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Environ. Sci. Technol. 2006, 40, 6324-6329

Prion Degradation in Soil: Possible Role of Microbial Enzymes Stimulated by the Decomposition of Buried Carcasses DELPHINE RAPP, PATRICK POTIER, LUCILE JOCTEUR-MONROZIER, AND A G N EÅ S R I C H A U M E * Ecologie Microbienne, Universite´ Claude Bernard Lyon 1 UMR CNRS 5557 - USC INRA 1193, bat. G. Mendel, 43 Bd du 11 Novembre 1918, 69 622 Villeurbanne Cedex, France

This study is part of a European project focused on understanding the biotic and abiotic mechanisms involved in the retention and dissemination of transmissible spongiform encephalopathies (TSE) infectivity in soil in order to propose practical recommendations to limit environmental contamination. A 1-year field experiment was conducted with lamb carcasses buried in a pasture soil at three depths (25, 45, and 105 cm). Microbial community response to carcasses was monitored through the potential proteolytic activity and substrate induced respiration (SIR). Soil above carcasses and control soil exhibited low proteolytic capacity, whatever the depth of burial. Contrastingly, in soil beneath the carcasses, proteolysis was stimulated. Decomposing carcasses also stimulated SIR, i.e., microbial biomass, suggesting that proteolytic populations specifically developed on lixiviates from animal tissues. Decomposition of soft tissues occurred within 2 months at subsurface while it lasted at least 1 year at deeper depth where proteolytic activities were season-dependent. The ability of soil proteases to degrade the β form of prion protein was shown in vitro and conditions of burial relevant to minimize the risk of prion protein dissemination are discussed.

Introduction Transmissible spongiform encephalopathies (TSE) epidemic in cattle is a major economic problem for the sustainability of breeding activities and a growing concern for public health with the transmission of TSE to human beings by consumption of contaminated meat (new variant of the CreutzfeldtJakob disease). Although several hypothesis concerning the exact nature of the causative agent of TSE have been advanced (1-4), it is widely accepted that an abnormal isoform of the prion protein (PrP) containing a high content in β-sheets is involved in infectiosity (5). This isoform has been shown to be partially resistant to proteolysis (6). The soil can be contaminated by causative agent as a result of (i) accidental dispersion from storage plants of meat and bone meal, (ii) incorporation of bone meal into fertilizers, (iii) spreading of effluents of slaughterhouses, or (iv) burial of carcasses of contaminated animals (7). In accordance with legislation, carcasses of TSE infected cows are nowadays * Corresponding author phone: 04 72 43 26 50; fax: 04 72 43 12 23; e-mail: [email protected]. 6324

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eliminated by incineration but on-site burial has long been used for both logistical and economical reasons. Sheep scrapie cases are still only partially registered and slaughtered contaminated animals are often left at field without any treatment but burial. Such practices represent a risk in terms of (i) persistence of the infective agent and (ii) dissemination in the environment. Persistence of infectious units over a 3-year period of time was shown in soil after addition of supernatant fluid from scrapie-infected hamster brain homogenates (8). Horizontal scrapie transmission from sheep to sheep by soil or pasture fauna has also been suggested (9-13). Decomposition of buried carcasses mainly relies on the activity of microorganisms (14) producing extracellular proteolytic enzymes which break polymers of organic matter to oligomeric and monomeric molecules. In this case, PrP is released in the environment by degradation of the surrounding tissues, i.e., brain and nervous tissues, and the surrounding soil can be contaminated and become a potential reservoir of TSE infectivity. This study is part of a European project (TSE-soil fate) which aims to understand the biotic and abiotic mechanisms involved in the retention and dissemination of TSE infectivity in soil in order to propose practical recommendations to limit contamination. In this context, the objectives of the present work were to determine (i) the consequences of carcass decomposition on the kinetics of proteolytic activities of soil microorganisms and (ii) if soil proteases synthesized in response to animal organic matter could be active against prion protein. We took the opportunity to study microbial activities in soil surrounding buried carcasses by taking advantage afforded by the experimental burying of lambs in a 1-year forensic study. We investigated the influence of burial depth and of seasonal variations on two relevant microbial activities that can affect the rate of carcass decomposition: proteolysis which is a direct reflection of microbial decomposers activity, and respiration as a global indicator of microbial metabolic status and biomass. Finally, we demonstrated that soil microbial proteases synthesized in response to an animal organic matter supply were able to degrade prion protein in vitro.

