt and Bluestem clay columns. Theinability to account for all of the PrPTSE may have been dueto time-dependent decline in the extractability of the proteinfrom soil as previously reported (24, 25) and as observed insoil persistence experiments conducted in our laboratory.Degradation of PrPTSE within the columns cannot be ruledout. However, past studies have demonstrated that PrPTSE isremarkably stable (7, 8), and preliminary experimentsindicated that PrPTSE does not degrade in landfill leachate inthe time frame studied. Thus, degradation within the columnsis considered unlikely.

Our finding of negligible PrPTSE transport in soil is consistentwith previous observations of low migration of infectivity (6)

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and prion protein (25) in soil persistence experiments. Limitedmobility in soil was also noted for a recombinant, noninfectiousform of the prion protein that is folded in a considerably differentmanner (26). The aggregate state in which prions are releasedfrom decomposing carcasses is not presently known. Theprocedure we used to isolate PrPTSE in this study produces rod-shaped prion aggregates that are larger than those present ininfected tissues. This may have promoted retention in theseporous media.

Transport of PrPTSE through MSW. PrPTSE was detectedin effluent samples from both the fresh and aged MSWcolumns (Figure 2). Relative to the aged MSW, the break-through observed for the fresh MSW included a largerpercentage of the loaded protein (28% versus 0.3%) and wasspread over a larger number of pore volumes (8 vs 3 PV)(Figure 2a,d). For both materials, no PrPTSE was detectedbeyond 8 PV. When sectioned (Figure 2b,e), both columnsshowed PrPTSE distributions similar to those of the soil andquartz columns, with all detectable protein residing in thetopmost sections. These results suggest that PrPTSE maymigrate through MSW. Migration of PrPTSE in MSW, but notin soils, may be due to the larger pores in MSW and thehigher porosity of MSW. Similarly, more migration may haveoccurred in fresh MSW relative to aged MSW because of thehigher porosity and larger pores of the former.

The MSW used in the transport experiments was shreddedto a maximum particle size of 10 mm to permit compactionin the columns. The pore spaces of actual (i.e., unshredded)MSW are expected to be considerably larger, potentiallypermitting migration of larger amounts of PrPTSE thanobserved in these experiments.

Transport of PrPTSE through Green Waste Residual.PrPTSE eluted in the first two pore volumes from the GWRcolumn (Figure 3a). The first PV sample produced a very

strong signal, while the second PV sample yielded a sub-stantially weaker signal (Figure 3a). A signal was not detectedin later samples. The total amount of PrPTSE detected in thefirst two pore volumes accounted for ∼2% of the PrPTSE loadedonto the column. Analysis of the column sections (Figure3b) showed a detectable signal in only the topmost section,suggesting that the majority of the PrPTSE did not migratethrough the column. These results indicate that, as with MSW,a fraction of PrPTSE can migrate through GWR. At present,the reason for larger migration through the GWR relative tothe soils is not clear. However, the large pore size and higherorganic matter content (relative to the soils used) of the GWRmay have contributed to transport of PrPTSE.

Estimated PrPTSE Transport Parameters. Transport pa-rameters for PrPTSE in the MSW and GWR (Table 2) wereobtained by inversion of eq 1 using HYDRUS. Since no PrPTSE

was observed in the effluent of the column tests conductedwith quartz sand, Boardman silt, or Bluestem clay, eq 3 wasused to calculate katt for these media. These katt for quartzsand, Boardman silt, or Bluestem clay represent lower-boundestimates; the actual katt are expected to be larger. Theestimated katt are an order of magnitude higher thancalculated environmental katt for viruses (0.17-0.3 h-1; seerefs 27 and 28), the most comparable colloidal contaminants.

The kdet for MSW and GWR are small, and the uncertaintyin these values is as large as, or larger than, the meanestimates, suggesting that detachment for these materials isnegligible (Table 2). Similarly, katt for the GWR does not differfrom zero. Additionally, Kd is nil or near zero for all threematerials for which the effluent data could be analyzed withHYDRUS. This finding, along with the parametric analysesreported in the SI, suggests that attachment is the dominantmode of PrPTSE binding to the solid phase in porous media

FIGURE 1. PrPTSE extracted from (a) quartz sand, (b) Boardman, and (c) Bluestem soil columns. Each section represented ∼3 mm,12.5% of the column height. Section 1 is the topmost section of the column. The PrPTSE preparation (10 μL or ∼50 ng of protein) wasloaded onto the top of the column. Positive controls are given as a percentage of the total PrPTSE preparation initially applied to thecolumn. Protein molecular mass is indicated at the left. The quartz sand column experiment was repeated three times; the Boardmanand Bluestem soil columns were repeated twice.

