F-specific Bacteriophages Research Articles

Simultaneous Concentration of Enteric Viruses and Protozoan Parasites: A Protocol Based on Tangential Flow Filtration and Adapted to Large Volumes of Surface and Drinking Waters

A filtration system, based on tangential flow filtration (TFF) followed by ultracentrifugation was developed in order to concentrate simultaneously viruses and parasites from large volumes of water. For TFF, no pre-treatment of the membrane is performed but a post-rinsing step using high pH-beef extract-based eluant. Applying our protocol to 20 l of surface waters spiked with vaccinal poliovirus-1, ϕX174 and MS2 bacteriophages resulted in an averaged viral recovery of 75% by TFF and 91% by ultracentrifugation (total viral recovery of 70%). Our protocol was further applied to 31 environmental samples including surface, ground and drinking waters from the Grand Duchy of Luxembourg in order to assess the occurrence of protozoan parasites (Cryptosporidium parvum and Giardia lamblia (oo)cysts), pathogenic viruses (enterovirus, norovirus and adenovirus) and infectious bacteriophages (somatic coliphages and F-specific phages) in these samples. High viral recovery rates of > 70% were confirmed concentrating environmental strains of somatic and F-specific coliphages from non-spiked surface waters. Parasites and enteric viruses were detected in 86 and 40% of the surface waters used for drinking water production, respectively. Infectious bacteriophages were isolated from all surface waters and in two out of seven (29%) groundwaters revealing a susceptibility of the corresponding wells to viral pollution. TFF-based method proved to be efficient for surveying the occurrence of non-bacterial pathogens such as enteric viruses and protozoan parasites in large volumes of environmental waters.

下水処理場における病原ウイルスの低減効果の解明

The occurrence of pathogenic viruses and indicator microorganisms in water samples from a wastewater treatment plant in Japan was investigated monthly from July 2003 to June 2004. Forty-eight samples of raw sewage, treated sewage before chlorination, effluent after chlorination, and reclaimed water after advanced wastewater treatment using sand filtration and ozonation were subjected to a virus concentration method using an HA electronegative membrane, followed by virus detection using a TaqMan PCR method. Noroviruses of genogroups I and II were detected in all 12 raw sewage samples, showing much higher concentrations in winter, an epidemic season. Corresponding to the occurrence in raw sewage, the concentrations of noroviruses in treated sewage and effluent also increased in winter. The concentrations of enteroviruses and adenoviruses in the tested samples were relatively constant compared with those of noroviruses. Noroviruses and adenoviruses were still detected even after advanced wastewater treatment, but the concentrations of these viruses were much lower than those in the effluent samples. Viruses were removed by wastewater treatment as effectively as indicator microorganisms (total coliforms, Escherichia coli, and F-specific phages), suggesting that sewerage systems can contribute to reducing the load of pathogenic viruses discharged into aquatic environments.

Comparative reductions of bacterial indicators, bacteriophage-infecting enteric bacteria and enteroviruses in wastewater tertiary treatments by lagooning and UV-radiation

A two-year monitoring program of microbiological and physical-chemical parameters at 2 waste water treatment plants (WWTPs) in Mallorca (Spain) was performed in order to (1) evaluate the efficiency of lagooning and UV radiation as tertiary treatment processes; (2) determine the characteristics of wastewater effluent for its potential agricultural reuse; and (3) establish correlations between bacteriological and virological parameters. The presence of currently established bacterial indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci, and spores of sulphite-reducing clostridia), virological (enteroviruses, somatic coliphages, F-specific coliphages, and phages infecting Bacteroides fragilis and Bacteroides thetaiotaomicron), and helminth eggs were tested during this study. Bacterial and viral indicators were removed at least with one log reduction in the lagooning system, and to a lesser extent with UV-radiation treatment. The lagooning system was less efficient in removing phages and viruses than were bacterial indicators, with the exception of F-specific phages. Phages of B. fragilis and B. thetaiotaomicron were less removed than all of the other microbiological parameters. In the UV-radiation treatment, however, the faecal coliforms proved the most sensitive, while clostridial spores, somatic coliphages, Bacteroides phages, and enteric viruses were the more resistant. Helminth eggs were not detected in any samples from effluents of either the secondary or tertiary treatments.Indicator levels in both treatments met the established regulations of both local and national authorities for the disposal or reuse of wastewater in irrigation for non-human crop. We demonstrate that somatic coliphages are effective indicators of enteric viruses in both of the WWTPs studied.