Materials and Methods Site and Soil Description. The study site was a 38 × 10 m plot located in Nouzilly (Indre et Loire, France). The soil was classified as Alfisol developed on loam deposits. Texture and chemical characteristics at 10, 30, and 90 cm depths were determined on representative composite samples by the Centre Scientifique Agro-alimentaire Re´gional (Ceyze´riat, France) according to normalized ISO techniques. Soil was a silty-loam showing slight textural differences according to depth (7.8% sand, 72.2% loam, 20% clay at 10 cm depth; 9.9% sand, 73.7% loam, 16.4% clay at 30 cm depth; and 5.6% sand, 65.1% loam, 29.3% clay at 90 cm depth). Organic carbon and nitrogen contents were 13.4, 4.7, and 3.5 g‚kg-1 of carbon and 1.5, 0.3, and 0.6 g‚kg-1 of nitrogen at 10, 30, and 90 cm depths, respectively. Soil pH was 6.3, 6.9, and 7.0 at 10, 30, and 90 cm depths, respectively. Rain data and external temperature were recorded with the local weather station “Meteo-France”. Soil temperature was recorded every 2 h during 1 year using three thermal probes (Testo 177-T1) placed between 10 and 90 cm depth. Data were analyzed with the software Testo Logiciel-Comfort Pro 2002 V.3. Experimental Design: Animal Burial and Exhumation. Lambs (Ovis aries) exempt of TSE were provided by the INRA 10.1021/es060943h CCC: $33.50

 2006 American Chemical Society Published on Web 09/09/2006

unit of physiology (Institut National de Recherche Agronomique, Centre de Tours, France). Lambs were 4 months old, weighed about 35 kg, and were not sheared. In May 2004, seventeen animals were slaughtered individually using the bull fighter gun technique, and carcasses were transported within 1 h to be buried. Seven carcasses were buried at 525 cm depth, 7 carcasses were buried at 25-45 cm depth, and 3 were buried at 85-105 cm depth. The graves were at least 5 m away from one another. One, 2, 3, 4, 6, 9, and 12 months after burying, one carcass buried at 5-25 cm depth and one at 25-45 cm depth were exhumed. All carcasses buried at 85-105 cm depth were exhumed at month 12. For each burial depth, three samples were collected directly in contact with carcasses (above or below) and homogenized to obtain a composite sample to be analyzed. For each sampling time, three samples were collected at 5-25 cm depth between the graves and homogenized to be used as a temporal control. At month 12, soil was also sampled along a depth gradient (0-15, 15-25, 45-60, 80-95, and 95-110 cm) and was used as a spatial control. All composite samples were sieved (2 mm) and analyzed 1 day after sampling. Extracellular Proteolytic Activity. Soil extracts were prepared as follows: soil samples (10 g equivalent dry weight) were blended for 1 min 30 sec into 50 mL of sterile 0.5 M KCl (Waring Blendor, Eberbach Corp.) before being centrifuged at 5525g for 30 min at 15 °C to discard coarse particles. Supernatants were filtered at 0.2 µm to separate extracellular enzymes from microbial bodies. Each extraction was performed in triplicate. The potential proteolytic activity contained in the filtered supernatants was assessed using azocasein as dye-stained protein substrate (Sigma). The reaction mixture was prepared by mixing 300 µL of supernatant with 900 µL of 0.1 mM Tris-HCl (pH 8.0) containing 0.1% (w/v) azocasein (final concentration). This procedure provided excess azocasein to ensure zero-order kinetics. Assays were performed at 38 °C for 2 h; then, a volume (300 µL) of 30% tricloracetic acid (TCA) was added to stop the reaction and precipitate nonhydrolyzed azocasein. Samples were kept at 4 °C for 15 min and precipitates were removed by centrifugation at 13 000g for 10 min. Dye release due to proteolytic activity was monitored at 450 nm using a UVIKON 810 spectrophotometer (Kontron Instruments, Switzerland) after addition of 500 µL of 0.1 M NaOH to 500 µL of supernatant volume. Substrate Induced Respiration (SIR). Soil samples (2 g equivalent dry weight) were placed in 125-mL plasma flasks before being supplemented with 6 mg of D-glucose and appropriate amounts of water in order to reach the soil water holding capacity. Each preparation was performed in triplicate. Flasks were sealed and incubated at 28 °C for 3, 5, and 7 h. For each incubation time, gas samples were analyzed for CO2 production with a gas chromatograph (P200, AGILENT Technologies, Inc., Wilmington, USA) equipped with a microcatharometer detector. Respiratory activity was expressed as µg of CO2-C produced per hour per g of soil (equivalent dry weight). In vitro recPrP Degradation by Soil Proteases. Soil extracts used to test the ability of soil proteases to degrade prion protein were obtained as described above from soil microcosms previously amended with animal organic matter in order to stimulate proteolytic activities. Three soil microcosms containing 10 g equivalent dry weight of 0-20 cm freshly collected soil in 125-mL plasma flasks (Verre Equipement SA, Collonges au Mont d’or, France) were supplied with a filtrate of TSE-free crushed lamb brains prepared as followed: fresh lamb brains were manually crushed in 13 mL of 0.8% NaCl and homogenized by mixing in a blender. The resulting mixture was filtered through gauze dressing to eliminate coarse debris. Protein concentration of the brain filtrate was determined by the bicinchoninic acid (BCA)

