2007 Progress Report: Quantitative Assessment of Pathogens in Drinking Water
EPA Grant Number:
Quantitative Assessment of Pathogens in Drinking Water
Schwab, Kellogg J.
, Graczyk, Thaddeus
, Halden, Rolf U.
The Johns Hopkins University
EPA Project Officer:
Klieforth, Barbara I
August 25, 2006 through
August 24, 2009
(Extended to September 30, 2010)
Project Period Covered by this Report:
August 25, 2006 through August 24,2007
Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005)
A major limiting factor in assessing the human health risk of microbial pathogens in raw and finished drinking water is the lack of robust, efficient methods for concentrating, identifying, and quantifying low levels of bacteria, viruses, and protozoa simultaneously, effectively, and rapidly. We will develop a microbial isolation and detection protocol capable of qualitative and quantitative identification of waterborne microbial pathogens by combining the latest high-efficiency filtration technology with rapid and sensitive molecular detection techniques, including quantitative PCR (qPCR), quantitative reverse transcription-PCR (qRT-PCR), fluorescent in situ hybridization (FISH), and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The sensitivity and specificity of the proposed pathogen recovery and detection approach will be directly compared to current USEPA methods via spiking and analysis of raw and finished drinking water samples collected from various water resources and distribution systems. Following method validation, a series of unspiked raw or finished waters (including waters from distribution systems) will be monitored for pathogenic microorganisms to demonstrate the utility of the approach in real world situations.
Research during the first 12 months of this project include the optimization and streamlining of several fluorescence in situ hybridization (FISH) methods into two primary assays for the simultaneous detection of 1) Cryptosporidium parvum oocysts, and Giardia lamblia cysts and 2) human-virulent microsporidia spores such as Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi. Efforts have also been made towards the further development of mass spectrometry methods for the detection and quantification of virus capsid proteins, which could allow for estimation of virus concentrations within a sample. Additionally, a bench-scale tangential flow ultrafiltration system has been developed to evaluate the ability of commercially available hollow fiber membranes to simultaneously recover viruses and bacteria from 3 different water types. Research in the development of methods for downstream concentration and detection of these recovered microorganisms is also ongoing. A summary of the research that has either been initiated and/or completed during the current reporting period (August 2006-August 2007) is presented below.
Multiplexed FISH assay for detection of C. parvum oocysts and G. lamblia cysts: The assay was developed based on C. parvum oocysts and G. lamblia cysts obtained from the experimental infection of calves. Drinking water samples (10 L) were spiked in duplicate with a total of either 1x103 or 1x102 C. parvum oocysts and G. lamblia cysts, and processed by U.S. EPA Method 1623.
Oligonucleotide probes and monoclonal antibodies. An oligonucleotide probe specific to C. parvum (Cry 1) and two probes to specific to G. lamblia (Giar-4 and Giar-6) were used (4, 6, 13). All probes were synthesized in 1.0 µM scale, purified by HPLC, and labeled with a single molecule of a fluorochrome, hexachlorinated 6-carboxyfluorescein (Hex) by the Johns Hopkins University DNA Analysis Facility in Baltimore, MD. A FITC-conjugated combination of monoclonal antibodies (mAb) against the cell wall antigens of Cryptosporidium and Giardia were obtained from the MERIFLUORTM Cryptosporidium/Giardia test kit (Meridian Diagnostic, Inc., Cincinnati, OH) (8).
FISH and FITC-conjugated mAb. All FISH and FITC-conjugated mAb experiments were carried out in Eppendorf tubes in a total volume of 100 µl of hybridization buffer, at 48 o C, for 1 hr (6). Concentration of each oligonucleotide probe was 1 mM, and the mAb were diluted 1:1 (4, 6). After incubation, the tubes were centrifuged twice at 4 oC (8,000g, 2 min), and the pellets were resuspended in 100 µl of phosphate buffered saline (PBS). Five 20µl samples were transferred onto individual lysine-coated wells (5mm in diameter) on a Teflon-coated glass slide (Carlson Scientific, Inc., Peotone, IL) and air-dried. The wells were examined with an Olympus BH2-RFL epifluorescent microscope, dry 60X objective, and BP-490 exciter filter.
