Multilevel analysis of childhood nonviral gastroenteritis associated with environmental risk factors in Quebec, 1999-2006.

Childhood nonviral gastroenteritis is a priority for various public health authorities. Given that waterborne transmission is sometimes incriminated during investigation of gastroenteritis outbreaks, the authors hypothesized that watershed characteristics may influence the occurrence of this disease and could contribute additional insights for better prevention and control. The study described here aimed to investigate watershed characteristics in relation to nonviral gastroenteritis and specifically three bacterial and parasitic forms of childhood gastroenteritis to assess their relative importance in the province of Quebec, Canada. Information on children aged 0-4 years with bacterial or parasitic enteric infections reported through ongoing surveillance between 1999 and 2006 in the province of Quebec was collected. Factors measured at the municipal and watershed levels were analyzed using multilevel models with a Poisson distribution and log link function. Childhood nonviral gastroenteritis, giardiasis, and campylobacteriosis were positively associated with small ruminants and cattle density. Childhood salmonellosis was positively associated with cattle density. Also, childhood campylobacteriosis incidence was positively associated with larger watershed agricultural surface. In addition to local agroenvironmental factors, this analysis revealed an important watershed effect.

Removal of human enteric viruses and indicator microorganisms from domestic wastewater by aerated lagoons.

Aerated lagoons offer a low-cost and simple approach to treating domestic wastewater in small municipalities. The objective of the current study was to evaluate, for each cell in the lagoons, the removal of indicator microorganisms and human enteric viruses under warm (summer) and cold (early spring) conditions. The two sites are located in southwest Quebec, Canada. Samples were assayed for thermotolerant coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, and culturable human enteric viruses (HEV). The results show higher removal under warm ambient conditions for all microorganisms. Thermotolerant coliforms and enterococci were removed to a greater extent than C. perfringens and HEV. HEV removal was only observed in warm ambient conditions. The removal of coliphages was different from the observed removal of HEV. The use of coliphages as surrogates for HEV has been proposed, but this does not seem appropriate for aerated lagoons, as the removal of coliphages overestimates the removal of HEV. Given the low observed removal of HEV during this study, the effluents remain a significant source of pathogens that can affect drinking water treatment plants drawing their raw water from receiving streams. Ultraviolet disinfection of treated wastewater effluent is a possible solution.

Universal mitochondrial PCR combined with species-specific dot-blot assay as a source-tracking method of human, bovine, chicken, ovine, and porcine in fecal-contaminated surface water.

Mitochondrial (mt) DNA has the potential to be used as an animal-specific genetic marker for source-tracking of fecal contamination in surface waters and groundwaters. In this report, we describe the development of a method to detect in a single assay human, bovine, ovine, porcine and chicken mitochondrial (mt) DNA in water. Consensus nucleic sequences were found between human, bovine, porcine, ovine and chicken mtDNA to design three sets of PCR universal primers. Upon polymerase chain reaction (PCR) with a digoxigenin-labeled nucleotide and the universal primers, species determination was carried out by dot-blotting membranes containing specific oligonucleotides for these five animals. Our method was carried out with three river samples and three wastewater samples, and the results were compared with those obtained by multiple nested PCR with specific primers for these five species. Our results showed that the dot-blot assays were as specific and sensitive as the nested-PCR approach. The proposed method has the advantage that it requires the use of only one PCR per sample and very little amounts of DNA. Finally, it is an alternative to multiplex PCR approach which is less sensitive, and shows the way for the development of DNA arrays for source-tracking of many more animal species in fecal-contaminated water.

Risk of diarrhea with adult residents of municipalities with significant livestock production activities.

