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Department of Biological and Agric. Eng., Univ. of Idaho, Moscow, ID 83844-2060
* Corresponding author (jboll{at}uidaho.edu)
Received for publication February 18, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Abbreviations: AF beads, AlignFlow beads • ES, oocysts from Excelsior-Sentinel, NY • FL1 or FL4, fluorescence detectors on flow cytometer • FSC, forward scatter detector on flow cytometer • PRL, (oo)cysts from Parasitology Research Laboratory, MO • (oo)cysts, oocysts and cysts • PHF, oocysts from Pleasant Hill Farm, ID • SSC, side scatter detector on flow cytometer • YG beads, Yellow-Green beads
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Cryptosporidium parvum and G. lamblia are ubiquitous in surface waters in the USA (Rose, 1988; LeChevallier et al., 1991). These two protozoan parasites exist as oocysts (4–6 µm) and cysts (7–11 µm), respectively, in the environment. The encystment protects them from environmental stresses and disinfection processes in conventional drinking water treatment. Possible sources of (oo)cysts include feces from infected domestic animals [e.g., cattle (Bos taurus), cats (Felis catus), mice (Mus musculus)] and wildlife (Garber et al., 1994, Ong et al., 1996; Walker et al., 1998), and sewage discharges.
A large body of literature exists on (oo)cyst occurrence in water (Ongerth et al., 1995; Hansen and Ongerth, 1991; Rose, 1988), detection methodologies (Ongerth and Stibbes, 1987; Rose et al., 1989; Vesey et al., 1994; Watanabe, 1996; Bukhari et al., 1998; Esch et al., 2001; Baeumner et al., 2001), and transmission (Fayer et al., 1997; Craun, 1990; Rose, 1988). Survival and transport of (oo)cysts in the aquatic environment, however, are poorly understood (Walker et al., 1998; Anguish and Ghiorse, 1997; Harvey et al., 1995). Information about survival and transport is essential to actual risk assessment and to development of effective control practices.
While several researchers have shown that transport of C. parvum oocysts through soil occurs (Mawdsley et al., 1995, 1996a, 1996b; Brush et al., 1999), transport by overland flow (i.e., runoff) has received little attention. One basic question regarding (oo)cyst transport by overland flow is whether the (oo)cysts travel freely in water or are attached to sediment particles. This question has not yet been answered conclusively, but has consequences for control practices and modeling approaches. If (oo)cysts travel freely in water, control practices reducing overland flow need to be used and modeling of their transport will be based on hydrologic modeling using, for example, the Saint-Venant Equations (Henderson and Wooding, 1964). If (oo)cysts are attached to sediment particles, control practices designed to reduce sediment transport can be applied, and modeling of their transport could be achieved using erosion modeling as in Hairsine and Rose (1992).
Interactions between small colloid particles the size of C. parvum oocysts, G. lamblia cysts and small soil particles, typically the clay sized fraction (<2 µm), depend to a large extent on electrostatic and other surface forces. Hence, surface charge, characterized as electrophoretic mobility or zeta potential, may govern the interaction between (oo)cysts and soil particles. This interaction is explained by the double layer theory as the interplay between van der Waals attraction forces produced by molecules on the surface of each particle and electrostatic repulsion forces between neighboring particles. Particles in solution carrying opposite charges tend to agglomerate, while particles with like charges repel and remain discrete.
Brush et al. (1999), Ongerth et al. (1995), and Hayes (2002) found that C. parvum oocysts and G. lamblia cysts were near neutral or negatively charged over a wide range of pH values, depending on the purification method used. An assumption from their data is that there is little chance of attachment to negatively charged soil particles. Yet, as stated by Walker et al. (1998), this assumption has to be tested. In this paper, we present mechanistic approaches to test the hypothesis that the (oo)cysts do not attach to natural soil particles, which is an important step toward answering the question if (oo)cysts travel freely in water or attached to suspended soil particles. One of the challenges in studying attachment of (oo)cysts to sediments has been to obtain accurate counts of (oo)cysts in presence of high turbidity (Anguish and Ghiorse, 1997; Brush et al., 1999; Esch et al., 2001; Baeumner et al., 2001).