The multiplexed FISH assay clearly identified C. parvum oocysts and G. lamblia cysts in all experiments (Figures 1 and 2). Ongoing research includes spiking 10 L drinking water samples with 10 oocysts and cysts, processing the water samples according to the U.S. EPA Method 1623, and then applying the multiplexed FISH assay for detection of oocysts and cysts.
Figure 1. Fluorescence in situ hybridization (FISH) images of Cryptosporidium parvum oocysts obtained in the multiplexed FISH assay. Scale bar, 5 micrometers.
Figure 2. Fluorescence in situ hybridization (FISH) image of Giardia lamblia cyst obtained in the multiplexed FISH assay. Scale bar, 7 micrometers.
Multiplexed FISH assay for detection of human-virulent microsporidia spores (i.e., E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi): The assay was developed using microsporidian spores obtained from in vitro cell line infections (i.e. E. intestinalis, E. hellem, and E. cuniculi) or purified from a human fecal sample (i.e. E. bieneusi). Briefly, the multiplexed FISH assay was carried out in quadruplicate in Eppendorf tubes with a total volume of 100 µl of hybridization buffer at 57 oC for 3 hrs, using 1 mM concentrations of each oligonucleotide probe (all probes were placed into the same Eppendorf tube) (8, 9, 12). The spores were permealized by incubation for 15 minutes in acetone (11). The E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi-specific oligonucleotide probes (8, 9, 11, 12) were synthesized in 1.0 µM scale, purified by HPLC, and 5'-labeled with a single molecule of a fluorochrome, Hex, 6-Fam, Tet, and Cy3 by the Johns Hopkins University DNA Analysis Facility in Baltimore, MD (12). A total of 1x104 spores were added for each species of microsporidian. The multiplexed FISH assay for the detection of human-virulent microsporidian spores was able to clearly identify, by color, the various species of microsporidian spores (Figure 3).
For confirmation purposes, samples were also assayed by polymerase chain reaction (PCR) using microsporidian species-specific primers based on the small subunit rRNA gene of E. bieneusi, E. hellem, E. intestinalis, and E. cuniculi (2, 5, 14). All PCR products were analyzed by gel electrophoresis and stained with ethidium bromide for visualization.
Experiments currently ongoing include spiking 10 L drinking water samples with 1x102 and 10 spores, processing the water samples according to the U.S. EPA Method 1623 and then applying the multiplexed FISH assay for the detection of human-virulent microsporidia spores.
Figure 3. Images of four human-virulent microsporidian spores species (i.e., E. hellem, E. bieneusi, E. intestinalis, and E. cuniculi) obtained in multiplexed fluorescence in situ hybridization (FISH) assay. Scale bar, 2 micrometers.
Mass Spectrometry Detection of Virus-like Particles: The initial proposal to incorporate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) into our research was to facilitate the detection of protein biomarkers from pathogens including bacteria, virus and protozoa in a number of settings. Our earlier methods involving MALDI-TOF MS utilized simple, rapid preparation and analysis strategies (1). However, this analysis was limited by high titer requirements, and potential co-contaminants in the final preparation often interfered with the MS analysis. We have therefore applied and utilized a more targeted approach, monitoring for specific peptide fragments (short sequences of amino acids) from the target virus capsid protein using liquid chromatography tandem MS (Figure 4). By using this method, we reduced the data burden by monitoring only for select biomarkers, which is accomplished with a method that is similar to traditional environmental chemistry methods (10). In addition, this method allows for the inclusion of isotopically labeled internal standards for absolute quantification (7). By adding these standards into the sample post-processing, we can measure quantitative levels of the target peptide and thus potentially estimate the concentration of virus in a given sample.
By using methods, such as liquid chromatography tandem MS, we are able to detect and quantify viral capsid protein targets down to the low femtomolar (10-15 M) to high attomolar (10-18 M) range, which is an increase in sensitivity of three orders of magnitude from our previous efforts with the MALDI-TOF. While the development of this technique is still in the early stages, we have successfully improved the sensitivity such that this method has the potential to detect relevant levels of viruses in the matrix of interest (i.e. drinking water) making it increasingly significant to the field of water research.
