Infectivity of Giardia lamblia cysts obtained from wastewater treated with ultraviolet light

Several waterborne outbreaks of giardiasis have been linked to discharge of wastewater effluents into surface water. Little is known about the infectivity of Giardia lamblia cysts present in UV treated wastewater effluents. In this study, the infectivity of G. lamblia cysts, recovered from primary effluent and secondary effluent, both upstream and downstream of operating full-scale UV reactors at four wastewater treatment plants, was assessed using the Mongolian gerbil model. Infectivity of cysts obtained from the primary effluents was scored as either strong or moderate for induction of infection in gerbils at three out of four wastewater treatment plants. G. lamblia recovered from secondary effluent both upstream and downstream of the UV reactors caused weak infections in the gerbils. The probability of weak infections caused by inoculums of 50-1400 cysts per gerbil was, on the average, reduced by approximately 10% at the four wastewater UV installations with coliform reduction equivalent doses ranging from 6 to 18 mJ/cm². The UV systems provided considerably less inactivation of the parasite than expected based on the UV dose response of Giardia reported in the literature.     

Infection risk assessment of diarrhea-related pathogens in a tropical canal network

A quantitative microbial risk assessment (QMRA) of Cryptosporidium, Giardia and diarrhegenic Escherichia coli (DEC) infection was performed using Monte Carlo simulations to estimate the human health risks associated with the use of canal water for recreational purposes, unrestricted and restricted irrigation in a tropical peri-urban area. Three canals receiving municipal, agricultural, and, predominantly, industrial wastewater were investigated. Identification of pathogenic protozoans revealed the major presence of Cryptosporidium hominis and both assemblages A and B of Giardia lamblia. The highest individual infection risk estimate was found to be for Giardia in an exposure scenario involving the accidental ingestion of water when swimming during the rainy season, particularly in the most polluted section, downstream of a large wholesale market. The estimated annual risks of diarrheal disease due to infection by the protozoan parasites were up to 120-fold greater than the reported disease incidence in the vicinity of the studied district and the entire Thailand, suggesting a significant host resistance to disease beyond our model's assumptions. In contrast, annual disease risk estimates for DEC were in agreement with actual cases of diarrhea in the study area.     

Role of predation by zooplankton in transport and fate of protozoan (oo)cysts in granular activated carbon filtration

The significance of zooplankton in the transport and fate of pathogenic organisms in drinking water is poorly understood, although many hints of the role of predation in the persistence of microorganisms through water treatment processes can be found in literature. The objective of this study was to assess the impact of predation by natural zooplankton on the transport and fate of protozoan (oo)cysts in granular activated carbon (GAC) filtration process. UV-irradiated unlabelled Cryptosporidium parvum and Giardia lamblia (oo)cysts were seeded into two pilot-scale GAC filtration columns operated under full-scale conditions. In a two-week period after seeding, a reduction of free (oo)cysts retained in the filter bed was observed. Zooplankton was isolated from the filter bed and effluent water on a 30 μm net before and during the two-week period after seeding; it was enumerated and identified. Rotifers, which are potential predators of (oo)cysts, accounted for the major part of the isolated zooplankton. Analytical methods were developed to detect (oo)cysts internalized in natural zooplankton isolated from the filter bed and effluent water. Sample sonication was optimized to disrupt zooplankton organisms and release internalized microorganisms. (Oo)cysts released from zooplankton after sonication were isolated by IMS and stained (EasyStain™) for microscopic counting. Both Cryptosporidium and Giardia (oo)cysts were detected in association with zooplankton in the filter bed samples as well as in the effluent of GAC filters. The results of this study suggest that predation by zooplankton can play a role in the remobilization of persistent pathogens such as Cryptosporidium and Giardia (oo)cysts retained in GAC filter beds, and consequently in the transmission of these pathogens in drinking water.     

Detection of pathogen based on the catalytic growth of gold nanocrystals

A homogenous detection of pathogen (Giardia lamblia cysts) based on the catalytic growth of gold nanoparticles (AuNPs) has been studied. In this study, centrifugal filters were employed as tools to concentrate and separate the pathogen cells, and moreover amplify the detection signal. The catalytic growth of gold nanoparticles was verified to be positively related to gold seeds concentration. On this basis, homogenous detection of the pathogenic bacteria in liquid phase was established by means of conjugating antibody to gold seeds. Under the given experimental condition, detection limit of G. lamblia cysts was determined as low as 1.088 × 10³ cells ml⁻¹. The additional nonspecific binding tests were also conducted to verify the detection specificity. This sensing platform has been proved to be a sensitive, reliable and simple method for large-scale pathogen detection, and provide valuable insight for the development of gold nanocrystals based colorimetric biosensors.     

