Infectious Cryptosporidium parvum Oocysts in Final Reclaimed Effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Development of a Rapid Method for Simultaneous Recovery of Diverse Microbes in Drinking Water by Ultrafiltration with Sodium Polyphosphate and Surfactants

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.5-μm microspheres, Salmonella enterica serovar Typhimurium, Bacillus globigii endospores, and echovirus 1. Ten-liter tap water samples were concentrated to ~250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2, C. parvum, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.     

Ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples

This study focused on ultrafiltration as a technique for simultaneously concentrating and recovering viruses, bacteria and parasites in 100-L drinking water samples. A chemical dispersant, sodium polyphosphate, and Tween 80 were used to increase microbial recovery efficiencies. Secondary concentration was performed to reduce sample volumes to 3-5 mL for analysis using tissue culture, microscopy, and real-time PCR and RT-PCR. At seeding levels of 100-1000 (CFU, PFU, oocysts, or particles), a "high-flux" ultrafiltration procedure was found to achieve mean recoveries of 51-94% of simultaneously seeded MS2 bacteriophage, echovirus 1, Salmonella enterica subsp. enterica serovar Typhimurium, Bacillus atrophaeus subsp. globigii endospores, Cryptosporidium parvum oocysts, and 4.5-mum microspheres. When 4-7% of the final sample concentrate volume was assayed using real-time PCR and RT-PCR, overall method sensitivities were <100 C. parvum oocysts, <240 PFU echovirus 1, <100 CFU Salmonella and approximately 160 CFU B. atrophaeus spores in 100-L drinking water samples. The "high-flux" ultrafiltration procedure required approximately 2 h, including time required for backflushing. Secondary concentration procedures required an additional 1-3 h, while nucleic acid extraction and real-time PCR procedures required an additional 2-2.5 h. Thus, this study demonstrated that efficient recovery and sensitive detection of diverse microbes in 100-L drinking water samples could be achieved within 5-8 h using ultrafiltration, rapid secondary processing techniques, and real-time PCR.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Dead-End Hollow-Fiber Ultrafiltration for Recovery of Diverse Microbes from Water