We are currently preparing to challenge the method with well-characterized, real-world samples to demonstrate the efficacy and reproducibility of mass spectrometry for the detection of pathogens of public health concern.
Figure 4. Representative data from mass spectrometric detection of norovirus virus-like particles. Detection using whole peptide scanning (A) resulted in insufficient sensitivity, so targeted monitoring of specific peptide fragment biomarkers (B) was utilized. The resulting targets, and thus the viral capsid, were detectable and quantifiable in the low femtomolar range (C).
Microbial Stock Production and Detection Methods: The recovery and downstream detection of several microorganisms will be used in the laboratory studies evaluating the tangential flow ultrafiltration system. All of these microorganisms have been used extensively in our laboratories and can be successfully propagated and analyzed by conventional diagnostic assays as described below.
A. MS2 Bacteriophage
1. MS2 stock generation: To generate high titer (1013 pfu) bacteriophage stocks, a bacterial host (i.e. Escherichia coli Famp) is incubated in a flask with tryptic soy broth (TSB) plus 1% ampicillin/streptomycin solution at 37°C with shaking for 4 hours to produce a log phase growth. The bacterial host at log phase growth is then used to propagate MS2 using a soft agar overlay method. In this method, 75ml of prepared bacterial host and 100ml of diluted MS2 stock are added to a sterile tube containing 5 mL of 0.7% tryptic soy agar (TSA). The contents of the tube are mixed gently by ‘rolling’ between the analyst’s palms and then carefully poured onto a Petri dish containing 15 mL of 1.5% TSA+1% ampicillin/streptomycin solution. These steps are repeated on a total of 4 plates. The plates are then incubated at 37°C, without inverting, for 16-18 hours. Using a cell scraper, harvest the MS2 from the plates by gently scraping the top, soft agar layers into a 50cc tube. Next, PBS is added to the 50cc tube to achieve a total volume of 23 mL followed by the addition of 23 mL of chloroform. This mixture is then vortexed for 5 minutes at maximum speed and centrifuged at 4,000xg for 30 minutes in order to separate the MS2 from the extracellular materials and soft agar. The MS2-containing aqueous phase is carefully removed so as not to disturb the interphase. To reduce the formation of aggregates, the stock is filtered through sequentially smaller (0.45 micron, 0.22 micron and 0.1 micron), low protein-binding filters pretreated with 5 mL of 0.1% Tween 80 followed by 5 mL PBS—multiple filters may be necessary to filter the entire MS2 stock solution. Aliquots of MS2 bacteriophage stocks are stored at –80°C.
2. Double agar overlay method for MS2 bacteriophage enumeration: MS2 bacteriophage are enumerated using the double agar overlay procedure in U.S. EPA Method 1602. In this method, 75ml of prepared bacterial host and 100ml of MS2 stock serially diluted in a buffer (PBS, 0.01% Tween 80 and 0.001% Antifoam-A) are added to a sterile tube containing 5 mL of 0.7% tryptic soy agar (TSA). The contents of the tube are mixed gently by ‘rolling’ between the analyst’s palms and then carefully poured onto a Petri dish containing 15 mL of 1.5% TSA+1% ampicillin/streptomycin solution. The sample is spread evenly over the surface of the plate by gently and quickly swirling the plate. The plate, which solidifies within 30 seconds, is then inverted and incubated at 370C for 16-18 hours. During the incubation time, the host bacteria form a confluent lawn over the surface of the Petri plate allowing the phage particles that are present in the sample to attach to and infiltrate the bacterial host cells. The MS2 bacteriophage then replicates within the host cells eventually causing it to lyse the bacterium. The destruction of the bacterial cells that make up the confluent lawn result in clear areas known as plaque forming units. The concentration of bacteriophage present in the sample is determined by visually counting the plaques (Figure 5).