protein assay reagent (Pierce Chemical, Rockford IL) using bovine serum albumin as standard (15). The volume of filtrate added to soil microcosms was determined in order to obtain a final concentration of 10 mg of proteins per g of soil, and the moisture content was adjusted to the water holding capacity with sterile water when necessary. Microcosms were incubated for 2 days at 28 °C in the dark after addition of organic matter. Lyophilized full-length ARQ recombinant sheep prion protein (R-coiled form) was provided by H. Rezaei (INRA, France) (16). Lyophilisate was solubilized in MOPS 20 mM buffer pH 7 at a final concentration of 2 mg‚mL-1 and was heated for 30 min at 70 °C to induce a change of conformation toward the β-sheeted form. β- sheeted recPrP was added to soil extracts or to a 0.5 M KCl solution (as control) at a final concentration of 0.2 mg‚mL-1. Mixtures were incubated for 0 and 8 days at 28 °C. At both sampling times, 82.5 µL was transferred into Eppendorf tubes, and total proteins were concentrated by acetone precipitation for 24 h at -20 °C. After centrifugation at 13 000g for 10 min, the pellets were dried under vacuum and resuspended in 25 µL of the denaturing sample buffer of Laemmli containing 4% sodium dodecyl sulfate (SDS). After boiling for 5 min, proteins were separated by standard SDS-PAGE (12% acrylamide) in a mini Protean III cell using the discontinuous buffer system of Laemmli (17). Total proteins were stained using the protein silver staining kit (Bexel Biotechnology, CA) according to the manufacturer’s instructions. Degradation of the recPrP was visualized by immunochemifluorescence after Western blotting as described elsewhere (18). The fluorescent signal was visualized after scanning with a FluorImager SI (Molecular Dynamics).

Results and Discussion Microbial activities were monitored in soil surrounding carcasses by taking advantage afforded by the experimental burial of lambs in a 1-year forensic study. Since long term measurements of microbial activities are difficult to handle directly in the field, potential proteolytic activity and substrate induced respiration (SIR) were monitored in soil surrounding buried carcasses as indicators of carcass decomposition and global metabolic status of microorganisms due to organic matter release, respectively. Potential activity measurements reflect the functioning of enzymes produced in situ, either in response to the presence of available nutrients or constitutively. The use of laboratory measurement of potential activities has been shown to be consistent with in situ activity of bacteria submitted to variations of environmental conditions (19). Spatial Variability of Microbial Activities in Soil Surrounding Carcasses. The experimental design used in this study was originally established for forensic purposes by partners involved in the European project. The number of sheep used did not provide us with sufficient carcasses for statistical analysis of activities measured for each exhumation time and depth. Nevertheless, the spatial variability due to both soil and carcass heterogeneity was estimated along a depth gradient in soil sampled above and below the three carcasses buried at 85-105 cm for 1 year. Results presented in Table 1 show that, for a given depth, proteolytic activity and SIR of soil sampled above carcasses were in the same range respectively, indicating a low level of variability among the three graves. Similar observations were already reported for respiration and N-mineralization rates in soil surrounding pig carcasses (20). Proteolytic activity and SIR responses of soil sampled below carcasses 1 and 3 were in the same range. The discrepancy with activities measured below carcass 2 may be attributed to the absence of adipocere on this carcass. VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Variability of Microbial Activities in Soil Surrounding 3 Carcasses Buried for 12 Months at 85-105 cm Deptha potential proteolytic activity (A 450 nm × 100)