In this study, we optimized flow cytometry in combination with epifluorescent and confocal microscopy to obtain reproducible (oo)cyst counts in water samples when soil particles were present. We observed pairs of like and opposite charged particles [i.e., beads, (oo)cysts, and soil], and performed batch experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Sources of C. parvum Oocysts and G. lamblia Cysts
We obtained C. parvum oocysts from Excelsior Sentinel (ES), Ithaca, NY; Parasitology Research Laboratory (PRL), Neosha, MO; and Pleasant Hill Farm (PHF), Troy, ID. Each supplier used a different purification method to extract the oocysts from fecal material of infected calves. Excelsior Sentinel used modified continuous-flow flotation described in Vetterling (1969). Parasitology Research Laboratory used NaCl floatation. Pleasant Hill Farm used ethyl ether to remove lipids of the feces and concentrated the oocysts by the modified sucrose floatation method of Riggs and Perryman (1987). Giardia lamblia cysts were obtained from PRL and were purified using zinc–sulfate and Percoll floatation. Based on information from suppliers, C. parvum oocysts were supplied at concentrations of 106/mL and G. lamblia cysts at approximately 105/mL. Excelsior Sentinel oocysts were stored in reversed osmosis water with an antibiotic-Antimycotic (penicillin G sodium, streptomycin sulfate, and amphotericin B). Pleasant Hill Farm oocysts were stored in phosphate buffer solution with antibiotics (penicillin and streptomycin). Both PRL C. parvum oocysts and G. lamblia cysts were stored in distilled water with Gentocin added as antibiotics. Giardia lamblia cyst stock was sometimes blurry and occasionally contained floating materials. Cryptosporidium parvum oocyst stocks from all three sources were quite clear. Generally, C. parvum oocysts were <6 mo old and G. lamblia cysts were about 3 to 6 mo old. Species information was provided by the suppliers.
Soil Particles
We collected topsoil classified as Palouse silt loam from the University of Idaho Plant Science Farm in Moscow, ID. The soil was air-dried, grounded and stored at 4°C until use. The soil consisted of 17.6% sand, 59.8% silt, and 22.6% clay. One gram of air-dried soil was sieved down to the fraction of <50 µm. We then used the USDA Pipet method at 5 cm to obtain a soil suspension, in which >78% of the particles were <2 µm. The suspension was centrifuged at 750 rpm for 3 min for further removal of the silt portion. The soil concentration in the final solution was 76 mg/L. More than 95% of the particles in the final solution were <2 µm diameter (Coulter Counter TAII, Coulter Electronics, Hialeah, FL). Chemical treatments were intentionally avoided to keep the soil in its natural condition including clay particles and organic matter, which are important for adhesion and may be high in water suspensions in the environment.
Mineralogical analyses were conducted by x-ray diffraction technique using a Siemens D5000 diffraktometer. Total organic carbon (TOC) was measured by methods in Nelson and Sommers (1982). The final soil suspension had the following characteristics: pH 7.06, mixed mineralogy with Vermiculite, Illite, and Kaolin as the primary minerals, and 5.8% TOC.
Two sands were used: a clean sand (South Wilk-1, ID) and a sand coated with metal ion oxyhydroxides (Santa-1, ID). They were sieved to 60 µm and washed with distilled water intensively to remove debris.
Beads
We obtained Fluoresbrite Yellow-Green (YG), negatively charged beads (2 µm diam.) from Polysciences (Warrington, PA), and AlignFlow (AF) negatively charged beads (0.2%, 6 µm, excited at 633 nm and emitted red at 645–680 nm) from Molecular Probes (Eugene, OR). The concentrated beads were sonicated before use. AlignFlow negative beads also were coated with a cationic polymer (polyquaternary ammonium resin; CYTEC, Bernadsville, NJ) to make them positively charged. These coated beads were centrifuged and washed to remove excess polymers before use.
Zeta Potential of Particles
Surface charges of C. parvum oocysts, G. lamblia cysts, and beads were measured as zeta potential using a Zeta meter with 95X magnification (ZM3-83, Zeta Meter, Staunton, VA). The solution used in the electrophoresis cell had a pH of 7.0. Zeta potential values reported are averages of 100 to 200 observations with more observations if (oo)cyst suspensions were not very clean (Hayes, 2002). The zeta potential of the soil particles was measured using a Zeta-sizer (Malvern 3000 Has) at a pH of 7.06 using three samples each providing an average reading from approximately 1000 particle scans. The point of zero net charge (PZNC) for the sands was determined according to methods described by Zelazny and He (1996).
Staining of (Oo)cysts
Cryptosporidium parvum oocysts and G. lamblia cysts were stained by FITC-MAb CRY10 and G203 (Flow Grid Group, Macquire University, Australia), respectively. The working antibody concentration added to each sample (1:25 volume ratio) was 50 µg/L. Samples were incubated on ice (4°C) for 15 to 30 min.