Figure 5. Typical results from dilution series of MS2 using the DAL method.
B. Escherichia coli CN-13
1. Production of Escherichia coli stock: Escherichia coli CN-13, a Nalidixic acid resistant strain, is generated by inoculating a flask containing 25 mL of TSB+1% Nalidixic acid solution with a 10 micron loop of frozen bacteria stock. The inoculated media is then incubated overnight at 37°C with shaking at 110 rpm. The overnight growth of bacteria is then stored at 4°C for future use.
2. Enumeration of overnight E. coli stock: E. coli CN-13 stocks are enumerated using a spread plate method. In this method, 100ml of bacteria serially diluted in a buffer (same as one used fro MS2) are pipetted onto a Petri dish containing 1% TSA+1% Nalidixic acid. The diluted sample is spread over the plate by adding 4-5 five mm glass beads to the dish and gently shaking back and forth to allow for even distribution of the bacteria. After allowing the inoculum to absorb into the agar, the plate is then inverted and incubated at 37°C for 16-18 hours. The concentration of E. coli present in the sample is determined by visually counting the colony forming units.
Figure 6. Typical results of dilution series with E. coli CN-13 using the spread plate method.
C. Enteric Viruses: The enteric viruses to be used in laboratory experiments are propagated and assayed using specific cell lines following similar protocols as represented by the outline for murine norovirus (MNV-1). Comparable procedures will be used for propagation and assaying of Hepatitis A virus, poliovirus, and adenovirus.
1. Propagation of MNV-1 stock: MNV-1 is replicated in a monolayer of RAW 264.7 cells (ATCC, Manassas, VA) which are grown in DMEM (Cellgro-Mediatech, Herndon, VA) supplemented with 10% low-endotoxin fetal bovine serum (Hyclone, Logan, UT), 100 U penicillin/mL, 100µg/mL streptomycin, 10 mM HEPES, and 2 mM L-glutamine (Invitrogen, Carlsbad, CA). Cell monolayers, at about 90% confluence, are inoculated at a multiplicity of infection (MOI) of .05 with MNV-1 and then allowed to incubate for 1 hr at 37ºC and 5% CO2 to enable viral adsorption. After infection, cells are incubated in maintenance media (DMEM with 2% low-endotoxin fetal bovine serum) and checked for cytopathic effects daily. Virus infected calls are then subjected to three freeze-thaw cycles; the cell lysates infected with virus are extracted and purified with an equal volume of Vertrel® XF (Miller-Stephenson), aliquoted, and stored at -80ºC for future use.
2. Enumeration of MNV-1 stock: MNV-1 stock titers are determined by plaque assay as previously described with some modifications (15). Briefly, 6-well tissue culture plates are seeded with RAW 264.7 cells at a concentration of 2 x 106 viable cells per well, and after twenty-four hours, viral stock dilutions are prepared in complete DMEM and plated in 500 μl volumes per well. Plates are incubated at 37ºC and 5% CO2 for 1 hr with continuous rocking followed by removal of the inoculum and application of 2 ml prepared overlay media per well. The overlaid plates are incubated at 37ºC and 5% CO2 for 24-hr and then each well is stained with an additional 2 ml of overlay media containing 1% neutral red (Invitrogen) for visualization of plaques. The concentration of MNV-1 present in the sample is determined by visually counting the plaque forming units (Figure 7).
1. Cryptosporidium parvum oocysts and Giardia lamblia cysts originate from experimental infection of calves. Infectious cystic stages of these pathogens are available in Dr. Graczyk’s laboratory in large quantities. Spores of human-virulent microsporidia are also available in large quantities in Dr. Graczyk's laboratory and originate from in vitro systems (E. intestinalis, E. hellem, and E. cuniculi) and from human fecal samples (E. bieneusi).