a

SIR (CO2-C µg‚g-1 soil‚h-1)

depth (cm)

carcass 1

carcass 2

carcass 3

carcass 1

carcass 2

carcass 3

0-15 45-60 80-95 95-110

0.4 ( 0.2 0.1 ( 0.1 0.1 ( 0.1 0.5 ( 0.3

0.5 ( 0.1 0.1 ( 0.1 0.2 ( 0.2 2.2 ( 0.3

0.3 ( 0.0 0.4 ( 0.3 0.1 ( 0.1 0.7 ( 0.3

9.14 ( 1.77 3.10 ( 0.85 nd 10.77 ( 2.65

5.60 ( 0.19 1.53 ( 0.34 1.08 ( 0.17 4.61 ( 0.57

7.10 ( 0.14 2.05 ( 0.07 1.86 ( 0.25 9.75 ( 1.79

Standard deviations were calculated from 3 independent measurements performed on each sample.

FIGURE 1. Proteolytic activities in soluble enzyme extracts (X axis, top) and SIR (X axis, bottom) in soil sampled 1 year after burial versus depth gradient (Y axis) in each grave: control soil (A), grave with carcass buried at 5-25 cm (B), grave with carcass buried at 25-45 cm (C), grave with carcass buried at 85-105 cm (D). Carcass positions are indicated by open banners. In D, bars represent standard error where n ) 3. Adipocere forms a soap-like substance resulting from the late post-mortem alteration of neutral fats in the soft tissues known to slow or inhibit carcass decomposition (21). Nevertheless, the trend of activity variations along the depth gradient followed the same pattern in the three graves with an enhancement below carcasses allowing further interpretations with a low number of replicates. Influence of the Depth of Burial on Microbial Activities. The primary factors affecting the decay rate of buried carcasses are those that affect microbial activity, as this is the primary way of decay (22, 23). Among these factors, the depth of burial is of particular importance due to modifications of physicochemical parameters and microbial community density, diversity, and functionality. Therefore, the influence of the depth of burial on microbial activities was evaluated by comparing potential microbial activities above and below carcasses buried for 1 year at increasing depths, i.e., 5-25, 25-45, and 85-105 cm. In control soil sampled between graves (Figure 1A), very low levels of potential proteolytic activity were observed down to 95-110 cm depth, suggesting naturally low contents of extra-cellular proteases in soil, probably due to low contents of inducing proteinaceous substrates (24). This result is in agreement with the low activities measured above carcasses (Table 1). We also observed a gradual decrease of SIR with depth. This is considered as a possible consequence of the decrease of organic-C content from 13.4 g‚kg-1 at 10 cm to 6326

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3.5 g‚kg-1 at 90 cm. Such a relationship between microbial respiration and organic-C content along a depth gradient is consistent with literature data (25). Proteolysis was enhanced beneath decaying carcasses in comparison to the overlying soil (Figure 1C and D). There was a trend for decreased potential proteolytic activity with depth of burial but results were highly variable. The observed trend in proteolytic activity was roughly correlated with increased microbial activity as determined by SIR, which was also highly variable. Decomposition of buried carcasses is likely to provide soil with a large amount of readily available organic resources for indigenous heterotrophic microorganisms together with a heavy microbial inoculum from the enteric community (14). Both communities should contribute to the enhancement of the proteolytic activity. The stimulation of proteolysis also suggests that specific enzyme production is an inducible response to the presence of complex substrates provided by leachates from decomposed carcasses. Our results are supported by the visual aspect of remains after 1 year of burial which was readily different according to depth. A large amount of organic material was still present for the deepest burials whereas only bones remained in the case of the carcass buried at 5-25 cm (data not shown). In this latter case similar activity levels were observed for soil sampled above and below the carcass (Figure 1B). This could be either due to an easier access of the carcass to necropha-