Particle Attachment Experiments
We used YG beads, AF beads, C. parvum oocysts (ES, PRL, PHF), G. lamblia cysts, and soil particles to visualize particle attachment by flow cytometer and confocal microscope. Mixtures of potentially adhering or nonadhering particles examined were: AF negative beads vs. YG negative beads, AF positive beads (cationic polymer-coated) vs. YG negative beads, AF negative beads vs. (oo)cysts, AF positive beads vs. (oo)cysts, and (oo)cysts vs. soil. All the mixtures were rotated overnight for complete mixing. (Oo)cysts were stained after mixing, but before analysis by flow cytometer (see below). Suspected attached pairs were sorted using the cell concentrator, and examined under epifluorescence and confocal microscopes (see below).
(Oo)cyst–Soil Batch Experiments
Cryptosporidium parvum oocysts (ES, PRL, PHF) and G. lamblia cysts were added to 9 mL soil suspensions each in a 15-mL centrifuge tube. Added amounts of (oo)cysts varied from 10 to 100 µL to obtain 104 to 105 (oo)cysts/tube. Two controls (pH = 7.06) were established: (oo)cysts mixed with distilled water (Control 1) and soil with distilled water (Control 2). The (oo)cyst–soil mixtures and both controls were rotated overnight to maximize interaction between particles. Tubes were placed vertically in racks on a vibration-free bench top after mixing to allow particles to settle. Since soil particles settled much faster than (oo)cysts, due to their different densities, the solution at the top of each tube was removed and analyzed for (oo)cyst amounts. If (oo)cysts were attached to soil particles, the number of (oo)cysts in Control 1 should be higher than in the samples removed from the (oo)cyst–soil batch. If (oo)cysts were not attached to soil particles, the number of (oo)cysts in Control 1 should be the same as in samples from the (oo)cyst–soil batch. Control 2 was used to correct flow cytometer counts for any soil particles in the (oo)cyst gates (as explained below).
One-milliliter samples (equivalent to a height of 6.4 mm) were repeatedly taken from each tube at pre-set time intervals. These intervals were determined by the settling velocities of C. parvum oocysts, G. lamblia cysts, and soil particles, respectively, using Stoke's Law. (Oo)cysts were assumed to behave as spherical or oval shaped particles with average size of 5.0 µm and 11.0 µm in diameter for C. parvum oocysts and G. lamblia cysts, respectively. Oocyst density was assumed to be 1.06 x 103 kg/m3 and cyst density was taken as 1.04 x 103 kg/m3 (Medema et al., 1998). At the ambient temperature of 21°C, a soil particle in the suspension will settle 6.4 mm in 30 min (avg.). Cryptosporidium parvum oocysts and G. lamblia cysts will settle the same 6.4 mm distance in 115 and 43 min, respectively. Therefore, oocyst–soil batches (including the controls) were sampled at 30-min intervals starting at time 0 (immediately after mixing) and ending at 120 min. Cyst–soil batches (including controls) were sampled at 15-min intervals from time 0 to 60 min. The 300-µL aliquots were withdrawn from each sample and stained with FITC-MAb CRY10 and G203 for oocysts and cysts, respectively. Three replicates were done for each sample. Batch experiments using PHF C. parvum oocysts also were conducted at pH = 4.50 and pH = 10.5, and ionic strength 10.0 mS/cm using a NaCl solution. Batch experiments with the two sands (South wilk-1 and Santa-1) were conducted in triplicate in the same manner as the soil suspensions, sampling once, 30 min after mixing.
Each 300-µL aliquot was analyzed by the flow cytometer as described below. The actual number of (oo)cysts in the 300-µL aliquot was determined as C = (A/B) x N, where A is the number of events within the (oo)cyst gate, B is the number of events within the AF beads gate, and N is the total number of AF beads added to the 300-µL sample. The total number (N) was obtained from hemacytometer counts. The number A was adjusted by subtracting the soil particles in Control 2 that appeared in the (oo)cyst gate. Recovery rate was calculated as (oo)cyst concentration from flow cytometry divided by the original (oo)cyst concentration in the tube.
Detection and Enumeration of (Oo)cysts
The FITC-labeled (oo)cysts were detected by flow cytometer FACSort equipped with four-color detectors, a cell concentrator, and CellQuest Version 3.1 (Becton Dickinson Immuno-cytometry Systems, CA). This instrument examined a stream of particles one-by-one using a laser beam. Several detectors were used simultaneously: forward scatter (FSC) for size information, side scatter (SSC) for internal complexity, and fluorescence (FL1 and FL4) for stained or fluorescing particles. Particle counts were visualized on two-dimensional dot plots, each showing counts of one detector on the x axis and counts of another detector on the y axis. Instrument settings included log scales on all detectors, and thresholds on FSC and SSC detectors. The following settings were used: FSC = E00, SSC = 365, FL1 = 539, FL4 = 327; thresholds: FSC = 321, SSC = 239; flow rate: 60 µL/min. AlignFlow beads were added to each sample immediately before use of the flow cytometer to adjust for flow variation during acquisition.