BACKGROUND: The intensification of livestock production has led to situations where the amount of manure that is produced exceeds the amounts needed in some areas. The objective of this study was to evaluate the relationship between the intensity of livestock activities and manure products, particularly in swine farms, and the prevalence of diarrhea in adults. METHODS: A survey was carried out on 8702 adults living in 161 municipalities in Quebec areas with intensive farming activities. Data were collected by a telephonic interview on diarrheal symptoms that occurred during the previous week of the interview, on water consumption and on selected risk factors. Statistical analysis was performed using a 'generalized estimating equations' model. RESULTS: Prevalence of diarrhea was found to be highest in adults aged between 25 and 34 years. No association was found between swine density or liquid manure application and diarrheal prevalence. There was also no association between cattle or total animal density and diarrheal prevalence. In the areas studied, there was no increase in risk associated with the consumption of tap water with suboptimal treatment and susceptible to microbiologic contamination. CONCLUSION: Significant livestock production and excess of manure were not associated with the risk of diarrhea in adults.

Impact of microparticles on UV disinfection of indigenous aerobic spores.

Numerous studies have shown that the efficacy of ultraviolet (UV) disinfection can be hindered by the presence of particles that can shield microorganisms. The main objective of this study was to determine to what extent natural particulate matter can shield indigenous spores of aerobic spore-forming bacteria (ASFB) from UV rays. The extent of the protective shielding was assessed by comparing the inactivation rates in three water fractions (untreated, dispersed and filtered on an 8 microm membrane) using a collimated beam apparatus with a low-pressure lamp emitting at 254 nm. Levels of inactivation were then related to the distribution and abundance of particles as measured by microflow imaging. Disinfection assays were completed on two source waters of different quality and particle content. A protocol was developed to break down particles and disperse aggregates (addition of 100mg/L of Zwittergent 3-12 and blending at 8000 rpm for 4 min). Particle size distribution (PSD) analysis confirmed a statistically significant decrease in the number of particles for diameter ranges above 5 microm following the dispersion protocol and 8 microm filtration. The fluence required to reach 1-log inactivation of ASFB spores was independent of particle concentration, while that required to reach 2-log inactivation or more was correlated with the concentration of particles larger than 8 microm (R(2)>0.61). Results suggest that natural particulate matter can protect indigenous organisms from UV radiation in waters with elevated particle content, while source water with low particle counts may not be subject to this interference.

Pathogen and indicator variability in a heavily impacted watershed.

Water samples were collected from 36 locations within the Grand River Watershed, in Southwestern Ontario, Canada from July 2002 to December 2003 and were analyzed for total coliforms, fecal coliforms, Escherichia coli, Escherichia coli O157:H7, and thermophilic Campylobacter spp. A subset of samples was also analyzed for Cryptosporidium spp., Giardia spp., culturable human enteric viruses, and Clostridium perfringens. Storm and snowmelt events were sampled at two locations including a drinking water intake. For the majority of the events, the Spearman rank correlation test showed a positive correlation between E. coli levels and turbidity. Peaks in pathogen numbers frequently preceded the peaks in numbers of indicator organisms and turbidity. Pathogen levels sometimes decreased to undetectable levels during an event. As pathogen peaks did not correspond to turbidity and indicator peaks, the correlations were weak. Weak correlations may be the result of differences in the sources of the pathogens, rather than differences in pathogen movement through the environment. Results from this investigation have implications for planning monitoring programs for water quality and for the development of pathogen fate and transport models to be used for source water risk assessment.

Use of eukaryotic mitochondrial DNA to differentiate human, bovine, porcine and ovine sources in fecally contaminated surface water.

A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.

An environmental survey of surface waters using mitochondrial DNA from human, bovine and porcine origin as fecal source tracking markers.

