Evaluation of different analysis and identification methods for Salmonella detection in surface drinking water sources

The standard method for detecting Salmonella generally analyzes food or fecal samples. Salmonella often occur in relatively low concentrations in environmental waters. Therefore, some form of concentration and proliferation may be needed. This study compares three Salmonella analysis methods and develops a new Salmonella detection procedure for use in environmental water samples. The new procedure for Salmonella detection include water concentration, nutrient broth enrichment, selection of Salmonella containing broth by PCR, isolation of Salmonella strains by selective culture plates, detection of possible Salmonella isolate by PCR, and biochemical testing. Serological assay and pulsed-field gel electrophoresis (PFGE) can be used to identify Salmonella serotype and genotype, respectively. This study analyzed 116 raw water samples taken from 18 water plants and belonging to 5 watersheds. Of these 116, 10 water samples (8.6%) taken from 7 water plants and belonging to 4 watersheds were positive for a Salmonella-specific polymerase chain reaction targeting the invA gene. Guided by serological assay results, this study identified 7 cultured Salmonella isolates as Salmonella enterica serovar: Alnaby, Enteritidis, Houten, Montevideo, Newport, Paratyphi B var. Java, and Victoria. These seven Salmonella serovars were identified in clinical cases for the same geographical areas, but only one of them was 100% homologous with clinical cases in the PFGE pattern.     

Occurrence and diversity of Arcobacter spp. along the Llobregat River catchment, at sewage effluents and in a drinking water treatment plant

The presence of Arcobacter species in faecally contaminated environmental waters has previously been studied. However, the ability to eliminate Arcobacter during the water treatment processes that produce drinking water has been little studied. We have investigated the prevalence and diversity of Arcobacter spp. throughout the year at 12 sampling points in the Llobregat River catchment (Catalonia, Spain) including 3 sites at a drinking water treatment plant. Positive samples for Arcobacter spp., came predominantly from the most faecally polluted sites. Recovery rates from all sites were greater in the spring (91.7%) and summer (83.3%) than in autumn and winter (75.0% in both cases), but this trend was not statistically evaluated due to the limited number of samples. Among the 339 colonies analyzed, the most prevalent species by multiplex PCR and 16S rDNA restriction fragment length polymorphism were Arcobacter butzleri (80.2%), followed by Arcobacter cryaerophilus (19.4%) and Arcobacter skirrowii (0.3%). Isolates showed a high genotype diversity as determined by the enterobacterial repetitive intergenic consensus PCR. In fact, 91.2% (309/339) of the colonies had different genotypes, i.e. 248 of them among the 275 isolates of A. butzleri and 60 among the 63 isolates of A. cryaerophilus and 1 genotype of A. skirrowii. Arcobacter was never detected or isolated from finished drinking water, demonstrating that water treatment is effective in removing Arcobacter species.     

Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality

This work aimed to understand the distribution of five bacterial pathogens in O’ahu coastal streams and relate their presence to microbial indicator concentrations, land cover of the surrounding watersheds, and physical-chemical measures of stream water quality. Twenty-two streams were sampled four times (in December and March, before sunrise and at high noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread —12 of 22 O’ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indicators and total vibrio with log mean ± standard deviation densities of 2.2 ± 0.8 enterococci, 2.7 ± 0.7 Escherichia coli, 1.1 ± 0.7 Clostridium perfringens, 1.2 ± 0.8 F⁺ coliphages, and 3.6 ± 0.7 total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed enterococci positively associated with the greatest number of bacterial pathogens. Higher concentrations of enterococci and higher incidence of Campylobacter were found in stream waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multivariate regression models of microbes as a function of land cover and physical-chemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physical-chemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O’ahu stream exposures and to mitigate these risks through control of land-based runoff sources.     

Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water

We have evaluated the use of a fluorescent in situ hybridization (FISH) technique for the detection of Helicobacter pylori in water (river and wastewater) samples. The assay was compared with PCR detection and isolation of cells on selective media. 16S rRNA and UreA+B sequence data were used as oligonucleotide probe and specific primers for FISH and PCR, respectively. Using FISH technique, H. pylori was detected in two river water and one wastewater samples, while PCR yielded only one positive result. H. pylori culture was not possible from any sample. According to these results, FISH technique has the potential to be used as a quick and sensitive method for detection of H. pylori in environmental samples.     

Detection of Toxoplasma gondii oocysts and surrogate microspheres in water using ultrafiltration and capsule filtration

While reports on waterborne infections with Toxoplasma gondii are emerging worldwide, detection of this zoonotic parasite in water remains challenging. Lack of standardized and quantitative methods for detection of T. gondii oocysts in water also limits research on the transport and fate of this pathogen through aquatic habitats. Here, we compare the ability of hollow-fiber ultrafiltration and capsule filtration to concentrate oocysts in spiked tap water, fresh surface water, and seawater samples. Detection of T. gondii oocysts in concentrated samples was achieved using molecular methods, as well as visually via epifluorescent microscopy. In addition to oocysts, water samples were spiked with T. gondii surrogate microspheres, and detection of microspheres was performed using flow cytometry and epifluorescent microscopy. Results demonstrate that both water concentration methods followed by microscopy allowed for quantitative detection of T. gondii oocysts and surrogate microspheres. For T. gondii oocysts, microscopy was more sensitive than TaqMan and conventional PCR, and allowed for detection of oocysts in all water samples tested. Compared with flow cytometry, microscopy was also a more cost-efficient and precise method for detection of fluorescent surrogate microspheres in tap, fresh and seawater samples. This study describes a novel approach for quantitative detection of T. gondii oocysts in drinking and environmental water samples. The techniques described for concentrating and detecting surrogate microspheres have broad application for evaluating the transport and fate of oocysts, as well as the efficiency of water treatment methods for removal of T. gondii from water supplies.     

Temporal flux and spatial dynamics of nutrients, fecal indicators, and zoonotic pathogens in anaerobic swine manure lagoon water

Confined animal feeding operations (CAFOs) often use anaerobic lagoons for manure treatment. In the USA, swine CAFO lagoon water is used for crop irrigation that is regulated by farm-specific nutrient management plans (NMPs). Implementation of stricter US environmental regulations in 2013 will set soil P limits; impacting land applications of manure and requiring revision of NMPs. Precise knowledge of lagoon water quality is needed for formulating NMPs, for understanding losses of N and C in ammonia and greenhouse gas emissions, and for understanding risks of environmental contamination by fecal bacteria, including zoonotic pathogens. In this study we determined year-round levels of nutrients and bacteria from swine CAFO lagoon water. Statistical analysis of data for pH, electrical conductivity (EC), inorganic and organic C, total N, water-soluble and total minerals (Ca, Cu, Fe, K, Mg, Mn, P, and Zn) and bacteria (Escherichia coli, enterococci, Clostridium perfringens, Campylobacter spp., Listeria spp., Salmonella spp., and staphylococci) showed that all differed significantly by dates of collection. During the irrigation season, levels of total N decreased by half and the N:P ratio changed from 9.7 to 2.8. Some seasonal differences were correlated with temperature. Total N and inorganic C increased below 19 °C, and decreased above 19 °C, consistent with summer increases in ammonia and greenhouse gas emissions. Water-soluble Cu, Fe, and Zn increased with higher summer temperatures while enterococci and zoonotic pathogens (Campylobacter, Listeria, and Salmonella) decreased. Although their populations changed seasonally, the zoonotic pathogens were present year-round. Increasing levels of E. coli were statistically correlated with increasing pH. Differences between depths were also found. Organic C, total nutrients (C, Ca, Cu, Fe, Mg, Mn, N, P, and Zn) and C. perfringens were higher in deeper samples, indicating stratification of these parameters. No statistical interactions were found between collection dates and depths.     