Dead-end ultrafiltration (DEUF) is an alternative approach to tangential-flow hollow-fiber ultrafiltration that can be readily employed under field conditions to recover microbes from water. The hydraulics of DEUF and microbe recovery for a new DEUF method were investigated using 100-liter tap water samples. Pressure, flow rate, and temperature were investigated using four hollow-fiber ultrafilter types. Based on hydraulic performance, the Asahi Kasei REXEED 25S ultrafilter was selected for microbe recovery experiments. Microbe recovery experiments were performed using MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts. Microbes were recovered from ultrafilters by backflushing using a surfactant solution. Average flow rates were 2.1 liters/min for 100-liter water samples having turbidities of 0.28 to 4.3 nephelometric turbidity units (NTU), and no evidence of appreciable filter clogging was observed. The DEUF average recovery efficiencies for each study analyte in tap water were as follows: for E. faecalis, 93% ± 16%; for MS2, 57% ± 7.7%; for C. perfringens spores, 94% ± 22%; and for C. parvum, 87% ± 18%. Average microbe recoveries for tap water amended with surface water (average turbidity = 4.3 NTU) were as follows: for E. faecalis, 78% ± 12%; for MS2, 73% ± 13%; for C. perfringens, 57% ± 21%; and for C. parvum, 83% ± 21%. These data demonstrate that DEUF is an effective method for recovering diverse microbes from water and should be a useful tool for field-based environmental investigations.There are an estimated 4 million to 33 million cases of acute gastrointestinal illness each year in the United States due to drinking water (3, 11). From 2005 to 2006, 20 reports of waterborne disease and outbreaks (WBDOs) associated with drinking water were submitted to the national Waterborne Disease and Outbreak Surveillance System (19). In addition, a record number (78 reports) of WBDOs associated with recreational water were also submitted to the Waterborne Disease and Outbreak Surveillance System in 2005 and 2006 (20). Detecting the etiologic agents for WBDOs is challenging due to such factors as the time delay between case exposure and water sampling, microbial die-off, and water dilution or treatment prior to sampling. Because it is likely that pathogens will be present at low concentrations in water sampled for outbreak investigations, relatively large volumes of water (e.g., 40 to 100 liters) should be collected. In addition, sampling water for a diverse array of microbes is sometimes a goal when multiple etiologic agents are suspected for a WBDO (13) or during emergency responses when the contaminant has not been identified.Hollow-fiber ultrafiltration (UF) has been shown to be an effective technique for collecting large-volume water samples for recovery of diverse microbes, including viruses, vegetative bacteria, bacterial spores, and parasites (5, 6, 10, 12, 14). However, most hollow-fiber UF techniques utilize a tangential-flow approach that requires comprehensive operator training and which is generally not conducive to rapid-response implementation for field sampling. While the tangential-flow (i.e., recirculating flow) UF technique has been shown to be effective for microbe recovery, it is a more complicated sampling technique than traditional direct-filtration techniques, such as membrane filtration for coliforms (1), microfiltration for Cryptosporidium and Giardia (oo)cysts (18), and adsorption-elution microfiltration for viruses (4). For emergency response, outbreak investigations, or other field investigations performed by personnel with limited training in water sample collection, a dead-end UF (DEUF) technique would be useful for capturing and recovering multiple microbe classes.Relatively few studies have reported using hollow-fiber UF in a DEUF configuration. Kearns et al. (7) reported using an automated DEUF system to recover Bacillus atrophaeus spores from tap water, with reported recovery efficiencies of 23 to 40% in ∼100-liter samples with low-level seeding (330 to 1,000 CFU). These researchers performed filter backflushing using a phosphate buffer containing either Tween 20 or sodium polyphosphate. Kearns et al. also reported suspected ultrafilter fouling based on measured reductions in flow rates for ∼100-liter samples (7). The Kearns et al. observations indicate that the ultrafilter pore size and filter area are important hydraulic performance variables for the DEUF technique. Leskinen and Lim (9) reported using hollow-fiber DEUF for recovery of enterococci from 100-liter samples of beach water. These researchers used a urea-lysine solution to elute (instead of backflush) enterococci from ultrafilters. Leskinen and Lim reported a wide range of recovery efficiencies (4 to 708%; average = 251%) for their DEUF method but did not discuss whether water quality (other than a potential variability in microbe distribution in the 100-liter samples) could have contributed to ultrafilter fouling or variable method performance (9).The present study was designed to investigate DEUF using different commercially available hollow-fiber ultrafilters having a range of pore sizes and filter medium sizes. The parameters tested included the effect of the water sample flow rate and temperature on system pressure and the effect of turbidity on the permeate flow rate and microbial recovery efficiencies. A suite of four distinctly different microbes (MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts) was studied to determine the performance of the DEUF method for simultaneous recovery of diverse microbes.     

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of Cryptosporidium Oocysts and Giardia Cysts in Water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants

The ability to simultaneously concentrate diverse microbes is an important consideration for sample collection methods that are used for emergency response and environmental monitoring when drinking water may be contaminated with an array of unknown microbes. This study focused on developing a concentration method using ultrafilters and different combinations of a chemical dispersant (sodium polyphosphate [NaPP]) and surfactants. Tap water samples were seeded with bacteriophage MS2, Escherichia coli, Enterococcus faecalis, Cryptosporidium parvum, 4.5-microm microspheres, Salmonella enterica serovar Typhimurium, Bacillus globigii endospores, and echovirus 1. Ten-liter tap water samples were concentrated to approximately 250 ml in 12 to 42 min, depending on the experimental condition. Initial experiments indicated that pretreating filters with fetal bovine serum or NaPP resulted in an increase in microbe recovery. The addition of NaPP to the tap water samples resulted in significantly higher microbe and microsphere recovery efficiencies. Backflushing of the ultrafilter was found to significantly improve recovery efficiencies. The effectiveness of backflushing was improved further with the addition of Tween 80 to the backflush solution. The ultrafiltration method developed in this study, incorporating the use of NaPP pretreatment and surfactant solution backflushing, was found to recover MS2, C. parvum, microspheres, and several bacterial species with mean recovery efficiencies of 70 to 93%. The mean recovery efficiency for echovirus 1 (49%) was the lowest of the microbes studied for this method. This research demonstrates that ultrafiltration can be effective for recovering diverse microbes simultaneously in tap water and that chemical dispersants and surfactants can be beneficial for improving microbial recovery using this technique.     

Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.     

Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ~40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.     

Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents

This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.     