Fecal contamination of surface waters is one the major sources of waterborne pathogens and consequently, is an important concern for public health. For reliable fecal source tracking (FST) monitoring, there is a need for a multi-marker toolbox as no single all-encompassing method currently exists. Mitochondrial DNA (mtDNA) as a source tracking marker has emerged as a promising animal-specific marker. However, very few comprehensive field studies were done on the occurrence of this marker in surface waters. In this report, water samples were obtained from 82 sites in different watersheds over a six year period. The samples were analyzed for the presence of human, bovine and porcine mtDNA by endpoint nested PCR, along with the human-specific Bacteroidales HF183 marker. These sites represented a mix of areas with different anthropogenic activities, natural, urban and agricultural. The occurrences of mitoHu (human), mitoBo (bovine), mitoPo (porcine) and HF183 specific PCR amplifications from the samples were 46%, 23%, 6% and 50%, respectively. The occurrence of mitoHu and HF183 was high in all environment types with higher occurrence in the natural and urban areas, whereas the occurrence of mitoBo was higher in agricultural areas. FST marker concentrations were measured by real-time PCR for samples positive for these markers. The concentration of the mitoHu markers was one order of magnitude lower than HF183. There was co-linearity between the concentrations of the mitoHu and HF183 markers. Co-linearity was also observed between HF183 concentration and fecal coliform levels. Such a relationship was not observed between the mitoHu concentration and fecal coliform levels. In summary, our results showed a high incidence of human fecal pollution throughout the environment while demonstrating the potential of mtDNA as suitable FST markers.

Rare occurrence of heterotrophic bacteria with pathogenic potential in potable water.

Since the discovery of Legionella pneumophila, an opportunistic pathogen that is indigenous to water, microbiologists have speculated that there may be other opportunistic pathogens among the numerous heterotrophic bacteria found in potable water. The US Environmental Protection Agency (USEPA) developed a series of rapid in vitro assays to assess the virulence potential of large numbers of bacteria from potable water to possibly identify currently unknown pathogens. Results of surveys of potable water from several distribution systems using these tests showed that only 50 of the approximately 10,000 bacterial colonies expressed one or more virulence characteristics. In another study, 45 potable water isolates that expressed multiple virulence factors were tested for pathogenicity in immunocompromised mice. None of the isolates infected mice that were compromised either by treatment with carrageenan (CG), to induce susceptibility to facultative intracellular pathogens, or by cyclophosphamide (CY), to induce susceptibility to extracellular pathogens. These results indicate that there are very few potential pathogens in potable water and that the currently developed in vitro virulence screening tests give an overestimation of the numbers of heterotrophic bacteria that may be pathogens. Current efforts are focused on using the animal models to screen concentrated samples of waters known to contain large numbers of heterotrophic bacteria and newly discovered Legionella-like organisms that parasitize amoebae.

Disinfection efficiency of peracetic acid, UV and ozone after enhanced primary treatment of municipal wastewater.

The City of Montreal Wastewater Treatment Plant uses enhanced physicochemical processes (ferric and/or alum coagulation) for suspended solids and phosphorus removal. The objective of this study was to assess the ability of peracetic acid (PAA), UV, or ozone to inactivate the indicator organisms fecal coliforms, Enterococci, MS-2 coliphage, or Clostridium perfringens in the effluent from this plant. PAA doses to reach the target fecal coliform level of 9000 CFU/100mL exceeded 6 mg/L; similar results were obtained for enterococci, and no inactivation of Clostridium perfringens was observed. However a 1-log reduction of MS-2 occurred at PAA doses of 1.5 mg/L and higher. It was expected that this effluent would have a high ozone demand, and would require relatively high UV fluences, because of relatively high effluent COD, iron and suspended solids concentrations, and low UV transmittance. This was confirmed herein. For UV, the inactivation curve for fecal coliforms showed the typical two-stage shape, with the target of 1000 CFU/100 mL (to account for photoreactivation) occurring in the asymptote zone at fluences >20 mJ/cm(2). In contrast, inactivation curves for MS-2 and Clostridium perfringens were linear. Clostridium perfringens was the most resistant organism. For ozone, inactivation was already observed before any residuals could be measured. The transferred ozone doses to reach target fecal coliform levels ( approximately 2-log reduction) were 30-50 mg/L. MS-2 was less resistant, but Clostridium perfringens was more resistant than fecal coliforms. The different behaviour of the four indicator organisms studied, depending on the disinfectant, suggests that a single indicator organism might not be appropriate. The required dose of any of the disinfectants is unlikely to be economically viable, and upstream changes to the plant will be needed.