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and that distribution coefficients reported in previous batchstudies (e.g., ref 11) likely reflect attachment rather thansorption.

Implications for Landfill Disposal of Prion-Contami-nated Waste. To evaluate the implications of our experi-mental results for the disposal infected carcasses in MSWlandfills, we simulated PrPTSE transport in a profile charac-teristic of a landfill setting and estimated PrPTSE concentra-tions in the leachate collection system. The disposal scenariosimulated was consistent with management and disposalpractices anticipated in Wisconsin, a state with a significantneed for safe and economical disposal of CWD-infectedmaterials. Disposal of infected carcasses was assumed tooccur annually in a single pit excavated within MSW at anoperating landfill. The annual carcass volume requiringdisposal was estimated to be 450 m3 based on the mass ofcarcasses collected each year in Wisconsin (130 Mg) andrecommendations for mass livestock burial (29). The disposalpit was assumed to be 15-m × 15-m × 2-m (Figure S2) andunderlain by a 0.5-m layer of burial soil. The burial soil wasseparated from the leachate collection system by 2 m of MSW.

This geometry is consistent with operations in practice, anddisposal requirements under consideration in Wisconsinstipulate even larger separation using MSW (>6 m). Leachatewas assumed to contact carcasses directly from MSW placedover the pit. This assumption overestimates contact betweenleachate and the carcasses, because disposal practices requirethat carcasses be encased in burial soils to reduce thelikelihood for contact with leachate and to provide contain-ment of PrPTSE should flows occur laterally within and throughthe sides of the disposal pit.

Transport Model. One-dimensional transport of PrPTSE inthe assumed landfill profile (Figure S2) was predicted usingeq 3 and with HYDRUS. The PrPTSE mass initially assignedto each disposal pit was 1.5 g PrPTSE. This value correspondsto the amount of PrPTSE estimated to be disposed annuallyin Wisconsin (see the SI for detailed discussion). All PrPTSE

was assumed to be instantaneously released from thedisposed CWD-contaminated material. This assumptionoverestimates PrPTSE release, because the agent would bemobilized more slowly in a landfill as carcasses degrade,

FIGURE 2. PrPTSE in fresh and aged MSW column eluents and extracted from MSW. (a) Immunoblot of the initial pore volumes elutedfrom the fresh MSW column (one pore volume ) 62.8 mL). No further PrPTSE was detected in subsequent pore volumes. (b)Comparison of observed PrP concentrations with those predicted from the transport parameters in the fresh MSW column. Observedprotein concentrations were determined by immunoblotting diluted samples and comparison with samples of known concentrations(not shown). (c) Immunoblot of the distribution of PrP extracted from the fresh MSW column. Each section represented ∼10 mm, 19%of the column height. (d) Immunoblot of the breakthrough of PrP from the aged MSW column (one pore volume ) 51.1 mL). Nofurther PrPTSE was detected in subsequent pore volumes. (e) Comparison of observed PrP concentrations and concentrationspredicted by the transport parameters in the aged MSW column. (f) Immunoblot of the distribution of PrP extracted from the agedMSW column. Bands at ∼50 kD and ∼37 kD were due to nonspecific binding of the secondary antibody to constituents of theleachate used. Left-hand labels indicate protein molecular mass as determined using molecular mass standards. PVF are equal topore volumes of flow. In all immunoblots, positive controls (PrPTSE spiked into leachate) are given as percentages of the volume ofPrPTSE enriched preparation initially applied to the top of the columns (300 μL or ∼1500 ng of protein).

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resulting in gradual release into underlying materials. Theremainderofthedomainwasassignedzeroinitialconcentration.

For analyses including burial soils (quartz sand, Boardmansilt, or Bluestem clay), the PrPTSE concentration at the bottomof burial soil was calculated using eq 3. This approach wasused because the large katt of these materials (Table 2) resultedin numerical instability in HYDRUS. When the burial soilwas GWR, transport through the GWR and MSW was analyzeddirectly using HYDRUS. Transport parameters obtained fromtracer tests (Table 1) and the experiments conducted withPrPTSE (Table 2) were assigned as input. A 46-y time periodwas used for modeling (6 y as an open cell, 40 y of postclosurecare as stipulated by Wisconsin regulations).

