Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and persistence of bacterial pathogens and indicator organisms in beach sand along the California coast

This report documents the presence of fecal indicators and bacterial pathogens in sand at 53 California marine beaches using both culture-dependent and -independent (PCR and quantitative PCR [QPCR]) methods. Fecal indicator bacteria were widespread in California beach sand, with Escherichia coli and enterococci detected at 68% and 94% of the beaches surveyed, respectively. Somatic coliphages and a Bacteroidales human-specific fecal marker were detected at 43% and 13% of the beaches, respectively. Dry sand samples from almost 30% of the beaches contained at least one of the following pathogens: Salmonella spp., Campylobacter spp., Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), which were detected at 15%, 13%, 14%, and 3% of tested beaches, respectively. Fecal indicators and pathogens were poorly correlated to one another and to land cover. Sands were dry at the time of collection, and those with relatively high moisture tended to have higher concentrations or a more frequent occurrence of both indicators and pathogens. Using culture-dependent assays, fecal indicators decayed faster than pathogens in microcosm experiments using unaltered beach sand seeded with sewage and assessed by culture-dependent assays. The following order of persistence was observed (listed from most to least persistent): Campylobacter > Salmonella > somatic coliphages > enterococci > E. coli > F(+) phages. In contrast, pathogens decayed faster than fecal indicators in culture-independent assays: enterococci > Bacteroidales human-specific marker > Salmonella > Campylobacter. Microcosm experiments demonstrated that both indicators and pathogens were mobilized by wetting with seawater. Decay rates measured by QPCR were lower than those measured with culture-dependent methods. Enterococcal persistence and possible growth were observed for wetted microcosms relative to unwetted controls.     

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2?kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E.?coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G.?duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.     

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

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

Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Correlation between bacterial indicators and bacteriophages in sewage and sludge

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.     

A study to assess the microbial contamination of Mya arenaria clams from the north shore of the St Lawrence River estuary, (Québec, Canada)

The aims of the present study were to assess the microbial quality of Mya arenaria clams from the north shore of the St. Lawrence River estuary and to validate various microbial indicator microorganisms of bivalve mollusks contamination. Clams were collected from nine sites, including four harvesting sites closed by virtue of the Canadian Shellfish Sanitation Program (CSSP). Six contamination indicators (fecal coliforms, somatic coliphages, F-specific coliphages, fecal streptococci, Clostridium perfringens, and Escherichia coli) and four pathogens (Campylobacter sp., Cryptosporidium parvum, Giardia sp., and Salmonella sp.) were identified in the clams. Indicators sensibility, specificity and predictive values with respect to the presence of pathogens were calculated. Pathogenic microorganisms detection frequency in clams was important (92%). Globally, pathogens tend to be less frequently detected in opened harvesting sites (p = 0.086). Although the assessed indicators were not perfect, when F-specific coliphages are associated with E. coli or fecal coliforms, a good sensibility (62%-64%) and good positive predictive value (88%) with respect to the investigated pathogens are obtained.     

Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges

A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1.1 x 10(5) pfu g-1) and the lowest being that of Bacteroides fragilis phages (110 pfu g-1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others.     

Enumeration of pathogenic bacteria from sewage sludge in Kuwait

Domestic sewage in Kuwait is mainly treated by an activated sludge process. Sewage sludges at different treatment stages and the digested sludge were studied for their content of pathogenic bacteria. Two Gram-positive cocci and eight Gram-negative bacilli were isolated and enumerated in the sewage sludges and in raw sewage. During March, Aeromonas hydrophila was the dominant bacterium isolated. Serratia liquefaciens in raw sewage and sludge-2, and Salmonella species in digested sludge were dominant during April. Enterobacter aerogenes was dominant in sewage samples in May. Various types of bacteria isolated were tested for their sensitivity to a wide range of antibiotics.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

Human enteropathogen load in activated sewage sludge and corresponding sewage sludge end products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Male-specific coliphages as indicators of thermal inactivation of pathogens in biosolids

Male-specific (F+) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F+ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F+ DNA and F+ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F+ RNA phages. Separate experiments on individual isolates at 53 degrees C confirmed the apparent heat resistance of group I F+ RNA coliphages as well as the susceptibility of group III F+ RNA coliphages. Although few F+ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F+ RNA phages. Hence, F+ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F+ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.     

Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.     

Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.     

The effect of sewage sludge treatment processes on oocysts of Cryptosporidium parvum

T.N. WHITMORE AND L.J. ROBERTSON. 1995. The effect of common sewage sludge treatment processes on oocysts of the coccidian protozoan Cryptosporidium was evaluated in laboratory simulations. The ability of primary sewage sedimentation to remove Cryptosporidium oocysts was found to be poor. Thermophilic (55°C) aerobic digestion and sludge pasteurization at the same temperature were found to be effective treatments to inactivate Cryptosporidium oocysts. Approximately 10% of the oocyst population were found to be viable after 18 d exposure to mesophilic (35°C) anaerobically digesting sludge. The viability of Cryptosporidium oocysts decreased within the range 20–40% in sludge-treated soil mesocosms over 30 d. The survival results obtained, however, indicated that oocysts would survive well beyond this period.     

The occurrence of intestinal parasites in swine slurry and their removal in activated sludge plants

Thirteen intensive pig farms and two activated sludge treatment plants for pig slurry in north-western Spain were studied from April 2005 to June 2006 in order to evaluate the presence of enteric pathogens (Cryptosporidium, Giardia and helminths) and the efficiency with which they were removed. These parasites were present on 53%, 7% and 38% of the farms studied, respectively, with concentrations of 10(4)-10(5) oocysts per litre (/L) for Cryptosporidium, 10(3)cysts/L for Giardia and 10(2)-10(3) eggs/L for helminths. The overall removal of parasites in the pig slurry treatment plants ranged from 86.7% to over 99.99%. The results revealed a constant reduction at each stage of the treatment system, with activated sludge processes being the most effective treatment in reducing pathogens in pig slurry, 78-81% for Cryptosporidium oocysts and over 99.9% for helminth eggs. A heat drying procedure for sludge removed 4.3 log units of Cryptosporidium oocysts, demonstrating the excellent effectiveness of this treatment for reducing pathogens in sludge intended to be applied to land.     

Human Enteropathogen Load in Activated Sewage Sludge and Corresponding Sewage Sludge End Products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Occurrence of airborne bacteria and pathogen indicators during land application of sewage sludge.

Glass impingers (AGI-30) were used at a commercial sludge application site to determine the levels of airborne bacteria and pathogen indicators. Even though heterotrophic bacteria averaged 10(5) CFU/m3, none of the sites showed the presence of Salmonella spp. or indicators such as fecal coliforms or coliphages. Indicators such as H2S producers and pathogenic clostridia were present in locations having significant physical agitation of the sludge material. PCR-based ribotyping using the 16S-23S interspacer region is a promising method to identify the genetic relatedness and origins of airborne clostridia.     

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).