Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters

Recreational beach water samples collected on weekends and weekdays during 11 consecutive summer weeks were tested for potentially viable Cryptosporidium parvum oocysts and Giardia lamblia cysts using the multiplexed fluorescence in situ hybridization (FISH) method. The levels of oocysts and cysts on weekends were significantly higher than on the weekdays (P<0.01). Concentrations of oocysts in weekend samples (n=27) ranged from 2 to 42 oocysts/L (mean: 13.7 oocysts/L), and cyst concentration ranged from 0 to 33 cysts/L (mean: 9.1 cysts/L). For the samples collected on weekdays (n=33), the highest oocyst concentration was 7 oocysts/L (mean: 1.5 oocysts/L), and the highest cyst concentration was 4 cysts/L (mean: 0.6 cysts/L). The values of water turbidity were significantly higher on weekends than on weekdays, and were correlated with the number of bathers and concentration of C. parvum oocysts and G. lamblia cysts (P<0.04). The study demonstrated positive relationships between number of bathers and levels of waterborne C. parvum oocysts and G. lamblia cysts in recreational beach water. It is essential to test recreational waters for Cryptosporidium and Giardia when numbers of bathers are greatest, or limit the number of bathers in a recreational beach area.     

Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst

Cryptosporidium parvum and Cryptosporidium hominis are obligate enteric protozoan parasites which infect the gastrointestinal tract of animals and humans. The mechanism(s) by which these parasites cause gastrointestinal distress in their hosts is not well understood. The risk of waterborne transmission of Cryptosporidium is a serious global issue in drinking water safety. Oocysts from these organisms are extremely robust, prevalent in source water supplies and capable of surviving in the environment for extended periods of time. Resistance to conventional water treatment by chlorination, lack of correlation with biological indicator microorganisms and the absence of adequate methods to detect the presence of infectious oocysts necessitates the development of consistent and effective means of parasite removal from the water supply. Additional research into improving water treatment and sewage treatment practices is needed, particularly in testing the efficiency of ozone in oocyst inactivation. Timely and efficient detection of infectious C. parvum and C. hominis oocysts in environmental samples requires the development of rapid and sensitive techniques for the concentration, purification and detection of these parasites. A major factor confounding proper detection remains the inability to adequately and efficiently concentrate oocysts from environmental samples, while limiting the presence of extraneous materials. Molecular-based techniques are the most promising methods for the sensitive and accurate detection of C. parvum and C. hominis. With the availability of numerous target sequences, RT-PCR will likely emerge as an important method to assess oocyst viability. In addition, a multiplex PCR for the simultaneous detection of C. parvum, C. hominis and other waterborne pathogens such as Giardia lamblia would greatly benefit the water industry and protect human health.     

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.     

Prevalence of Cryptosporidium oocysts and giardia cysts in the drinking water supply in Japan

A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of 13 samples of 50 L river source water and 26 samples of 2,000 L-filtered water, treated by coagulation flocculation, sedimentation and rapid filtration, were tested. Prior to conducting a survey of a water purification plant, we developed a method for concentrating Cryptosporidium oocysts from a large volume of raw or filtered water using a hollow fiber ultrafiltration (UF) membrane, and this procedure was adapted to survey a water purification plant. Cryptosporidium oocysts were detected in all of the 13 raw water samples. The geometric mean concentration was 40 oocysts 100 L. Giardia cysts were detected in 12 of 13 raw water samples (92%) and the geometric mean concentration was 17 cysts/100 L. Probability distributions of both Cryptosporidium oocyst and Giardia cyst concentration in raw water were nearly lognormal. In filtered water samples, Cryptosporidium oocysts were detected in 9 of the 26 samples (35%) with the geometric mean concentration of 1.2 oocysts /1,000 L and Giardia cysts in 3 samples (12%) with 0.8 cysts/1,000 L. The estimated log10 removal efficiency of Cryptosporidium oocysts and Giardia cysts by rapid-sand filtration was 2.47 and 2.53, respectively. Empty particles were removed at a higher log10 than intact oocysts and cysts. The efficiency of particle removal in the rapid sand filtration process tends to be reduced under cold-water conditions. Close management is necessary in the winter when the water temperature is low.     