Changes in Escherichia coli to Cryptosporidium ratios for various fecal pollution sources and drinking water intakes.

Assessing the presence of human pathogenic Cryptosporidium oocysts in surface water remains a significant water treatment and public health challenge. Most drinking water suppliers rely on fecal indicators, such as the well-established Escherichia coli (E. coli), to avoid costly Cryptosporidium assays. However, the use of E. coli has significant limitations in predicting the concentration, the removal and the transport of Cryptosporidium. This study presents a meta-analysis of E. coli to Cryptosporidium concentration paired ratios to compare their complex relationships in eight municipal wastewater sources, five agricultural fecal pollution sources and at 13 drinking water intakes (DWI) to a risk threshold based on US Environmental Protection Agency (USEPA) regulations. Ratios lower than the USEPA risk threshold suggested higher concentrations of oocysts in relation to E. coli concentrations, revealing an underestimed risk for Cryptosporidium based on E. coli measurements. In raw sewage (RS), high ratios proved E. coli (or fecal coliforms) concentrations were a conservative indicator of Cryptosporidium concentrations, which was also typically true for secondary treated wastewater (TWW). Removals of fecal indicator bacteria (FIB) and parasites were quantified in WWTPs and their differences are put forward as a plausible explanation of the sporadic ratio shift. Ratios measured from agricultural runoff surface water were typically lower than the USEPA risk threshold and within the range of risk misinterpretation. Indeed, heavy precipitation events in the agricultural watershed led to high oocyst concentrations but not to E. coli or enterococci concentrations. More importantly, ratios established in variously impacted DWI from 13 Canadian drinking water plants were found to be related to dominant fecal pollution sources, namely municipal sewage. In most cases, when DWIs were mainly influenced by municipal sewage, E. coli or fecal coliforms concentrations agreed with Cryptosporidium concentrations as estimated by the meta-analysis, but when DWIs were influenced by agricultural runoff or wildlife, there was a poor relationship. Average recovery values were available for 6 out of 22 Cryptosporidium concentration data sets and concomitant analysis demonstrated no changes in trends, with and without correction. Nevertheless, recovery assays performed along with every oocyst count would have enhanced the precision of this work. Based on our findings, the use of annual averages of E. coli concentrations as a surrogate for Cryptosporidium concentrations can result in an inaccurate estimate of the Cryptosporidium risk for agriculture impacted drinking water intakes or for intakes with more distant wastewater sources. Studies of upstream fecal pollution sources are recommended for drinking water suppliers to improve their interpretation of source water quality data.

Fecal source tracking in water using a mitochondrial DNA microarray.

A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

Fecal coliforms, caffeine and carbamazepine in stormwater collection systems in a large urban area.

Water samples from streams, brooks and storm sewer outfall pipes that collect storm waters across the Island of Montréal were analyzed for caffeine, carbamazepine and fecal coliforms. All samples contained various concentrations of these tracers, indicating a widespread sanitary contamination in urban environments. Fecal coliforms and caffeine levels ranged over several orders of magnitude with a modest correlation between caffeine and fecal coliforms (R(2) value of 0.558). An arbitrary threshold of 400 ng caffeine L(-1) allows us to identify samples with an elevated fecal contamination, as defined by more than 200 colony-forming units per 100 mL (cfu 100 mL(-1)) of fecal coliforms. Low caffeine levels were sporadically related to high fecal coliform counts. Lower levels of caffeine and fecal coliforms were observed in the brooks while the larger streams and storm water discharge points contained over ten times more. The carbamazepine data showed little or no apparent correlation to caffeine. These data suggest that this storm water collection system, located in a highly urbanized urban environment, is widely contaminated by domestic sewers as indicated by the ubiquitous presence of fecal contaminants as well as caffeine and carbamazepine. Caffeine concentrations were relatively well correlated to fecal coliforms, and could potentially be used as a chemical indicator of the level of contamination by sanitary sources. The carbamazepine data was not significantly correlated to fecal coliforms and of little use in this dataset.

Pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples.