Flow in the profile was defined by Richards’ equation

∂θ∂t

) ∂

∂z(K(h)∂h∂z )- ∂K(h)

∂z(4)

where h ) pressure head and K ) hydraulic conductivity.Equation 5 was solved by HYDRUS using a constant fluxboundary at the surface and a unit gradient boundary at thebase of the MSW layer. When the cell was open, the surfaceflux was assumed to be 0.3 m ·y-1, an upper bound leachategeneration rate in the eastern United States (30). For closedconditions, the surface flux was assumed to be 0.003 m ·y-1,

characteristic of percolation rates from landfill final coverswith composite barriers in the eastern United States (13).

For all simulations, van Genuchten’s equation was usedto describe the relationship between θ and pressure head

θ) θr + (θs - θr)1

[1+ (Rh)n]1-1⁄n(5)

where θr is the residual volumetric water content, θs is thesaturated water content, and R and n are the van Genuchtenparameters describing the water retention function. Param-eters assigned to eq 6 for GWR and MSW in HYDRUS aresummarized in Table 1.

Estimated Concentration of PrPTSE in the Leachate Col-lection System. For the quartz sand, Boardman silt, andBluestem clay, PrPTSE concentrations at the bottom of burialsoil were at the computational limit of the computer usedfor the simulations and, therefore, ∼0 ng ·L-1. Consequentlythe concentration in the leachate was ∼0 ng ·L-1. Penetrationinto the burial material over the 46-y simulation period was<20 mm. Simulations were also conducted for GWR as theburial material and for disposal directly on the existing MSW(no burial soil or GWR). For these cases, the concentrationof PrPTSE at the bottom of the profile (i.e., at the interface ofthe MSW and leachate collection system) was also zero. These

FIGURE 3. (a) Immunoblot of the first seven pore volumes eluted from the green waste residual (GWR) column (one pore volume )0.85 mL). No further PrPTSE was detected in subsequent pore volumes. MeOH Precip ) methanol-precipitated. (b) PrPTSE extractedfrom the GWR solids. Each section represented ∼3 mm, 12.5% of the column height. In both immunoblots, positive controls (PrPTSE

spiked into leachate) are given as percentages of the volume of PrPTSE enriched preparation initially applied to the top of thecolumn (10 μL). Left-hand labels indicate protein molecular mass as determined using molecular mass standards.

TABLE 2. PrPTSE Transport Parameters from Column Tests

porous medium amount of loaded PrPTSE eluted (%) analysis method Kd (L ·kg -1) katt (h-1) kdet (h-1) MSEb

quartz sand 0 eq 3a - >2.9 - -Boardman silt 0 eq 3 - >2.6 - -Bluestem clay 0 eq 3 - >3.3 - -

fresh MSW 28 eq 1 0.15 ( 0.05 0.55 ( 0.04 0.003 ( 0.003 4.0 × 10-20

aged MSW 0.3 eq 1 4.5 ( 0.2 6.6 ( 0.3 0.001 ( 0.002 2.9 × 10-23

green waste residual 2 eq 1 0.0 ( 1.2 1.9 ( 1.8 0.01 ( 0.38 2.3 × 10-18

a Analysis with eq 3 permits lower-bound estimate of katt only. b Mean square error of effluent concentration (g ·mL-1) vstime (h) function.

VOL. 43, NO. 6, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2027

analyses indicate that concentrations of PrPTSE in a leachatecollection system should be effectively zero for the conditionsthat were simulated, regardless of whether the CWD wasteis encased in burial soil. These simulations were based oncolumn experiments that used PrPTSE enriched from braintissue using a method that causes the protein to aggregateto a larger extent than appears to occur in vivo. Futureexperiments should employ partially disaggregated (31) andN-terminally truncated (32) PrPTSE and track the migrationof prion infectivity.

As noted above, however, the transport parameters forMSW were derived for experiments conducted with shreddedMSW. The pore spaces of actual, unshredded MSW areexpected to be considerably larger, potentially permittingmigration of larger amounts of PrPTSE than observed in thisexperiment. Thus, while this analysis may suggest that PrPTSE

may not migrate even if the CWD waste is disposed directlyon MSW, encasement in burial soil is still recommendeduntil data from larger-scale experiments are available. As afurther safeguard, PrPTSE migration may be further limitedby the use of reactive burial materials (33).