Populations of (oo)cysts and beads were tested separately to identify where on the dot plots they appeared, and which combination of detectors had to be used. (Oo)cyst populations were best defined by combination of FSC, SSC, and FL1. AlignFlow beads were defined by a combination of FSC, SSC, and FL4. These populations were then marked using a gate, which is a region drawn around the population on each dot plot. When more than one particle type was present in solution, particles falling within these gated areas were counted. For each sample, particles were detected for a period of 102 s, which is equivalent to a sample size of approximately 100 µL. Finally, populations of interest were sorted by the cell concentrator onto a 25-mm Nuclepore polycarbonate membrane (Whatman, Newton, MA) and verified by Leica DMR epifluorescence microscope.
Confocal Microscopic Confirmation
Suspected attached particle pairs were sorted into centrifuge tubes and the solutions were scanned by a BioRad 1024 confocal microscope. Each of these samples was dispersed on a no. 1 thickness 22 mm cover slip and examined in the z direction, which eliminated potential errors caused by particle overlapping on a fixed slide or membrane. The excitation wavelength from the Krypton/Argon laser was 488 nm. For yellow-green (FITC) and red emissions, 522 and 605 nm filters were selected, respectively. An overall magnification of 1000x was achieved using an eyepiece with 10x magnification and an oil immersion lens with 100x magnification. Images were captured by BioRad Lasersharp software.
Statistical Analysis
We analyzed differences in recovered (oo)cyst amounts from the control and soil suspensions by ANOVA using the SAS program (v 8.0, SAS Inst., Cary, NC). Our null hypothesis was that there is no difference between (oo)cyst recovery between the (oo)cyst-distilled water batches (Control 1) and the (oo)cyst–soil batches. The null hypothesis was tested at 
= 0.05 significant level. Any comparison with p values >0.05 failed to reject the null hypothesis and indicated no significant difference existed between the amount of (oo)cysts recovered from Control 1 and (oo)cyst–soil suspensions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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The suspected attached populations in Fig. 2a–e were sorted and examined by epifluoresence and confocal microscopes. Figure 3 shows images captured from the confocal microscope. Visualization of the attached pairs confirmed the flow cytometry findings. In addition, we did not observe attachment in the mixtures of negative beads and (oo)cysts using epifluoresence and confocal microscopes. Figure 3f shows an image of negative beads and PHF C. parvum oocysts in suspension without attachment. Because samples under the confocal microscope were in suspension form, beads and (oo)cysts could be moving when the sequence of images were taken, showing a streak instead of a still image (see Fig. 3a, b, f).
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The recovery rates of (oo)cysts in samples removed from Control 1 were very similar to the recovery rates of (oo)cysts in samples removed from the (oo)cyst–soil batches (Table 2). These rates were calculated as the percentage of (oo)cyst concentration determined by the flow cytometer to the (oo)cyst concentration just after mixing. (Oo)cyst counts in samples from the (oo)cyst–soil batches were corrected for soil particles that appeared in the (oo)cyst gates using samples from Control 2, which contained soil particles in water only. ANOVA tests showed that p values for all comparisons were greater than the 0.05 significant level (Table 2), confirming that no significant difference exists between recovered (oo)cyst amounts in Control 1 and (oo)cyst–soil batches. Variations of recovery rates in (oo)cyst–soil batches generally were higher than in Control 1, which may be due to variations in soil particles in the (oo)cyst gates in Control 2 vs. the (oo)cyst–soil batches. When mixtures of (oo)cyst–soil were examined under both microscopes, no attachment was observed.
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Batch experiments using the South Wilk-1 and Santa-1 sands at pH = 7.06 showed similar recovery rates for Control 1 and (oo)cyst–soil batches (Table 3). The sands were nearly neutral as the NZPCs were 7.1 and 6.9 for South Wilk-1 and Santa-1, respectively. Hence, no attachment was observed between (oo)cysts and sand particles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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The number of attached pairs depicted in Fig. 2a–e appeared low relative to the total number of particles used in the mixtures. The reason for this incomplete attachment is not obvious from our experiments. Possible explanations may be the low density of particles in the overall solution, so that full contact was not achieved during overnight mixing, weak or reversible interaction between particles, and incomplete cover of the cationic polymer on the AF beads.