Detection and genotyping of Giardia duodenalis in wastewater: relation between assemblages and faecal contamination origin

Among the seven assemblages identified in Giardia duodenalis species, only assemblages A and B infect humans and numerous other mammals as well. On the other hand, assemblage E is considered to be host restricted to livestock. The aim of the present study was to compare the presence of G. duodenalis assemblages A, B and E in wastewater samples from two municipal treatment plants (n=24) and one slaughterhouse (n=12). Thus, PCR assays targeting the tpi gene were developed to detect specifically these three G. duodenalis assemblages. Assemblages A and B were detected in urban wastewater with a predominance of assemblage A, especially for one treatment plant. Concerning slaughterhouse wastewater, assemblage A was found in 58% of the samples, whereas assemblage B was not detected. Assemblage E was not detected in urban wastewater, but was found in 92% of the samples from slaughterhouse. Thus, combination of assemblages A and B seemed to indicate a human contamination origin, while combination of assemblages A and E appeared to correspond to a livestock contamination origin.     

Cryptosporidium spp. and Giardia duodenalis in two areas of Galicia (NW Spain)

The aim of the present study was to investigate the environmental dispersal of Cryptosporidium spp. and Giardia duodenalis in two distinct areas (coastal and inland) in Galicia (NW Spain). Faecal samples were collected from healthy asymptomatic domestic (cows and sheep) and wild animals (deer and wild boars) in the selected areas. In each of the selected areas, samples of untreated water (influent) and of treated water (final effluent) were collected from each of the 12 drinking water treatments plants (DWTPs) and 12 wastewater treatment plants (WTPs) under study. Analysis of a single sample from each of the 635 (coastal) and 851 (inland) domestic and wild animals selected at random revealed that the prevalences of cryptosporidiosis and giardiosis in coastal area were 9.2% and 15.9% respectively, and in inland area, 13.7% and 26.7% respectively. In the coastal area, Cryptosporidium spp. oocysts were detected in influent and effluent samples from 2/12 (16.6%) DWTPs and 8/12 (66.6%) WTPs, while G. duodenalis cysts were detected in influent and effluent samples from 3/12 (25.0%) DWTPs and 12/12 (100%) WTPs. The concentrations were notably higher in WTPs; the mean parasite concentrations in the final treated effluent were 10 oocysts per litre and 137.8 cysts per litre for Cryptosporidium and Giardia, respectively. The mean concentration of G. duodenalis cysts per litre was significantly higher (P < 0.05) than the mean concentration of Cryptosporidium spp. oocysts per litre in both the influent and the effluent samples from all the treatment plants. In the coastal area, C. parvum, C. hominis and G. duodenalis assemblages A (I and II) and E were most repeatedly detected. In the inland area, C. parvum, C. andersoni and G. duodenalis assemblages A (I and II), B and E were most frequently identified.     

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.     

Improvements in Cryptosporidium recovery and variability through modifications to United States Environmental Protection Agency Method 1623

The use of United States Environmental Protection Agency (USEPA) Method 1623 for the detection of Cryptosporidium in water often results in low and variable rates of recovery. According to data disseminated in Method 1623, less than a quarter of tested surface water samples had Cryptosporidium recoveries of 60% or greater. This research explored various method modifications in order to produce a method with higher recovery and less variability. Once method modifications were implemented, recoveries of C. parvum in reagent water increased by greater than 20% to an average recovery of 76%. In surface water, a significantly higher (P = 0.049) mean recovery of 67% for C. parvum was achieved with the modified method. Giardia is simultaneously detected when using Method 1623. The method modifications significantly increased the recovery of Giardia in reagent water by 37% (P = 0.0002) while also allowing for a 17% higher recovery in surface water samples (P = 0.21).     

提高过滤浓缩阶段对两虫回收率的试验研究

针对美国EPA1623检测方法中过滤浓缩、免疫磁分离和荧光免疫镜检这3个主要步骤进行了分段回收率的研究。结果表明,过滤浓缩为导致两虫回收率损失的关键步骤,因此对过滤浓缩中的洗脱和分离过程进行了相应的改进和替代。以超声洗脱替代振荡洗脱,用滤膜抽滤/超声洗脱替代离心分离,构成优化的过滤浓缩工艺,具有较高的回收率,操作简单且成本较低。其对卵囊和孢囊的初始回收率分别为61%和55%,远高于EPA1623方法的最低要求,适宜作为国内自来水厂的常规检测方法。     

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p < 0.05, r_s range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.     

Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: A health risk for humans and animals

The objective of the present study was to determine the degree of contamination by Cryptosporidium spp. and Giardia duodenalis in a river basin in a livestock farming area in Galicia (NW, Spain). Water samples (50 l) were collected at 22 points in the main basin (including 5 recreational areas), and at the source and mouth of the 3 most important rivers and at the mouth of a smaller, secondary river. Faecal samples were collected from dairy cattle selected at random from 18 herds farmed in the area. A total of 139 neonatal calves, 480 heifers and 697 cows were sampled. The prevalence, intensity of infection and the risk associated with the spread of infection by both enteropathogens were determined. Water and faecal samples were collected in spring, summer, autumn and winter of 2007. The species and genotypes of these parasites present in the water samples were identified. In both water and faecal samples, more parasitic stages were collected in spring and summer than in autumn and winter. In spring, Cryptosporidium spp. oocysts were detected in 33 (9.4%) cows from 13 (72.2%) herds, and G. duodenalis cysts were detected in 56 (16.0%) cows from 15 farms (83.3%); the intensity of infection ranged from 5 to 7895 G. duodenalis cysts per gram of faeces. Infective stages of Cryptosporidium spp. and G. duodenalis were also detected in respectively 26 (89.6%) and 27 (93.1%) water samples, in spring. The mean concentrations of parasites ranged from 2 to 1200 Cryptosporidium spp. oocysts per litre and from 2 to 400 G. duodenalis cysts per litre. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The presence of both protozoans must be monitored in cattle, in sources of water used for recreational purposes and in artificial waterways used by farmers (water channels, animal drinking water and drainage systems).     

Identification of opportunistic parasites and helminth ova in concentrated water samples using a hollow-fibre ultrafiltration system

In the present study, we applied an ultrafiltration process to detect Blastocystis spp., Cryptosporidium parvum, Giardia lamblia and helminth ova during a dry season with cold and warm periods. Samples of wastewater, reuse water for the irrigation of green areas and groundwater were collected on the campus of the National Autonomous University of Mexico. Physicochemical parameters were measured. Blastocystis spp. and its subtypes (ST) were identified using polymerase chain reaction (PCR) and indirect immunofluorescence (IFA); G. lamblia and C. parvum were identified using IFA, and helminth ova were searched for using flotation/sedimentation and microscopic observation. Blastocystis ST3, C. parvum and G. lamblia were detected in wastewater and reuse water, but only G. lamblia was detected in all of the samples of reuse water. Helminth ova were not detected. Statistical analysis showed a dependency between total dissolved solids and protozoa. Our data suggest that G. lamblia detection can be performed as part of reuse water quality assessments for irrigation systems.     

密度梯度分离纯化/免疫荧光技术检测饮用水中“两虫”

采用膜溶解、密度梯度分离纯化结合免疫荧光染色法对饮用水中的“两虫”（隐孢子虫和贾第鞭毛虫）进行定性与定量检测，以大幅降低检测成本，简化操作。结果表明，使用Percoll-蔗糖介质可取得良好的分离纯化效果，其卵囊和孢囊初始回收率分别为56％和35％，远高于EPA1623方法的要求。利用该方法对河网型水源水及水厂滤后水进行了检测，发现10L水源水中有1～3个隐孢子虫和0～2个贾第鞭毛虫，但在50L滤后水中均未检出“两虫”。     

Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.     

GAC adsorption filters as barriers for viruses, bacteria and protozoan (oo)cysts in water treatment

Granular Activated Carbon (GAC) adsorption filtration is commonly used in drinking water treatment to remove NOM and micro-pollutants and on base of the process conditions a certain capacity to eliminate pathogenic micro-organisms was expected. The experiences with the mandatory quantitative microbial risk assessment of Dutch drinking water revealed a lack of knowledge on the elimination capacity of this process for pathogens. The objective of the current study was to determine the capacity of GAC filtration to remove MS2, Escherichia coli and spores of Clostridium bifermentans as process indicators for pathogens and more directly of (oo)cysts of Cryptosporidium parvum and Giardia lamblia. Challenge tests with fresh and loaded GAC were performed in pilot plant GAC filters supplied with pre-treated surface water at a contact time which was half of the contact time of the full-scale GAC filters. MS2 phages were not removed and the removal of E. coli and the anaerobic spores was limited ranging from ≤0.1-1.1 log. The (oo)cysts of C. parvum and G. lamblia, however, were removed significantly (1.3-2.7 log). On base of the results of the experiments and the filtration conditions the removal of the indicator bacteria and (oo)cysts was largely attributed to attachment. The model of the Colloid Filtration Theory was used to describe the removal of the dosed biocolloids in the GAC filters, but the results demonstrated that there is a lack of quantitative knowledge about the influence of collector characteristics on the two major CFT parameters, the single collector and the sticking efficiency.     