AcknowledgmentsThis research was supported, in part, by grants from theWisconsin Department of Natural Resources, National Sci-ence Foundation (CBET-0547484 (CAREER) and CBET-0826204), Department of Defense (DAMD17-03-1-0369), andU.S. Environmental Protection Agency (4C-R070-NAEX).K.H.J. was supported by a National Institutes of Healthtraining grant (NIH 5 T32 GM08349). C.H.B. was partiallysupported by his Wisconsin Distinguished Professorship. Wegratefully acknowledge Richard Rubenstein (SUNY Down-state Medical Center) for the gift of mAb 3F4, Harry Read fordetermining the composition of the MSW, Glen Hinckely forassistance with the column experiments, and Xiaodong Wangfor characterization of the porous media. We thank GeneMitchell, Alan Crossley, Fran Kremer, and Susan Mooney forhelpful discussions and three anonymous reviewers for theirconstructive comments. Endorsement by the sponsors is notimplied and should not be assumed.

Supporting Information AvailableText, tables, and figures. This material is available free ofcharge via the Internet at http://pubs.acs.org.

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(11) Ma, X.; Benson, C. H.; McKenzie, D.; Aiken, J. M.; Pedersen, J. A.Adsorption of pathogenic prion protein to quartz sand. Environ.Sci. Technol. 2007, 41, 2324–2330.

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S-1

Supporting Information

Transport of the Pathogenic Prion Protein through Landfill Materials

Kurt H. Jacobson1, Seunghak Lee1, McKenzie2, †, Craig H. Benson1, ‡, Joel A. Pedersen1,3,* 1 Department of Civil and Environmental Engineering, 2 Department of Comparative Biosciences, and 3 Department

of Soil Science, University of Wisconsin, Madison, WI 53706

*Corresponding author address: Department of Soil Science, University of Wisconsin, 1525 Observatory Drive, Madison, WI 53706-1299; phone: (608) 263-4971; fax: (608) 265-2595; e-mail: japedersen@soils.wisc.edu

† Present address: Alberta Centre for Prions and Protein Folding Diseases, 2-04 Environmental Engineering Building, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada ‡ Present address: Department of Civil & Environmental Engineering, University of Washington, Seattle, WA

Text S1. Parametric simulations to evaluate Kd, katt, and kdet for cases where no PrPTSE was detected in column effluent.

Text S2. Estimation of PrPTSE disposed annually in Wisconsin, USA.

Figure S1. Results of forward simulation with variable Kd. Figure S2. Schematic of disposal pit and underlying materials used in simulations.

Table S1. Mineralogy of porous media used in column tests.

Table S2. Properties of MSW leachate.

Table S3. Estimation of infectious material disposed annually

Text S3. Literature cited in the Supporting Information

S-2

Text S1. Parametric Simulations to Evaluate Kd, katt, and kdet for Cases where No PrPTSE was Detected in Quartz Sand Column Effluent. Forward simulations of PrPTSE transport in the quartz sand column experiments were

conducted with HYDRUS to evaluate the relevance of Kd, katt, and kdet for cases where effluent

concentrations were below detection limits throughout the duration of the experiments. All

conditions were identical to those used for the column tests. A constant flow rate boundary was

applied (0.098 m·d-1), and the sand was assigned a porosity of 0.45 and dispersivity of 0.11 mm

(Table 1).

Predicted PrPTSE concentrations in effluent from a column are shown in Figure S1 for katt

= 0 and Kd = 0.0 and 3.0 L·kg-1. These distribution coefficients bracket the range reported by Ma

et al. (ref. 1) for PrPTSE binding to quartz sand in batch tests. For these conditions, PrPTSE

concentrations in effluent above the detection limit were predicted, whereas no concentrations

exceeding the detection limit were observed in the column experiments. In contrast, when katt =

2.60 h-1 and Kd = 0 L·kg-1, the peak predicted effluent concentration is at the detection limit at

one PV (not shown). This suggests that attachment is a more likely binding mechanism than

instantaneous linear adsorption, and that the binding observed in the batch tests by Ma et al. (ref.

1) was likely attributable to attachment rather than adsorption. This conclusion is supported by

the breakthrough observed from the column tests where PrPTSE was detected in the effluent.

When PrPTSE was detected, breakthrough was first observed at < 1 PV, suggesting minimal

retardation due to adsorption.

The effect of kdet on PrPTSE transport was evaluated by varying kdet while setting Kd = 0.0

L·kg-1 and katt = 2.6 h-1. When kdet = 0.0, these input parameters yield PrPTSE concentrations equal

to the detection limit in the column effluent. In contrast, increasing kdet only slightly above zero

(viz. 0-13 h-1) resulted in effluent concentrations exceeding the detection limit in less than 10 PV.