Use of a Salmonella typhimurium hilA fusion strain to assess effects of environmental fresh water sources on virulence gene expression

Many fruits and vegetables are irrigated with water from rivers, lakes and even wastewater systems. Irrigation may be a route for the introduction of Salmonella. Our objectives in this study were to determine survivability and virulence expression in a strain of Salmonella typhimurium when exposed to environmental water sources. Virulence expression was measured using a beta-galactosidase assay on a hilA:lacZY fusion strain of S. typhimurium. Water samples for environmental impact studies were taken from a local pond and specific sites along the Rio Grande River, which serves as a source of irrigation water in southern Texas. There was a significant difference (p<0.05) of virulence expression among the water sites. Certain regions along the Rio Grande River yielded greater amounts of beta-galactosidase activity than others. All sites yielded at least a two-fold greater virulence response than S. typhimurium grown in brain heart infusion. Salmonella survivors were enumerated as colony forming units (CFU)/ml as plated on a selective medium for the duration of 1 week and beta-galactosidase assays were performed to determine a possible correlation between culturable cells and virulence gene expression. Bacterial cells remained viable but decreased after 7 days incubation. In conclusion, water sampled at specific locations and at different times water samples exhibited differences in virulence expression in S. typhimurium.     

Occurrence of Mycobacterium avium subsp. paratuberculosis in raw water and water treatment operations for the production of potable water

Mycobacterium avium subsp. paratuberculosis (Map) causes Johne’s disease of cattle and is implicated as a cause of Crohn’s disease in humans. The organism is excreted in animal faeces and can contaminate water catchment areas. This coupled with Map’s survival in the environment means that water destined for domestic use may be a source of exposure. This work was designed to determine the occurrence of Map in Lough Neagh (the largest freshwater lake in the British Isles), used as a reservoir, and in two water treatment works (WTW1 and WTW2) which abstract from the lough and which have slow sand filtration (SSF) and dissolved air flotation respectively as their principal treatment regimes. The organism was not detected in lough water samples by culture (n = 70) but 29% (20/70) were positive by PCR. In the raw water to WTW1 and WTW2 no culture positives were detected but 54% (13/24) and 58% (14/24) respectively were PCR positive. In WTW1 there were no culture positives at the SSF or final water but 31% (8/26) and 45% (9/20) respectively were PCR positive. In WTW2 similar results were obtained with 26% (6/23) and 48% (11/23) in the floccules and final water respectively. At WTW2 however one culture positive was detected in the final water. This latter finding is of concern. The inability to reach definitive conclusions indicates the need for further research, particularly in the detection methods for viable Map.     

Quantitative risk assessment of Cryptosporidium in tap water in Ireland

Cryptosporidium species are protozoan parasites associated with gastro-intestinal illness. Following a number of high profile outbreaks worldwide, it has emerged as a parasite of major public health concern. A quantitative Monte Carlo simulation model was developed to evaluate the annual risk of infection from Cryptosporidium in tap water in Ireland. The assessment considers the potential initial contamination levels in raw water, oocyst removal and decontamination events following various process stages, including coagulation/flocculation, sedimentation, filtration and disinfection. A number of scenarios were analysed to represent potential risks from public water supplies, group water schemes and private wells. Where surface water is used additional physical and chemical water treatment is important in terms of reducing the risk to consumers. The simulated annual risk of illness for immunocompetent individuals was below 1 × 10^− 4 per year (as set by the US EPA) except under extreme contamination events. The risk for immunocompromised individuals was 2-3 orders of magnitude greater for the scenarios analysed. The model indicates a reduced risk of infection from tap water that has undergone microfiltration, as this treatment is more robust in the event of high contamination loads. The sensitivity analysis highlighted the importance of watershed protection and the importance of adequate coagulation/flocculation in conventional treatment. The frequency of failure of the treatment process is the most important parameter influencing human risk in conventional treatment. The model developed in this study may be useful for local authorities, government agencies and other stakeholders to evaluate the likely risk of infection given some basic input data on source water and treatment processes used.     