用PCR技术检测水中隐孢子虫

介绍了一种检测水中隐孢子虫的巢式PCR方法：根据Cryptosporidium parvum 18s rRNA基因序列选用了两对引物，特异性扩增了该基因可变区中1056bp和435bp目标片段。加标试验表明，该方法的检测限为10个卵囊。     

Detection of enteric viruses, Giardia and Cryptosporidium in two different types of drinking water treatment facilities

In this study, two types of drinking water treatment facilities (two conventional drinking water treatment plants (DWTPs) and two compact units (Cus)) were compared referring to their production capacity. Water samples were collected from three main points: (a) different water treatment steps (b) washings of sand filters and (c) distribution system at different distances from the water treatment plants. Both viruses and protozoa were concentrated from each water sample by adsorption and accumulation on the same nitrocellulose membrane filters (0.45 microm pore size). Enteroviruses were detected by plaque infectivity assay in BGM cells and HAV, HEV and Norovirus were detected by RT-PCR. Giardia and Cryptosporidium were detected by conventional staining methods and PCR. The results revealed that enterovirus load at the intake ranged between 10-15 PFU/L for the two compact units and between 4.5 and 75 PFU/L for the two conventional DWTPs. The virus load in distribution system of the first type DWTPs at 1 km from the plant was the same as that of the intake. Viruses in the other type of treatment plants CUs at 1, 5 and 7 km, were much reduced. Investigation of raw water sediments of the two DWTPs showed enterovirus counts between 12 and 17.5 PFU/L. Virus count was reduced in sand of filters after washing. Giardia cysts were equally detected by microscopy and PCR in only intake samples of EL-Hawamdia CU (33.3%) and Meet Fares DWTP (50%). Cryptosporidium oocysts were equally detected by microscopy and PCR in intake samples of Abo EL-Nomros CU (100%), EL-Hawamdia CU (66.7%) and Fowa DWTP (50%). At Meet Fares DWTP three positive intake samples for Cryptosporidium were detected by PCR, compared with only two positive samples by microscopy. Giardia cysts and Cryptosporidium oocysts were detected in raw water sediment and sand of filters before washing. Only one sample from Meet Fares DWTP sand of filters after washing was positive for both Giardia and Cryptosporidium. It can be concluded that the poor microbial quality of the water may be due to improper operational skills and management of the various water treatment plants (especially at the two high capacity treatment plants).     

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.     

上海市浦东自来水厂净水工艺对两虫去除效果的研究

采用免疫磁分离及荧光染色法（EPA1623法）对上海市浦东地区4家自来水厂的原水、出厂水、管网水中贾第鞭毛虫和隐孢子虫的分布状况进行了分析，并考察了水厂净水过程中不同操作单元对两虫的去除效果。研究表明：原水中存在贾第鞭毛虫与隐孢子虫，但其密度分别维持在0～8个／10L和0～6个／10L的低水平，而在出厂水与管网水中均未检出两虫；水厂的混凝、沉淀单元对两虫具有明显的去除效果。     

Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters

The effect of sequential exposure to ozone followed by monochloramine on inactivation of Cryptosporidium parvum oocysts suspended in untreated natural surface water from two different sources was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. A statistically significant synergistic effect on oocyst inactivation was measured in both natural water samples studied. The magnitude of the effect measured in the natural water with lower turbidity, colour, and organic carbon concentration was comparable to that previously reported for oocysts suspended in buffered de-ionized water but was reduced considerably in the natural water with higher turbidity, colour and organic carbon concentration. Synergy increased with initial pH and with the degree of ozone pre-treatment but was independent of temperature. For water treatment plants with adequate disinfectant contact times, ozone followed by monochloramine may be a practical means of achieving additional C. parvum inactivation, however, the influence of water quality characteristics should be considered.     

Evaluation and optimization of a reusable hollow fiber ultrafilter as a first step in concentrating Cryptosporidium parvum oocysts from water.

Experiments with a small-scale hollow fiber ultrafiltration system (50,000 MWCO) was used to characterize the filtration process and identify conditions that optimize the recovery of Cryptosporidium parvum oocysts from 2 L samples of water. Seeded experiments were conducted using deionized water as well as four environmental water sources (tap, ground, Arkansas river, and Rio Grande river; 0-30.9NTU). Optimal and consistent recovery of spiked oocysts was observed (68-81%), when the membrane was sanitized with a 10% sodium dodecyl sulfate (SDS) solution and then blocked with 5% fetal bovine serum (FBS).     