Contribution of treated wastewater to the microbiological quality of Seine River in Paris

Urban part of Seine River serving as drinking water supply in Paris can be heavily contaminated by Cryptosporidium spp. and Giardia duodenalis. In the absence of agricultural practice in this highly urbanized area, we investigated herein the contribution of treated wastewater to the microbiological quality of this river focusing on these two parasites. Other microorganisms such as faecal bacterial indicators, enteroviruses and oocysts of Toxoplasma gondii were assessed concurrently. Raw wastewaters were heavily contaminated by Cryptosporidium and Giardia (oo)cysts, whereas concentrations of both protozoa in treated wastewater were lower. Treated wastewater, flowed into Seine River, had a parasite concentration closed to the one found along the river, in particular at the entry of a drinking water plant (DWP). Even if faecal bacteria were reliable indicators of a reduction in parasite concentrations during the wastewater treatment, they were not correlated to protozoal contamination of wastewater and river water. Oocysts of T. gondii were not found in both raw and treated wastewater, or in Seine River. Parasitic contamination was shown to be constant in the Seine River up to 40 km upstream Paris. Altogether, these results strongly suggest that treated wastewater does not contribute to the main parasitic contamination of the Seine River usually observed in this urbanized area.     

Presence of Cryptosporidium spp. and Giardia duodenalis through drinking water

To evaluate the presence of Cryptosporidium spp. and Giardia duodenalis in the influent and final effluent of sixteen drinking water treatment plants located in a hydrographic basin in Galicia (NW Spain) - in which the principal river is recognised as a Site of Community Importance (SCI) - estimate the efficiency of treatment plants in removing these protozoans and determine the species and genotypes of the parasites by means of a molecular assay. All plant samples of influent and final effluent (50-100 l) were examined in the spring, summer, autumn and winter of 2007. A total of 128 samples were analysed by method 1623, developed by US Environmental Protection Agency for isolation and detection of both parasites. To identify the genotypes present the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and b-giardina (G. duodenalis). The mean concentrations of parasites in the influent were 0.0-10.5 Cryptosporidium spp. oocysts per litre and 1.0-12.8 of G. duodenalis cysts per litre. In the final treated effluent, the mean concentration of parasites ranged from 0.0-3.0 oocysts per litre and 0.5-4.0 cysts per litre. The distribution of results by season revealed that in all plants, the highest numbers of (oo)cysts were recorded in spring and summer. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. Cryptosporidium spp. and G. duodenalis were consistently found at high concentrations in drinking water destined for human and animal consumption in the hydrographic basin under study, in Galicia (NW Spain). It is important that drinking water treatment authorities rethink the relevance of contamination levels of both parasites in drinking water and develop adequate countermeasures.     

水中隐孢子虫和贾第鞭毛虫的替代检测指标

美国国家环保局（USEPA）制定并发布的1623方法是目前国际上常用的水中隐孢子虫和贾第鞭毛虫标准检测方法，该方法存在成本高、回收率低等缺陷。各国研究者试图通过考察其他水质指标与两虫的相关关系来寻找两虫密度和活性的有效替代指标，以间接反映两虫在水体中的存在水平，其中包括浊度、颗粒计数等物理学指标以及细菌、细菌孢子、病毒、噬菌体等生物学指标。虽然还未得出具有普遍性的结论，但研究表明颗粒计数、好氧孢子等有可能成为有效的两虫替代指标。     

Metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea

When evaluating metal accumulation patterns in parasite-host assemblages species specific metal requirements should be taken into account. The aim of the present study was therefore to determine the metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea and to determine the effect of the parasites on the metal balance of the fish. To this purpose the host-parasite assemblage was analysed for several metals at sites in the coastal zone of Lake Victoria differing in metal contamination. Our results showed that some elements (Ca, Sr, and Mg) reflected the physiological differences of bone formation and ionic balance and pointed to physiological disturbances of infested R. argentea. Other essential metals including Cu and Co were subject of element competition between fish and parasite, while only a micro-element (Cr) and a non-essential metal (Cd) displayed a partitioning with high concentration in the parasite. The present study clearly demonstrated the impact of the large cestodes on their small fish hosts and it is concluded that the partitioning of metals in the assemblage of R. argentea and L. intestinalis is subject to metal specific mechanisms for essential and non-essential elements.