2. Detection of these protozoa follows the multiplex FISH assay protocols described previously in this report.
Bench-scale Ultrafiltration Preliminary Findings: A bench-scale tangential flow ultrafiltration system has been developed, and we have begun to evaluate the ability of a commercially available ultrafilter to simultaneously recover viruses and bacteria from 3 different water types. The capsule filter, available through Fresenius Medical Care (Waltham, MA), contains polysulfone, hollow fiber membranes with an average molecular weight cut-off (MWCO) of 80kDa and a total surface area of 1.8m2. These filters are disposable and are subject to one-time use during bench-scale evaluations. The ultrafiltration system is operated at a flow rate of 1,700 ml/min under 7-8 lb/in2 pressure with an average flow of 800 ml/min and 900 ml/min for the permeate rate and cross-flow rate, respectively. These parameters are achieved using a digital peristaltic pump with a high-performance pump head (Cole Parmer, Vernon Hills, IL).
Ultrafiltration experiments thus far have been conducted with 10L lab-controlled water samples spiked with MS2 bacteriophage, E. coli
CN-13, and MNV-1. The filters are pretreated with 500 ml of a 5% bovine serum solution (0.22µm sterile-filtered) (Sigma, St. Louis, MO) the night before an experiment and then allowed to incubate at room temperature overnight with continuous rotation. After filtration of the 10L sample, the retentate (78 to 200 ml) is collected, and the filter is then flushed with 500 ml of an elution buffer containing 0.5% Tween 80, 0.001% Antifoam-A, and 0.01% Sodium Polyphosphate (NaPP) (Sigma). This elution buffer is recirculated through the system—without the application of backpressure—at a flow rate of 1,000-1,500 ml/min until roughly 100 ml of the buffer, or eluant, remains (i.e. 400 ml has been filtered). Throughout the filtration process, quality control samples are collected and assayed along with the spiked water samples, retentate, and eluant. A summary of the preliminary data is shown in Table 1 and is based on results for microbial detection assays described
previously in this report.
Ongoing experiments include the evaluation of various methods for the secondary concentration of microorganisms from the retentate and eluant. Methods being explored include polyethylene glycol concentration of the sample as well as the use of 100kDa centrifugal filtration devices available through Millipore (Centricon Plus-70). Preliminary findings indicate that the Centricon device is better suited for our proposed workflow scheme. In addition, lower concentrations of microorganisms are being added to challenge the system even further.
The following research will be conducted during the next project period (August 2007 to August 2008):
- Further evaluations of the multiplexed FISH assays will be conducted by spiking 10 L water samples with low concentrations of cysts, oocysts, and spores (1-10) for the detection of G. lamblia, C. parvum, and human-virulent microsporidia, respectively.
- Our mass spectrometry protocol will be tested further for its ability to detect enteric viruses, like norovirus, within a complex matrix such as a human stool extract. Once optimized, MS will be utilized in the detection of microorganisms from the ultrafiltration process in combination with the established culture methods of detection as well as quantitative molecular analysis.
- Evaluation and optimization of the bench-scale ultrafiltration system will continue. The following parameters will be involved:
- The system will be challenged by spiking progressively lower concentrations of E. coli, MNV-1, and MS2 into 10 L and 100 L samples of sterile dechlorinated tap water.
- The retentate, eluant, and secondary concentrate will be analyzed for the microorganisms by both culture and quantitative molecular techniques which have been established in our lab.
- Additional microorganisms will be added to the water samples including poliovirus, norovirus, Hepatitis A virus, adenovirus, enterococci, Clostridium perfringens, Pseudomonas diminuta, Cryptosporidium and Microsporidia. This will help to evaluate the ability of the ultrafilter to recovery a wide variety of microorganisms ranging in size, shape, and type.
- Large volume surface, ground, and distribution system water samples will be collected and spiked with previously mentioned microorganisms. These samples will then be processed using the ultrafiltration setup and detection methods described within this report.
1. Colquhoun, D. R., K. J. Schwab, R. N. Cole, and R. U. Halden. 2006. Detection of norovirus capsid protein in authentic standards and in stool extracts by matrix-assisted laser desorption ionization and nanospray mass spectrometry. Appl Environ Microbiol 72:2749-55
2. DaSilva, A. J., S. B. Slemenda, G. S. Visvesvara, D. A. Schwartz, C. M. Wilcox, S. Wallace, and N. J. Pieniazek. 1997. Detection of Septata intestinalis (Microsporidia) Cali et al. 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol. Diagn. 2:47-52.