Intensive exploitation of a karst aquifer leads to Cryptosporidium water supply contamination

Groundwater from karst aquifers is an important source of drinking water worldwide. Outbreaks of cryptosporidiosis linked to surface water and treated public water are regularly reported. Cryptosporidium oocysts are resistant to conventional drinking water disinfectants and are a major concern for the water industry. Here, we examined conditions associated with oocyst transport along a karstic hydrosystem, and the impact of intensive exploitation on Cryptosporidium oocyst contamination of the water supply. We studied a well-characterized karstic hydrosystem composed of a sinkhole, a spring and a wellbore. Thirty-six surface water and groundwater samples were analyzed for suspended particulate matter, turbidity, electrical conductivity, and Cryptosporidium and Giardia (oo)cyst concentrations. (Oo)cysts were identified and counted by means of solid-phase cytometry (ChemScan RDI^®), a highly sensitive method. Cryptosporidium oocysts were detected in 78% of both surface water and groundwater samples, while Giardia cysts were found in respectively 22% and 8% of surface water and groundwater samples. Mean Cryptosporidium oocyst concentrations were 29, 13 and 4/100 L at the sinkhole, spring and wellbore, respectively. Cryptosporidium oocysts were transported from the sinkhole to the spring and the wellbore, with respective release rates of 45% and 14%, suggesting that oocysts are subject to storage and remobilization in karst conduits. Principal components analysis showed that Cryptosporidium oocyst concentrations depended on variations in hydrological forcing factors. All water samples collected during intensive exploitation contained oocysts. Control of Cryptosporidium oocyst contamination during intensive exploitation is therefore necessary to ensure drinking water quality.     

Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water

A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential.     

Removal of viable and inactivated Cryptosporidium by dual- and tri-media filtration

The limited efficacy of disinfectants, other than ultraviolet irradiation and ozonation, as a barrier against Cryptosporidium parvum in drinking water treatment has underscored the increased importance of oocyst removal by filtration. Currently, no reliable surrogates have been identified for C. parvum removal by filtration. As a result, evaluations of the Cryptosporidium removal by treatment operations have been performed using oocysts. It has typically been assumed that chemically inactivated oocysts are suitable surrogates for viable oocysts. Measurements of electrophoretic mobility, however, have shown that chemical inactivation changes the surface charge of Cryptosporidium oocysts. The present bench-scale research indicated that formalin-inactivated oocysts are reliable surrogates for viable oocysts during both stable filter operation and periods where filtration processes are challenged, such as coagulation failure. This finding is important because of the practical difficulties associated with using viable oocysts in filtration investigations. Poor coagulation conditions severely compromised removal of viable and inactivated oocysts by dual- and tri-media filters compared to stable operating conditions and filter ripening, emphasizing the importance of optimized chemical pre-treatment (coagulation) for the successful removal of oocysts during filtration. The treatment optimization experiments also indicated that tri-media filters offered only marginally higher oocyst removals than dual-media filters.     

Influence of land use and watershed characteristics on protozoa contamination in a potential drinking water resources reservoir

Relative changes in the microbial quality of Lake Texoma, on the border of Texas and Oklahoma, were investigated by monitoring protozoan pathogens, fecal indicators, and factors influencing the intensity of the microbiological contamination of surface water reservoirs. The watershed serves rural agricultural communities active in cattle ranching, recreation, and is a potential drinking water source. A total of 193 surface water samples were tested over a 27-month period to determine levels of parasite contamination. The overall occurrence of Cryptosporidium oocysts was higher in both frequency and concentration than Giardia cysts. Cryptosporidium oocysts were found in 99% and Giardia cysts in 87% of the samples. Although Cryptosporidium and Giardia occurrence were significantly but not strongly correlated, all other correlation coefficients including turbidity and total dissolved solids were non-significant. Statistically supportable seasonal variations were found suggesting that Cryptosporidium and Giardia were higher in summer and fall than in other seasons of the year. While Cryptosporidium levels were correlated with rainfall, this was not the case with Giardia. The maximum numbers for both protozoan parasites were detected from a site impacted by cattle ranching during calving season. Restriction fragment length polymorphism analysis was used for confirmation of Cryptosporidium in surface waters influenced by agricultural discharges. As we had expected, oocysts were of the bovine type indicating that the Cryptosporidium parvum detected in surface waters perhaps came from cattle living in the watershed.     

The prevalence and characterisation of Cryptosporidium spp. in beef abattoir water supplies

The prevalence of Cryptosporidium spp. in 50 l samples of water used to wash beef carcasses at (a) an abattoir with a borehole water (BH) supply (n = 46) and (b) an abattoir with a river water (RW) supply (n = 48) was determined. In addition, a 100 l water sample and post-wash carcass samples (n = 24) were collected from the RW supply on a single day in July. Cryptosporidium spp. was detected in 0% and 26.1% of samples from the BH and RW supply abattoirs, respectively, with oocyst concentrations ranging from 0.02 to 8.6/l. Cryptosporidium spp. was not isolated from post-wash beef carcasses, while it was detected in water samples from that day at a concentration of 0.06 oocysts/l. The species of 3/5 isolates were identified as C. parvum, and the remaining were C. andersoni. This study has demonstrated that water used to wash beef carcasses can be contaminated with Cryptosporidium of human health importance and is a potential source of carcass contamination.     