FIGURE 2. Seasonal variations of microbial activities in soil surrounding carcasses. Proteolytic activity (black) and SIR (hatched lines). A, control soil at 5-25 cm depth; B, soil below carcasses buried at 5-25 cm; C, soil below carcasses buried at 25-45 cm. gous insects (23, 26) and/or higher microbial activities. However, the amount of time required for buried carcasses to decompose also depends on other factors deeply influenced by seasonal variations susceptible to affect microbial activities, such as temperature, rainfall, dryness. This is why kinetics of microbial activities was monitored around carcasses over a 1 year period of time. Kinetics of Microbial Activities in Soil Surrounding Carcasses Buried at Different Depths. Seasonal variations of proteolysis and SIR below carcasses buried at 5-25 and 25-45 cm depth are shown in Figure 2. Rainfall and soil temperature were recorded over the duration of the experiment. At each sampling date, soil sampled immediately above carcasses exhibited up to 8-fold lower potential proteolytic activities than soil sampled below carcasses but followed the same stimulation patterns whereas SIR was in the same range as control soil (data not shown). Proteolysis was enhanced within the first 2 months in soil below carcasses buried at 5-25 cm depth (Figure 2B) followed by a rapid decrease down to values equivalent to those recorded in control soil (Figure 2A). This evolution, together with an increase of SIR in July, is likely to be due to changes in the nature and amount of readily available nutriments. Such a flush of microbial activity following substrate input has already been reported (27-29) and was attributed to the response of the zymogenous microbial community to highly decomposable substrates such as blood, brain, and soft tissues (30). A similar response pattern was observed below carcasses buried at 25-45 cm with a rough correlation between proteolytic activity and SIR (Figure 2C). However, the highest potential proteolytic activity did not peak at the same time. Although very little information is available about the microbially mediated decomposition processes and their regulation, it has long been known that temperature can directly influence metabolic activities while affecting the soil microhabitat via changes in soil physicochemical properties (e.g., redox potential, pH, and moisture content, etc.). However, differences in soil temperature recorded within

this period (June to August) were too low to account for the differences observed for proteolytic activities between carcasses buried at 5-25 and 25-45 cm. Thus, changes in the number and/or the composition of the proteolytic microbial community according to depth may contribute to the differences observed in the kinetics of proteolytic activities during the 3 months following burial. Microbial activities decreased during late fall and winter, where average monthly temperatures fell down to 6.0 °C at 10 cm depth. The lowering of activity could be due to a cold-induced repression of hydrolytic enzymes involved in decomposition as suggested by Carter and Tibbett (30). In the case of carcasses buried at 25-45 cm depth, this seasonal effect was followed by a resumption of proteolytic activity in May. Remains of soft tissues on carcasses together with a return of soil temperatures to values favoring both microbial physiological activities and enzyme functioning may be responsible for the observed resumption. The current results indicate that the depth and the season of burial are important parameters that prevail in the control of microbial activities responsible for the degradation of soft tissues susceptible to contain the prion protein. The release of the prion protein should occur in spring and summer. In the case of carcasses buried below 25 cm, an additional release could also occur during the following spring as suggested by the presence of remaining soft tissues. The fate of prion protein in soil is controversial and recommendations for the disposal of contaminated carcasses will largely depend on (i) the efficiency of soil microbial proteases, and (ii) the protection of prion protein against proteolytic attack. Efficiency of Soluble Soil Proteases against Prion Protein. According to literature, the β-form of prion protein is known to be very recalcitrant to degradation. Nevertheless, recent studies evidenced that Streptomycetales, Bacillales, and anaerobic thermophilic prokaryotes can produce proteases able to degrade the β-form of prion protein but only under harsh conditions; the protein being degraded within 3 min at 60-80 °C and pH 11 (31-33). Such conditions are unlikely to correspond to natural situations normally encountered in soil. However, Mu ¨ller-Hellwig et al. (34) recently identified several protease-secreting foodborne bacteria active against prion protein at milder conditions (30 °C, pH 8). Out of nearly 700 tested microbial cultures, only 6 strains were found to secrete proteases able to degrade the prion protein in hamster brain homogenates. Therefore, bacteria secreting proteases competent to degrade PrP appear to be rare. Considering the huge diversity of micro-organisms and activities in soil, microbial proteases active toward prion protein are likely to exist, although their presence and efficiency have not yet been reported. Since we showed that buried carcasses stimulated proteolytic activities in the surrounding soil, we aimed to investigate whether soluble proteases extracted from three independent soil microcosms previously amended with animal organic matter could degrade the β-form of prion protein. The presence of active proteases in filtered soil extracts obtained from soil microcosms amended with crushed lamb brain was attested by measuring proteolytic activity. Silver staining of SDS-PAGE showed numerous bands in soil extracts indicating the presence of different indigenous soil proteins even after 8 days of incubation (Figure 3A, lane 4 to 7). Freshly prepared β-form of prion protein gave 3 bands with apparent molecular weights of 15, 22, and 39 KDa, corresponding to the globular C-terminal domain, the fulllength protein, and the dimeric form, respectively (Figure 3B, lane 1). We did not observe immunoblot signal in soluble extracts of stimulated soil incubated for 8 days without recPrP (Figure 3B, lane 6 and 7), which excludes nonspecific interferences of the monoclonal recPrP 2D6 antibody used to detect the recPrP with soil and/or brain homogenates VOL. 40, NO. 20, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Literature Cited