Practical pre-analytical and analytical procedures were developed and validated for detection of enteric viruses in three water matrices. Both RNA viruses (norovirus, coxsackievirus, echovirus, and rotavirus) and DNA virus (adenovirus 41) were included in the study. The NanoCeram 90mm laminated disc with electropositive filter and procedures of filtration, elution and flocculation were utilized to concentrate known amount of viruses in different water matrices. Real time quantitative PCR was used to evaluate the recovery of virus and cell culture to assess viral infectivity. There was no PCR inhibition using various concentrations and pH of beef extract eluting buffer. A good recovery of the viruses spiked in 10L of deionized water was achieved for serial dilutions of coxsackievirus (41-67%), echovirus (22-90%), norovirus (23-44%) and rotavirus (24-46%). Relatively lower recovery was observed for adenovirus 41 (24-35%). There was no significant difference in viral recovery from deionized, tap and river water samples. The infectivity of recovered adenovirus, coxsackievirus and echovirus was demonstrated using in vitro cell culture. The pre-analytical and analytic procedures attained consistent recovery of RNA and DNA viruses both as infectious viral particles and viral genome, provided effective removal of inhibitory substances, achieved reliable reproducibility, and were relatively inexpensive for monitoring viruses in water.

Pathogens in water: value and limits of correlation with microbial indicators.

This article discusses the value and limitations of using microbial indicators to predict occurrence of enteric pathogens in water. Raw or treated sewage is a primary source of fecal contamination of the receiving surface water or groundwater; hence, understanding the relationship between pathogens and indicators in sewage is an important step in understanding the correlation in receiving waters. This article presents three different datasets representing different concentrations of pathogens and microbial indicators: sewage containing high concentrations of pathogens and indicators, surface water with variable concentrations, and groundwater with low concentrations. In sewage, even with very high levels of microorganisms, no mathematical correlation can predict the type or concentration of any pathogen. After discharge in the environment, direct correlation becomes biologically improbable as dilution, transport, and different inactivation rates occur in various environments. In surface waters, advanced statistical methods such as logistic regression have provided some level of predictability of the occurrence of pathogens but not specific counts. In groundwater, the continuous absence of indicators indicates an improbable occurrence of pathogen. In contrast, when these indicators are detected, pathogen occurrence probability increases significantly. In groundwater, given the nature and dissemination pattern of pathogenic microorganisms, a direct correlation with fecal microbial indicators is not observed and should not be expected. However, the indicators are still useful as a measure of risk. In summary, many pathogens of public health importance do not behave like fecal microbial indicators, and there is still no absolute indicator of their presence, only a probability of their co-occurrence.

A descriptive review of selected nonviral enteric illnesses reported in children in Quebec between 1999 and 2006.

OBJECTIVE: To review the epidemiology of selected nonviral enteric illnesses reported in children in Quebec between 1999 and 2006. METHODS: Incidence rates were calculated to describe age, sex, temporal and geographical characteristics of the selected nonviral enteric cases reported in children who were between zero and four years of age. Standard descriptive methods were used to analyze the temporal and geographical distributions of the incidence rates. RESULTS: A total of 5068 cases were reported. Of these, three pathogens accounted for the majority of the infections: Giardia (32.52%), Salmonella (30.98%) and Campylobacter (30.82%). Salmonella was most frequent in children younger than one year of age, whereas comparable incidence rates for the three pathogens were calculated for children between one and four years of age. For Giardia, the geographical distributions showed that the highest rates were in areas with more than 100,000 inhabitants (except Montreal, Quebec); for Salmonella, the highest rates were in Montreal; and for Campylobacter, the highest rates were in areas with fewer than 10,000 inhabitants. No detectable trends were seen over the study period for the three pathogens. Seasonal summer peaks were noted for Salmonella and Campylobacter, contrasting with late summer to early autumn peaks for Giardia. CONCLUSION: Findings suggest that Giardia, Salmonella and Campylobacter were the most common causes of nonviral enteric illnesses reported in children in Quebec. Giardia cases seemed to arise from different sources and transmission routes than the other two pathogens. Characteristics specific to Campylobacter infections in children, namely its predominance in areas with low population densities, and to Salmonella infections, namely predominance in the Greater Montreal area, should be further investigated to better guide prevention and control measures.