S-3

Given that PrPTSE was not observed in any effluent in any of the column tests on soil over 40 PV,

and that none of the columns where PrPTSE was observed in the effluent (MSW, GWR columns)

exhibited effluent concentrations with a rising tail, detachment of PrPTSE appears unlikely.

Consequently, kdet was set at 0 for all simulations reported in this manuscript.

Text S2. Estimation of PrPTSE Disposed Annually in Wisconsin.

Several assumptions were required to estimate the mass of PrPTSE disposed in each pit.

Levels of prion infectivity in deer tissues have not been reported to date. Therefore, the

distribution of prion infectivity in deer tissue was assumed similar to that of scrapie-infected

sheep displaying clinical symptoms. Qualitative studies on CWD-infected mule deer support this

assumption (2). Levels of infectivity per mass of various ovine tissue assayed by intercerebral

injection in mice (3) were scaled by 1000 to account for the mouse-sheep species barrier (4). The

remainder of the carcass was assumed to have infectivity at the limit of detection of the mouse

bioassay (102.1 infectious unit (IU50)·g-1, ref. 1). These values were then multiplied by the total

tissue mass of an average adult deer (full data for mule deer can be found in Hakonson et al. [5],

similar limited data for white tailed deer in Robinson [6]) to estimate the infectivity per deer.

This approach is believed to provide an upper bound estimate of the amount of infectious

material disposed. Many of the CWD-positive deer that are disposed do not manifest overt

disease symptoms and may contain lower levels of PrPTSE per unit mass of tissue than clinically

affected animals.

The mass requiring disposal was assumed to consist of 36% road kill, 47% butcher and

hunter waste, and 17% heads from deer obtained within a central region of Wisconsin where

practices are in place to eradicate CWD (Alan Crossley, Wisconsin Department of Natural

S-4

Resources, personal communication). Worst-case estimates of CWD incidence in the deer

populations responsible for each of these waste streams were 6% for road kill (7) and 3% for

heads (Alan Crossley, personal communication), and 1% for butcher and hunter waste. Because

some of the waste is processed (removal of meat) before disposal, each waste stream was broken

down further to estimate its tissue content. We assumed that 50% of the carcass mass was

removed from hunter and butcher waste, but all assayed organs remained. We also assumed road

kill was composed of whole deer, while the heads contained only the brain and 5 kg of minimally

infectious tissue (the remainder of the head). A summary of these estimates is presented in Table

S3. For each waste stream, the mass of each relevant tissue type was multiplied by the

corresponding level of infectivity per mass. The infectivity was summed across the waste

streams waste to obtain 3.0 × 1014 IU50 disposed annually. This amount of infectivity was

converted to PrPTSE mass by assuming a 105:1 ratio of PrPTSE molecules to IU50 (8) and an

average PrPTSE molecular mass of 30 kDa, resulting in 1.49 g PrPTSE disposed annually.

S-5

0

2 x 104

4 x 104

6 x 104

8 x 104

1 x 105

0 2 4 6 8 10 12

Kd=0.0 L kg-1

Kd=3.0 L kg-1

detection limit

Con

cent

ratio

n in

the

colu

mn

efflu

ent (

ng L

-1)

PV

detection limit (1100 ng L-1)

0

2 x 104

4 x 104

6 x 104

8 x 104

1 x 105

0 2 4 6 8 10 12

Kd=0.0 L kg-1

Kd=3.0 L kg-1

detection limit

Con

cent

ratio

n in

the

colu

mn

efflu

ent (

ng L

-1)

PV

detection limit (1100 ng L-1)

FIGURE S1. PrPTSE concentrations predicted in column effluent using HYDRUS for Kd = 0 and 3.0 L kg-1 and katt and kdet = 0.

S-6

2.0 m

2.0 m

Burial Soil

Municipal Solid Waste

(MSW)

0.5 m

Area : 225 m2

CWD infected Carcasses

(1.5 g of PrPTSE)

Granular LeachateCollection System (LCS)

2.0 m

2.0 m

Burial Soil

Municipal Solid Waste

(MSW)

0.5 m

Area : 225 m2

CWD infected Carcasses

(1.5 g of PrPTSE)

Granular LeachateCollection System (LCS)

FIGURE S2. Schematic of MSW landfill profile used in simulations of flow and transport in the disposal pit and underlying materials.