3. DaSilva, A. J., F. J. Bornay-Llinares, L. N. S. Moura, S. B. Slemenda, J. L. Tuttle, and N. J. Pieniazek.1999. Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Mol. Diagn. 4:57-64.
4. Deere D, Vesey G, Milner M, Williams K, Ashbolt N, Veal DA. 1998. Rapid method for fluorescent in situ ribosomal RNA labeling of Cryptosporidium parvum. J Appl Microbiol 85: 807-818.
5. deGroote, M. A., G. S. Visvesvara, M. L. Wilson, N. J. Pieniazek, S. B. Slemenda, A. J. DaSilva, G. J. Leitch, R. T. Bryan, and R. Reeves. 1995. Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in patient with AIDS: successful therapy with albendazole. J. Infect. Dis. 171:1375-1378.
6. Dorsch MR, Veal DA, 2001. Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization. J Appl Microbiol 90: 836-842.
7. Gerber, S. A., J. Rush, O. Stemman, M. W. Kirschner, and S. P. Gygi. 2003. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940-5.
8. Graczyk, T. K., J. Bosco-Nizeyi, A. J. DaSilva, L. N. S. Moura, N. J. Pieniazek, M. R. Cranfield, and H. D. A. Lindguist. 2002. A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats, Uganda. Parasitol. Res. 88:926-931
9. Graczyk, T. K., D. B. Conn, F. Lucy, D. Minchin, L. Tamang, L. N. S. Moura, and A. J. DaSilva. 2004. Human waterborne parasites in zebra mussels (Dreissena polymorpha) from the Shannon River drainage, Ireland. Parasitol. Res. 93:389-391
10. Halden, R. U., and D. H. Paull. 2004. Analysis of triclocarban in aquatic samples by liquid chromatography electrospray ionization mass spectrometry. Environmental Science & Technology 38:4849-4855.
11. Hester, F. D., H. D. A. Linquist, A. M. Bobst, and F. W. Schaffer. 2000. Fluorescent in situ detection of Encephalitozoon hellem spores with a 6carboxyfluorescein-labeled ribosomal RNA-targeted oligonucleotide probe. J. Eukaryot. Microbiol. 47:299-308.
12. Slodkowicz-Kowalska, A., T. K. Graczyk, L. Tamang, S. Jedrzejewski, A. Nowosad, P. Zduniak, P. Solarczyk, A. S. Girouard, and A. C. Majewska. 2006. Microsporidia species known to infect humans are present in aquatic birds; implications for transmission via water? Appl. Environ. Microbiol. 72:4540-4544.
13. Vesey G, Ashbolt N, Fricker EJ, Deere D, William KL, Veal DA, Dorsch M, 1998. The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts. J Appl Microbiol 85: 429-440.
14. Visvesvara, G. S., A. J. DaSilva, G. P. Croppo, N. J. Pieniazek, G. J. Leitch, D. Ferguson, H. Moura, S. Wallace, S. B. Slemenda, and I. Tyrrell. 1995. In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. J. Clin. Microbiol. 33:930-936.
15. Wobus, C. E., S. M. Karst, L. B. Thackray, K. O. Chang, S. V. Sosnovtsev, G. Belliot, A. Krug, J. M. Mackenzie, K. Y. Green, and H. W. Virgin. 2004. Replication of a Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2:e432.
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|| Graczyk TK, Sunderland D, Rule AM, da Silva AJ, Moura INS, Tamang L, Girouard AS, Schwab KJ, Breysse PN. Urban feral pigeons (Columba livia) as a source for air- and waterborne contamination with Enterocytozoon bieneusi spores. Applied and Environmental Microbiology 2007;73(13):4357-4358.
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|| Graczyk TK, Majewska AC, Schwab KJ. The role of birds in dissemination of human waterborne enteropathogens. Trends in Parasitology 2008;24(2):55-59.
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2008 Progress Report