Pressure monitoring and characterization of external sources of contamination at the site of the payment drinking water epidemiological studies.

The 1990s epidemiological studies by Payment and colleagues suggested that an increase in gastrointestinal illnesses observed in the population consuming tap water from a system meeting all water quality regulations might be associated with distribution system deficiencies. In the current study, the vulnerability of this distribution system to microbial intrusion was assessed by characterizing potential sources of contamination near pipelines and monitoring the frequency and magnitude of negative pressures. Bacterial indicators of fecal contamination were recovered more frequently in the water from flooded air-valve vaults than in the soil or water from pipe trenches. The level of fecal contamination in these various sources was more similar to levels from river water rather than wastewater. Because of its configuration, this distribution system is vulnerable to negative pressures when pressure values out of the treatment plant reach or drop below 172 kPa (25 psi), which occurred nine times during a monitoring period of 17 months. The results from this investigation suggest that this distribution system is vulnerable to contamination by intrusion. Comparison of the frequency of occurrence of negative pressure events and repair rates with data from other distribution systems suggests that the system studied by Payment and colleagues is not atypical.

Protection against UV disinfection of E. coli bacteria and B. subtilis spores ingested by C. elegans nematodes.

Nematodes, which occur abundantly in granular media filters of drinking water treatment plants and in distribution systems, can ingest and transport pathogenic bacteria and provide them protection against chemical disinfectants. However, protection against UV disinfection had not been investigated to date. In this study, Caenorhabditis elegans nematodes (wild-type strain N2) were allowed to feed on Escherichia coli OP50 and Bacillus subtilis spores before being exposed to 5 and 40 mJ/cm(2) UV fluences, using a collimated beam apparatus (LP, 254 nm). Sonication (15 W, 60s) was used to extract bacteria from nematode guts following UV exposure in order to assess the amount of ingested bacteria that resisted the UV treatment using a standard culture method. Bacteria located inside the gut of C. elegans were shown to benefit from a significant protection against UV. Approximately 15% of the applied UV fluence of 40 mJ/cm(2) (as typically used in WTP) was found to reach the bacteria located inside nematode guts based on the inactivation of recovered bacteria (2.7 log reduction of E. coli bacteria and 0.7 log reduction of B. subtilis spores at 40 mJ/cm(2)). To our knowledge, this study is the first demonstration of the protection effect of bacterial internalization by higher organisms against UV treatment, using the specific case of E. coli and B. subtilis spores ingested by C. elegans.

Protection of waterborne pathogens by higher organisms in drinking water: a review.

Higher organisms are ubiquitous in surface waters, and some species can proliferate in granular filters of water treatment plants and colonize distribution systems. Meanwhile, some waterborne pathogens are known to maintain viability inside amoebae or nematodes. The well-documented case of Legionella replication within amoebae is only one example of a bacterial pathogen that can be amplified inside the vacuoles of protozoa and then benefit from the protection of a resistant structure that favours its transport and persistence through water systems. Yet the role of most zooplankton organisms (rotifers, copepods, cladocerans) in pathogen transmission through drinking water remains poorly understood, since their capacity to digest waterborne pathogens has not been well characterized to date. This review aims at (i) evaluating the scientific observations of diverse associations between superior organisms and pathogenic microorganisms in a drinking water perspective and (ii) identifying the missing data that impede the establishment of cause-and-effect relationships that would permit a better appreciation of the sanitary risk arising from such associations. Additional studies are needed to (i) document the occurrence of invertebrate-associated pathogens in relevant field conditions, such as distribution systems; (ii) assess the fate of microorganisms ingested by higher organisms in terms of viability and (or) infectivity; and (iii) study the impact of internalization by zooplankton on pathogen resistance to water disinfection processes, including advanced treatments such as UV disinfection.