S-7

TABLE S1. Mineralogy of porous media used in column tests (mass percent)a

quartz sand Bluestem clay Boardman silt

green waste

residual quartz 85 67 34 70.4

K-feldspar 5.6 6.9 6.3 7.6 plagioclase 7.9 15 35 15.9 amphibole – 2.5 1.7 –

calcite – 0.4 2.3 – dolomite – – – – hematite – 1.2 0.5 –

mixed-layer illite/smectite – 4.4 5.9 1.8

illite + mica 0.4 1.3 10 2.7 kaolinite 0.7 1.7 1.7 0.7 chlorite 0.4 0.2 2.7 0.9

a Determined by X-ray diffraction analysis (K/T Geoservices Inc. Argyle, TX).

S-8

TABLE S2. Properties of MSW leachate

pH 7.7

alkalinity 1263 mg CaCO3·L-1 conductivity 3.84 mS·cm-1 ionic strength 37 mM reduction potential -70 mV versus SHE concentration

elementa (mg·L-1) P <0.05 K 117.38

Ca 80.12 Mg 93.90 S 11.37 Zn 0.02 B 3.10

Mn 0.06 Fe 1.95 Cu 0.01 Al <0.05 Na 340.27

anionb

F 5.4 Cl 829.2 Br 7.8

NO3 208.2

PO4 <0.02

SO4 100.2

a Determined by Inductively Coupled Plasma Optical Emission Spectroscopy (Wisconsin Soil and Plant Analysis Lab) b Determined by Ion Chromatography (Wisconsin Soil and Plant Analysis Lab)

S-9

TABLE S3. Estimation of infectious material disposed annually

mass of disposed infectious tissue per source (kg·y-1)

tissue

log10 estimated cervid infectivity

(ID50·g-1)a

roadkill (6%

infectious)

hunter and butcher waste (1% infectious)

disease erradiacation zone

heads (3% infectious)

log10 total infectivity disposed (ID50·y-1)

mass of PrPTSE

disposed (mg·yr-1)b

brains 9.9 7.8 3.4 26 14.5 1470

adrenal glands 6.4 0.2 0.1 - 8.9 <0.01

lymph nodes 7.6 7.9 3.4 - 11.7 2.24

pituitary glands 5.7 0.05 0.02 - 7.5 <0.01

spleen 7.8 7.8 3.4 - 11.8 3.51

other tissues 5.1 3000 630 670 11.7 2.70

aCervid infectivity estimated from ovine infectivity data (3) and reported mouse/sheep species barriers to infectivity (4); bMass of PrPTSE estimated from infectivity assuming a 105:1 ratio of PrPTSE molecules to IU50 [8] and an average PrPTSE molecular mass of 30 kDa.

S-10

Text S3. Literature Cited in the Supporting Information

(1) Ma, X.; Benson, C.H.; McKenzie, D.; Aiken, J.M.; Pedersen, J.A. Adsorption of pathogenic prion protein to quartz sand. Environ. Sci. Technol. 2007, 41, 2324-2330.

(2) Race, B.; Meade-White, K.D. ; Ward, A. ; Jewell, J. ; Miller, M.W. ; Williams, E.S. ; Chesebro, B. ; Race, R. Levels of abnormal prion protein in deer and elk with chronic wasting disease. Emerg. Infect. Dis. 2007, 13, 824-830.

(3) Hadlow, W.J.; Kennedy, R.C.; Race, R.E. Natural infection of Suffolk sheep with scrapie virus. J. Infect Dis. 1982, 146, 657-664.

(4) Kimberlin, R.H. Bovine spongiform encephalopathy and public health: some problems and solutions in assessing risks. In Transmissible Subacute Spongiform Encephalopathies: Prion Diseases. Court, L.; Dodet, B. (Eds.); Elsevier: Paris, France; 1996; pp. 487–503.

(5) Hakonson, T.E; Whicker, F.W. The contribution of various tissues and organs to total body mass in the mule deer. J. Mammology. 1971, 52, 628-630.

(6) Robinson, P.F. Organ: Body weight relationships in the white-tailed deer, Odocoileus virginianus. Ches. Sci. 1966, 7, 217-218.

(7) Krumm, C.E.; Conner, M.M.; Miller, M.W. Relative vulnerability of chronic wasting disease infected mule deer to vehicle collisions. J. Wildl. Dis. 2005, 41, 503-511.

(8) Masel, J.; Jansen, V.A.A.; Nowak, M.A. Quantifying the kinetic parameters of prion replication. Biophys. Chem. 1999, 77, 139-152