FIGURE 3. Degradation of the prion protein by soluble enzymes extracted from soil. A: Silver staining of total proteins obtained from PrP solution in MOPS (lane 1), PrP solution in KCl (lane 2, day 0; lane 3, 8 days of incubation), soil soluble enzyme extracts with PrP (lane 4, day 0; lane 5, 8 days), soil soluble enzyme extracts without PrP (lane 6, day 0; lane 7, 8 days). B: Immunoblotting of PrP in MOPS (lane 1), in KCl (lane 2, day 0; lane 3, 8 days of incubation), soil soluble enzyme extracts with PrP (lane 4, day 0; lane 5, 8 days), soil soluble enzyme extracts without PrP (lane 6, day 0; lane 7, 8 days) soluble enzymes. The degradation of the β-form of prion protein occurred within 8 days of incubation with soil extracted soluble enzymes, as indicated by the absence of immunoblot signal in lane 5 (Figure 3B). This conclusion is supported by the recovery of recPrP incubated after 8 days of incubation in KCl solution, thereby excluding their disappearance due to adsorption on tube walls or to the conditions used in these experiments (Figure 3B, lane 3). Similar immunoblot patterns were obtained with soluble enzymes extracted from the two other soil microcosms (data not shown). Our results suggest that proteases active toward prion protein are very likely to be produced in soil following stimulation by animal tissues. In the case of carcasses buried in spring, prion protein could be degraded rapidly during spring or summer, with the risk of potential dissemination of scrapie infectious agent in the environment being therefore limited. However, the strong adsorption of prion protein on clays and organic matter has been demonstrated (35). As observed for other proteins and DNA, such adsorption hampers the action of hydrolytic enzymes (36-37). The resumption of proteolytic activity observed after winter for carcasses buried below 45 cm depth offers an interesting perspective about the fate of adsorbed prion protein. Our results suggest that a further contact with proteases could occur, and that the deep burial of contaminated carcasses should trigger the degradation of prion protein.

Acknowledgments This work was funded by the European Project QLK4-CT2002-02493 “TSE Soil Fate”. We thank Emmanuel Gaudry and Thierry Pasquerault (IRCGN, Rosny-sous-Bois, France), and Thierry Chaumeil and Jacques Cabaret (INRA, Nouzilly, France) for the design and management of the field experiment. Human Rezaei and Yann Quenet (INRA, Jouy-en-Josas, France) are thanked for providing the recombinant PrP. Marjorie Malain and Claire Commeaux are greatly acknowledged for technical assistance. 6328

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Received for review April 19, 2006. Revised manuscript received July 21, 2006. Accepted July 27, 2006. ES060943H

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