Concentration and detection of cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4',6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Effect of Particles on the Recovery of Cryptosporidium Oocysts from Source Water Samples of Various Turbidities

Cryptosporidium parvum can be found in both source and drinking water and has been reported to cause serious waterborne outbreaks which threaten public health safety. The U.S. Environmental Protection Agency has developed method 1622 for detection of Cryptosporidium oocysts present in water. Method 1622 involves four key processing steps: filtration, immunomagnetic separation (IMS), fluorescent-antibody (FA) staining, and microscopic evaluation. The individual performance of each of these four steps was evaluated in this study. We found that the levels of recovery of C. parvum oocysts at the IMS-FA and FA staining stages were high, averaging more than 95%. In contrast, the level of recovery declined significantly, to 14.4%, when the filtration step was incorporated with tap water as a spiking medium. This observation suggested that a significant fraction of C. parvum oocysts was lost during the filtration step. When C. parvum oocysts were spiked into reclaimed water, tap water, microfiltration filtrate, and reservoir water, the highest mean level of recovery of (85.0% ± 5.2% [mean ± standard deviation]) was obtained for the relatively turbid reservoir water. Further studies indicated that it was the suspended particles present in the reservoir water that contributed to the enhanced C. parvum oocyst recovery. The levels of C. parvum oocyst recovery from spiked reservoir water with different turbidities indicated that particle size and concentration could affect oocyst recovery. Similar observations were also made when silica particles of different sizes and masses were added to seeded tap water. The optimal particle size was determined to be in the range from 5 to 40 μm, and the corresponding optimal concentration of suspended particles was 1.42 g for 10 liters of tap water.     

Succession of Bacterial Community Structure and Diversity in Soil along a Chronosequence of Reclamation and Re-Vegetation on Coal Mine Spoils in China

The growing concern about the effectiveness of reclamation strategies has motivated the evaluation of soil properties following reclamation. Recovery of belowground microbial community is important for reclamation success, however, the response of soil bacterial communities to reclamation has not been well understood. In this study, PCR-based 454 pyrosequencing was applied to compare bacterial communities in undisturbed soils with those in reclaimed soils using chronosequences ranging in time following reclamation from 1 to 20 year. Bacteria from the Proteobacteria, Chloroflexi, Actinobacteria, Acidobacteria, Planctomycetes and Bacteroidetes were abundant in all soils, while the composition of predominant phyla differed greatly across all sites. Long-term reclamation strongly affected microbial community structure and diversity. Initial effects of reclamation resulted in significant declines in bacterial diversity indices in younger reclaimed sites (1, 8-year-old) compared to the undisturbed site. However, bacterial diversity indices tended to be higher in older reclaimed sites (15, 20-year-old) as recovery time increased, and were more similar to predisturbance levels nearly 20 years after reclamation. Bacterial communities are highly responsive to soil physicochemical properties (pH, soil organic matter, Total N and P), in terms of both their diversity and community composition. Our results suggest that the response of soil microorganisms to reclamation is likely governed by soil characteristics and, indirectly, by the effects of vegetation restoration. Mixture sowing of gramineae and leguminosae herbage largely promoted soil geochemical conditions and bacterial diversity that recovered to those of undisturbed soil, representing an adequate solution for soil remediation and sustainable utilization for agriculture. These results confirm the positive impacts of reclamation and vegetation restoration on soil microbial diversity and suggest that the most important phase of microbial community recovery occurs between 15 and 20 years after reclamation.     

Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, approximately 40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.     

Effects of seeding procedures and water quality on recovery of Cryptosporidium oocysts from stream water by using U.S. Environmental Protection Agency Method 1623

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.     

Evaluation of Five Membrane Filtration Methods for Recovery of Cryptosporidium and Giardia Isolates from Water Samples

We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 ± 15.4 per 10 liters) and cysts (n = 59 ± 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 ± 8, P = 0.015; cysts, 49.8 ± 12.2, P = 0.4260), and SMF (oocysts, 16.2 ± 2.8, P = 0.0079; cysts, 35.2 ± 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.     

Development and Application of Different Methods for the Detection of Toxoplasma gondii in Water

Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T. gondii oocysts (1 × 10⁵ and 1 × 10⁴ oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe₂(SO₄)₃ and Al₂(SO₄)₃. The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al₂(SO₄)₃ (96.5% ± 21.7%) than by flocculation with Fe₂(SO₄)₃ (93.1% ± 8.1%) or by centrifugation at 2,073 × g (82.5% ± 6.8%). For the unsporulated oocysts, flocculation with Fe₂(SO₄)₃ was more successful (100.3% ± 26.9%) than flocculation with Al₂(SO₄)₃ (90.4% ± 19.1%) or centrifugation at 2,565 × g (97.2% ± 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water.