Molecular characterization of microbial populations in groundwater sources and sand filters for drinking water production

In full-scale drinking water production from groundwater, subsurface aeration is an effective means of enhancing the often troublesome process of nitrification. Until now the exact mechanism, however, has been unknown. By studying the microbial population we can improve the understanding of this process. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments of bacteria, archaea and ammonia-oxidizing bacteria was used to characterize the microbial populations in raw groundwater and trickling filters of an active nitrifying surface aerated system and an inactive non-surface aerated system. Only in the active filter were nitrifying microorganisms found above the detection limit of the method. In ammonia oxidation in this groundwater filter both bacteria and archaea played a role, while members belonging to the genus Nitrospira were the only nitrite-oxidizing species found. The subsurface aerated groundwater did not contain any of the nitrifying organisms active in the filter above the detection limit, but did contain Gallionella species that might play a major role in iron oxidation in the filter.     

Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation

Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.     

Occurrence, molecular characterization and antibiogram of water quality indicator bacteria in river water serving a water treatment plant

Water pollution by microorganisms of fecal origin is a current world-wide public health concern. Total coliforms, fecal coliforms (Escherichia coli) and enterococci are indicators commonly used to assess the microbiological safety of water resources. In this study, influent water samples and treated water were collected seasonally from a water treatment plant and two major water wells in a Black Belt county of Alabama and evaluated for water quality indicator bacteria. Influent river water samples serving the treatment plant were positive for total coliforms, fecal coliforms (E. coli), and enterococci. The highest number of total coliform most probable number (MPN) was observed in the winter (847.5 MPN/100 mL) and the lowest number in the summer (385.6 MPN/100 mL). Similarly E. coli MPN was substantially higher in the winter (62.25 MPN/100 mL). Seasonal variation of E. coli MPN in influent river water samples was strongly correlated with color (R² = 0.998) and turbidity (R² = 0.992). Neither E. coli nor other coliform type bacteria were detected in effluent potable water from the treatment plant. The MPN of enterococci was the highest in the fall and the lowest in the winter. Approximately 99.7 and 51.5 enterococci MPN/100 mL were recorded in fall and winter seasons respectively. One-way ANOVA tests revealed significant differences in seasonal variation of total coliforms (P < 0.05), fecal coliforms (P < 0.01) and enterococci (P < 0.01). Treated effluent river water samples and well water samples revealed no enterococci contamination. Representative coliform bacteria selected by differential screening on Coliscan Easygel were identified by 16S ribosomal RNA gene sequence analysis. E. coli isolates were sensitive to gentamicin, trimethoprim/sulfamethazole, ciprofloxacin, vancomycin, tetracycline, ampicillin, cefixime, and nitrofurantoin. Nonetheless, isolate BO-54 displayed decreased sensitivity compared to other E. coli isolates. Antibiotic sensitivity pattern can be employed in microbial source tracking.     

Landscape and seasonal factors influence Salmonella and Campylobacter prevalence in a rural mixed use watershed

Salmonella and Campylobacter prevalence in stream networks of the Satilla River Basin (SRB) were monitored monthly from August 2007 to August 2009 to study relationships between these pathogens and land use, presence of poultry houses and wastewater treatment plant (WWTP) discharge. Salmonella and Campylobacter were detected at all 10 stream sites and the three sites at the sole wastewater treatment plant (WWTP) in the study area. In all, 43% (129/299) and 62% (96/156) of samples were positive for Salmonella and Campylobacter, respectively, with detection frequency increasing in downstream sites with more poultry production and influence of WWTP discharge. Both Salmonella and Campylobacter detection frequencies were positively associated with the number of poultry houses in the subwatersheds, but agricultural land use as a proportion of the watershed was not a significant predictor of either pathogen. Fecal indicator bacterial levels were assessed and evaluated for their ability to predict the presence of pathogens. Of those examined, enterococci was most predictive; of the 129 samples positive for Salmonella, 88% (113/129) were detected when enterococci were above EPA single sample threshold (61 CFU 100 ml^-1); and of the 96 samples positive for Campylobacter, 90% (86/96) were detected when enterococci levels exceeded this level. Comparatively, Escherichia coli concentrations were above EPA single sample thresholds in 38% (49/129) of the positive Salmonella samples. Detection of the pathogens throughout the watershed indicated that there was potential for waterborne transmission especially in downstream areas that were more likely to have recreational users.     