Cryptosporidium parvum oocyst inactivation in field soil and its relation to soil characteristics: analyses using the geographic information systems

The need exists to understand the environmental parameters that affect inactivation of Cryptosporidium parvum oocysts in soil under field conditions. The inactivation of C. parvum oocysts placed in the natural environment was studied at a dairy farm in western New York State, USA. Seventy sampling points were arranged in a grid with points 150 m apart using the Geographic Information System. The sampling points were distributed among three distinct areas: woodland, corn field and pasture. Purified oocysts were inoculated into chambers filled with soil from each sampling point, and buried in the surface of each respective sampling point. To compare C. parvum oocyst survival with another organism known to survive environmental stresses, Ascaris suum eggs were also placed in soil contained in chambers and buried at the same sampling points as the oocysts. As controls oocysts and eggs in distilled water were also placed at each sampling point. Oocyst and egg viability, soil pH and percent gravimetric water content were measured at all sampling points at 0, 60 and 120 day sampling periods. Soil organic content was determined for each sampling point. At 120 days after placement, mean viability of C. parvum oocysts was 10% although at a few sampling points, 30% of oocysts were still potentially infective; whereas 90% of A. suum eggs were viable at all sampling points. Statistically significant differences were not observed among the three different sampling areas, and no statistically significant predictors were found by regression analysis. Results exemplified the heterogeneity of soil parameters and oocyst viability across a landscape; such results make predictive models for C. parvum inactivation problematical. The long-term survival of C. parvum oocysts in soil under field conditions, as this study demonstrated, emphasizes their potential as a risk to contaminate surface waters.     

Monitoring of Cryptosporidium and Giardia river contamination in Paris area

This study evaluates the protozoan contamination of river waters, which are used for drinking water in Paris and its surrounding area (about 615,000 m(3) per day in total, including 300,000 m(3) for Paris area). Twenty litre samples of Seine and Marne Rivers were collected over 30 months and analyzed for Cryptosporidium oocysts and Giardia cysts detection according to standard national or international methods. Cryptosporidium oocysts and Giardia cysts were found, respectively, in 45.7% and 93.8% of a total of 162 river samples, with occasional high concentration peaks. A significant seasonal pattern was observed, with positive samples for Cryptosporidium more frequent in autumn than spring, summer and winter, and positive samples for Giardia less frequent in summer. Counts of enterococci and rainfalls were significantly associated with Giardia concentration but not Cryptosporidium. Other faecal bacteria were not correlated with monitored protozoan. Marne seems to contribute mainly to the parasitic contamination observed in Seine. Based on seasonal pattern and rainfall correlation, we hypothesize that the origin of contamination is agricultural practices and possible dysfunction of sewage treatment plants during periods of heavy rainfalls. High concentrations of protozoa found at the entry of drinking water plants justify the use of efficient water treatment methods. Treatment performances must be regularly monitored to ensure efficient disinfection according to the French regulations.     

再生水中隐孢子虫和贾第鞭毛虫的检测方法研究

隐孢子虫和贾第鞭毛虫（简称“两虫”）是两种严重危害水质安全的病原性原生动物,但国际上常用的两虫检测方法——1623方法存在工作量大、成本高、回收率低等缺点。通过考察1623方法中浓缩、分离两个主要环节的替代方案,发现膜过滤—洗脱是比较理想的两虫浓缩方式,在免疫磁性分离（IMS）环节中酸解离比热解离的效果好。根据以上研究成果,建立了回收率较高（对两虫的回收率分别超过了30％和40％）而成本较低（为原方法的55％左右）的再生水两虫密度检测方法。进一步的研究表明,浓缩是限制低浊水（浊度约1NTU）回收率的主要步骤,而IMS是限制高浊水（浊度约4NTU）回收率的主要步骤。     

某市贾第鞭毛虫和隐孢子虫污染现状

利用免疫荧光分析技术对南方某市饮用水源水、自来水厂出水和污水处理厂进、出水中贾第鞭毛虫和隐孢子虫(两虫)的污染状况进行了调查,并就自来水厂常规处理工艺对两虫的去除特性进行了研究.结果显示:该市饮用水源水中的两虫密度分别为2～120个/100L和0～24个/100 L,自来水厂出水分别为0～12个/1000 L和0～8个/1000 L,污水处理厂进水中的两虫密度分别为7 200～18 300个/L和69～1 210个/L,二级处理出水的分别为6～153个/L和1～46个/L;混凝沉淀和过滤对两虫有较好的去除效果.     

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.