Mass Balance of Batch Experiments
Results from our batch experiments showed no attachment based on the comparison of Control 1 with the (oo)cyst–soil batches. In all experiments, however, full recovery was not achieved. To achieve 100% recovery, we gently resuspended the settled PHF C. parvum oocyst–soil mixtures and repeated the batch experiments as described above. PHF oocyst concentrations in resuspended samples were the same as in the original samples and the total number of oocysts recovered was the same as the initial amount injected in the tubes (data not shown). Thus, the resuspension experiments further showed that attachment of PHF oocysts to the soil particles did not happen.
Since 90% of the soil particles in the soil suspensions were <2 µm, some very small particles remained in suspension after the settling time. Attachment of (oo)cysts to these very small particles was not ruled out by our experiments. First, if attachment to these particles had occurred, the fluorescence of the (oo)cysts would have been diminished and, in turn, the flow cytometer counts would have been reduced. We did not observe such a reduction (Table 2). Second, the sand batch experiments further assessed this possibility using near neutral SW-1 and SD-1 sands. These sands outsized the (oo)cysts by a factor of 10 and settled 15 to 20 times faster than free C. parvum oocysts and 6 to 8 times faster than G. lamblia cysts. No small soil particles remained in suspension after settling during these experiments. We found that (oo)cysts remained in suspension by themselves and did not attach to the sands (Table 3). (Oo)cyst recovery rates were consistent with those in natural soil batch experiments.
Effects of Different Stocks of Oocysts
Differences in stock preparation and storage media of PHF, PRL, and ES oocysts appear to have had little effect on the outcomes of our experiments. Previous studies indicated that different purification methods may change oocysts' surface properties (Brush et al., 1999; Drozd and Schwartzbrod, 1996). Our measurements, however, showed that oocysts were negatively charged, regardless of purification method and storage media. Results from the particle attachment and oocyst–soil batch experiments showed very little difference between the PHF, PRL and ES oocysts. The null hypothesis was not rejected for either of these oocysts. These findings suggest that concerns about standardization of oocyst stock (Dufour et al., 1999) are not necessarily important for experiments performed to characterize surface charge interactions between soil particles and oocysts.
(Oo)cyst Detection in the Presence of Soil
In this study, we successfully detected C. parvum oocysts and G. lamblia cysts in the presence of soil particles using the flow cytometer. Conventional enumeration using an epifluorescence microscope has been difficult, resulting in poor recovery rates because soil particles blocked the view (Brush, 1997). Successful applications of flow cytometry on environmental water samples have been reported previously (Vesey et al., 1994, Schets et al., 1995), but there was concern of low recovery rate with other particles present. At our flow cytometer settings, we achieved stable counting results at approximate soil concentrations of 2 mg/L. This concentration is still relatively low compared with suspended sediment concentrations in environmental water samples. In this study, the flow cytometer consistently recovered >90% of the oocysts in original suspensions with soil concentrations of 76 mg/L, since samples were removed after 97% of the soil particles had settled. At soil concentrations above 2 mg/L, too many particles flowed past the detector during the 25 ms scanning period causing many particles to bypass the detector including (oo)cysts and beads.
Attachment to Soil versus Effluent
Our findings from the batch experiments that (oo)cysts do not attach to natural soil particles are different from those in Medema et al. (1998), who reported apparent attachment to effluent from a sewage treatment plant. Medema et al. (1998) mixed (oo)cysts with secondary effluent from a biological (active sludge) wastewater treatment plant. The authors reported that one-third of the added (oo)cysts attached to the effluent particles immediately after mixing and that a maximum of 70% was achieved after 24 h. However, they could not confirm actual attachment with complete certainty (F.M. Schets, personal communication, 1999). The difference in attachment results likely is due to the different media characteristics (i.e., soil with some organic matter vs. effluent particles). The majority of the particles in the secondary effluent consist of bioflocs from the active-sludge tank. Biological particles may have different charge characteristics and act differently than the soil particles used in this study.
Effects of Hydrophobicity in Batch Experiments
Recovery of C. parvum oocysts using the flow cytometer was very high as shown by >90% recovery rates in Control 1 and the oocyst–soil batches (Table 2). However, overall recovery of G. lamblia cysts was consistently lower (<50%) in Control 1 and cyst–soil batches. This reduced recovery for cysts may be due to the hydrophobic characteristics of G. lamblia cysts observed in our laboratory (Hayes, 2002) and by others (e.g., Drozd and Schwartzbrod, 1996).
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SUMMARY AND CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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