Molecular and phylogenetic approaches for assessing sources of Cryptosporidium contamination in water

The high sequence diversity and heterogeneity observed within species or genotypes of Cryptosporidium requires phylogenetic approaches for the identification of novel sequences obtained from the environment. A long-term study on Cryptosporidium in the agriculturally-intensive South Nation River watershed in Ontario, Canada was undertaken, in which 60 sequence types were detected. Of these sequence types 33 were considered novel with no identical matches in GenBank. Detailed phylogenetic analysis identified that most sequences belonged to 17 previously described species: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, muskrat I, muskrat II, deer mouse II, fox, vole, skunk, shrew, W12, W18, W19 and W25 genotypes. In addition, two new genotypes were identified, W27 and W28. C. andersoni and the muskrat II genotype were most frequently detected in the water samples. Species associated with livestock made up 39% of the total molecular detections, while wildlife associated species and genotypes accounted for 55% of the Cryptosporidium identified. The human pathogenic species C. hominis and C. parvum had an overall prevalence of 1.6% in the environment, indicating a small risk to humans from the Cryptosporidium present in the watershed. Phylogenetic analysis and knowledge of host-parasite relationships are fundamental in using Cryptosporidium as a source-tracking or human health risk assessment tool.     

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.     

Method for rapid detection of viable Escherichia coli in water using real-time NASBA

A rapid real-time NASBA method was developed for detection of Escherichia coli in water samples. In this method, a fragment of the clpB-mRNA is amplified and a specific molecular beacon probe is used to detect the amplified mRNA fragment during the NASBA reaction. The method was shown to be specific and sensitive (1 viable E. coli in 100 ml) and can be performed within 3-4 h. Different inactivation processes (starvation, heat, UV-irradiation and chlorine) were employed to study the relationship between culturability and the ability to detect E. coli using NASBA. Detection of clpB-mRNA correlated with culturability after starvation or chlorine treatment. After UV-irradiation or heat-inactivation, detection of the increase in production of clpB-mRNA in viable E. coli cells after heat-shock induction correlated with culturability. Application of the NASBA method on tap water, treated sewage and surface water samples showed that culture and NASBA yielded comparable results in these different matrices. This study demonstrates that the NASBA method has high potential as a rapid test for microbiological water quality monitoring.     

Die-off of enteric bacterial pathogens during mesophilic anaerobic digestion

Conventionally treated sewage sludge may contain high concentrations of potentially pathogenic microorganisms and additional treatment is required to minimise the risks to health if it is to be recycled to agricultural land. Mesophilic anaerobic digestion (MAD) is the most widely used process in the UK for stabilising sludge prior to agricultural recycling, but little is known about the fate of a number of enteric pathogens as the sludge passes through the treatment processes. The aim of this study was to determine the efficiency of MAD in removing the bacterial enteric pathogens, Salmonella senftenberg, Listeria monocytogenes and Campylobacter jejuni which were added as a spike to the digester feedstock, together with the die-off of indigenous Escherichia coli already present in the sludge. The primary sludge digestion stage of MAD was found to achieve a log removal of 1.66 for E. coli, 2.23 for L. monocytogenes and 2.23 for S. senftenberg. However, the extent of die-off was a function of the numbers of pathogens in the feed and as these increased the log removal also increased. The numbers of C. jejuni were not affected by primary sludge digestion. Additional die-off was provided by secondary sludge digestion with log removals of 1.70 for E. coli, 2.10 for S. senftenberg and 0.36 for C. jejuni.     

Antibiotic contamination and occurrence of antibiotic-resistant bacteria in aquatic environments of northern Vietnam

The ubiquitous application and release of antibiotics to the environment can result in bacterial antibiotic resistance, which in turn can be a serious risk to humans and other animals. Southeast Asian countries commonly apply an integrated recycling farm system called VAC (Vegetable, Aquaculture and Caged animal). In the VAC environment, antibiotics are released from animal and human origins, which would cause antibiotic-resistant bacteria (ARB). This study evaluated occurrence of ARB in the VAC environment in northern Vietnam, with quantitative analysis of antibiotic pollution. We found that sulfonamides were commonly detected at all sites. In dry season, while sulfamethazine was a major contaminant in pig farm pond (475-6662 ng/l) and less common in city canal and aquaculture sites, sulfamethoxazole was a major one in city canal (612-4330 ng/l). Erythromycin (154-2246 ng/l) and clarithromycin (2.8-778 ng/ml) were the common macrolides in city canal, but very low concentrations in pig farm pond and aquaculture sites. High frequencies of sulfamethoxazole-resistant bacteria (2.14-94.44%) were found whereas the occurrence rates of erythromycin-resistant bacteria were lower (< 0.01-38.8%). A positive correlation was found between sulfamethoxazole concentration and occurrence of sulfamethoxazole-resistant bacteria in dry season. The sulfamethoxazole-resistant isolates were found to belong to 25 genera. Acinetobacter and Aeromonas were the major genera. Twenty three of 25 genera contained sul genes. This study showed specific contamination patterns in city and VAC environments and concluded that ARB occurred not only within contaminated sites but also those less contaminated. Various species can obtain resistance in VAC environment, which would be reservoir of drug resistance genes. Occurrence of ARB is suggested to relate with rainfall condition and horizontal gene transfer in diverse microbial community.     

Removal of Giardia and Cryptosporidium in drinking water treatment: a pilot-scale study

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern for public health. The aim of this study is to examine both parasites in the water samples taken from three pilot-scale plant processes located in southern Taiwan, to upgrade the current facilities. Three processes include: conventional process without prechlorination (Process 1), conventional process plus ozonation and pellet softening (Process 2), and integrated membrane process (MF plus NF) followed conventional process (Process 3). The detection methods of both parasites are modified from USEPA Methods 1622 and 1623. Results indicated that coagulation, sedimentation and filtration removed the most percentage of both protozoan parasites. The pre-ozonation step can destruct both parasites, especially for Giardia cysts. The microfiltration systems can intercept Giardia cysts and Cryptosporidium oocysts completely. A significant correlation between water turbidity and Cryptosporidium oocysts was found in this study. The similar results were also found between three kinds of particles (phi=3-5,5-8 and 8-10 microm) and Cryptosporidium oocysts.     

Changes of physical and biochemical properties of Cryptosporidium oocysts with various storage conditions

Physical and biochemical properties of Cryptosporidium parvum oocyst were examined after storage under various conditions. Oocyst-positive-fecal samples recovered from calves were either stored in a 2.0% potassium dichromate solution (Cr) or deionized water (W), or kept as a fecal pellet (P), and stored at 4 or 18 degrees C for a maximum of 100 days. When stored in Cr at 4 degrees C, the morphology of oocysts and their ability to withstand ultrasonics was not affected by the storing media or the storage period. However, when stored at 18 degees C as a fecal pellet, the specific gravity of the oocysts increased and a significant decrease in the oocysts resistance to ultrasonics occurred. These changes in oocyst properties may affect the performance of methods used to detect oocysts in water samples. When using the current test methods or when developing a new test method, it is important to take these factors into consideration.