The use of Armored RNA as a multi-purpose internal control for RT-PCR

Real time reverse transcriptase-PCR (RT-PCR) is now used commonly for the detection of viral pathogens in respiratory samples. However, due to potential inhibition of the RT-PCR or inefficient extraction, this sample type can present significant challenges to accurate patient testing. The goal of this study was to create an internal control to be multiplexed in a real time RT-PCR assay for detecting a viral target in respiratory samples. This report describes an Armored RNA (aRNA) internal control developed originally to be multiplexed in a real time RT-PCR assay for detecting SARS-associated Coronavirus, but can be incorporated into any RT-PCR assay. The internal control primers and probe target a region in the coat protein gene of the E. coli F-specific bacteriophage ms2, which is contained within the aRNA.

Aggregation and surface properties of F-specific RNA phages: Implication for membrane filtration processes

We report an experimental investigation of the electrokinetic properties and size variations of four F-specific bacteriophages of the types MS2, GA, Qβ and SP (21–30nm in diameter) over a broad range of pH values (1.5–7.5) and NaNO3 electrolyte concentrations (1–100mM). The results obtained by dynamic light scattering show that the aggregation of SP and GA particles takes place over the whole range of pH and ionic strength conditions examined. For MS2 phages, the aggregation of MS2 particles is not observed for pH higher than the isoelectric point (pI) and large ionic strengths for which interparticular repulsive electrostatic interactions are however expected to be sufficiently screened. Aggregation of the MS2 phages, dispersed in 1 and 100mM electrolyte concentration, occurs at pH 4, which basically corresponds to the pI as determined by electrophoresis measurements. The Qβ particles suspended in solutions of low electrolyte concentrations aggregate at low pH values (pI∼3) and, unlike MS2, at large ionic strengths over the whole range of pH conditions considered in this study. These elements allow the determination of the hydrophobic sequence for the four phages SP∼GA>Qβ>MS2. Close inspection of the electrokinetic results reveals small to significant variations of the pI values—depending on the phage considered—with respect to the concentration of indifferent NaNO3 electrolyte. This indicates that features other than chemical and electrostatic in nature play a key role in determining the pI and more generally the electrophoretic mobility μ of viral particles. A qualitative interpretation is given and is based on the consideration of inner electro-osmotic flow within the isolated or aggregated particles. The impact of the flow properties within the particles is further in agreement with recent theoretical formalism developed for the electrokinetics of soft multiplayer particles, the phages analyzed here being some illustrative examples. The determination and qualitative interpretation of the surface properties of the viral particles as reported in the current study are commented within the context of water treatment especially concerning viral removal by membrane filtration processes.

Evaluation of Escherichia coli Host Strain CB390 for Simultaneous Detection of Somatic and F-Specific Coliphages

Escherichia coli WG5, the strain recommended by the International Organization for Standardization (ISO) to detect somatic coliphages, was transformed to F(+) by introducing the plasmid Famp, which rendered it capable of simultaneously detecting both somatic and F-specific coliphages. Indeed, this strain, CB390, proved as effective in detecting similar numbers of phages as the sum of somatic and F-specific bacteriophages detected by the host strains recommended by both the ISO and the U.S. Environmental Protection Agency standardized methods.

Occurrence and persistence of bacterial and viral faecal indicators in wastewater biofilms

Biofilms within wastewater treatment plants can capture enteric microorganisms initially present in the water phase immobilising them either definitively or temporarily. Consequently, fates of microorganisms may totally change depending on whether they interact or not with biofilms. In this study, we assessed the stability of wastewater biofilms comparing the evolution of the concentrations of bacteria (heterotrophic plate count [HPC], thermotolerant coliforms [TC]) and viral (somatic coliphages [SC] and F-specific phages [F +]) indicators in the biofilms and in the corresponding wastewaters at 4 and 20 dgrees C. Additionally, we assessed the monthly occurrence of these bacterial and viral indicators as well as of pathogenic protozoa (Cryptosporidium oocysts and Giardia cysts) in three native wastewater biofilms for four months. Our results show that viral indicators (SC and F + ) persist longer in biofilms than in the corresponding wastewaters at 4 degrees C as well as at 20 degrees C. In contrast, persistence of bacterial indicators (TC and HPC) depends on both the temperature and the matrix. Differences between viral and bacterial persistence are discussed. Monthly analysis of native wastewater biofilms shows that bacterial and viral indicators, as well as Cryptosporidium oocysts and Giardia cysts, attach to wastewater biofilms to a concentration that remains stable in time, probably as a result of a dynamic equilibrium between attachment and detachment processes.

Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan

The seasonal profiles of microorganisms in raw sewage, secondary-treated sewage, and final effluent at a wastewater treatment plant in Tokyo, Japan, were quantitatively determined each month for one year, from July 2003 to June 2004. Human noroviruses, which were determined by real-time PCR, in raw sewage varied from 0.17-260 copies/mL for genotype 1 and from 2.4-1900 copies/mL for genotype 2, showing much higher values in winter, the epidemic season. The concentration of total coliforms, Escherichia coli, or F-specific phages in raw sewage was almost constant throughout the year. Human noroviruses of genotype 2 were removed most effectively (3.69 log10 on average) at the wastewater treatment plant, followed by E. coli (3.37 log10), total coliforms (3.05 loglo), F-specific phages (2.81 log10), and human noroviruses of genotype 1 (2.27 log10). The removal ratio of human noroviruses was almost constant, independent of the initial concentration of the viruses in raw sewage, which led to the increasing concentration of human noroviruses in final effluent in winter. None of the tested bacteria was judged to be a reliable indicator of human noroviruses in final effluent.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Removal of Noro‐ and Enteroviruses, Giardia Cysts, Cryptosporidium Oocysts, and Fecal Indicators at Four Secondary Wastewater Treatment Plants in Sweden

The aim of this study was to investigate variation in the occurrence and removal of enteroviruses, noroviruses, Giardia cysts, Cryptosporidium oocysts, and the most commonly used fecal indicators in four Swedish secondary wastewater treatment plants (WWTPs). Paired samples were taken from the inlet and outlet of each WWTP. (Oo)cysts and indicators were enumerated with standard methods and viruses with a reverse transcriptase polymerase chain reaction. Giardia cysts and enteroviruses were constantly detected (mean numbers were 10(3.31) cysts and 10(4.44) polymerase chain reaction (PCR) units L(-1), respectively). Oocysts were found in 5 out of 19 samples (mean number was 20 L(-1)). Noroviruses were found between November and February, with an average titer of 10(3.29( L(-1). Mean cyst removal was 2.6 log, while noroviruses and enteroviruses were removed by 0.9 and 1.3 log, respectively. There was no correlation between the removals of pathogens and indicators (p > 0.05). Coliphage removal resembled human viral removal better than did F-specific phage.

Enteric viruses in inlet and outlet samples from sewage treatment plants

Samples collected every two weeks from the inlet and outlet of three sewage treatment plants were screened for the presence of noro-, rota-, astro-, adeno-, hepatitis A- and circoviruses by (RT)-nested PCR, and for F-specific bacteriophages by isolation in Escherichia coli Famp. Plants A and B were secondary treatment plants and plant C used primary treatment. Noroviruses were detected in 43%, 53% and 24% of the inlet samples and 26%, 40% and 21% of the outlet samples from plants A, B and C, respectively. Astroviruses, rotaviruses and adenoviruses were more prevalent. Adenoviruses were detected in 96% of inlet and 94% of outlet samples, supporting the potential of these viruses as indicators of viral contamination from sewage. Hepatitis A virus and circoviruses were found only rarely. Reduction of infective viral particles during sewage treatment was evaluated using F-specific bacteriophages. The phages were reduced by, respectively, 99%, 87% and 0% in plants A, B and C, which corresponded to the observed differences in reduction of norovirus positive samples between the same plants. The study shows that the high viral load in sewage results in a discharge to the environment of a large amount of virus despite sewage treatment. On the other hand, the advantage of a more advanced treatment is demonstrated.

Sanitary quality of sands from marine recreational beaches of São Paulo, Brazil

A sanitary evaluation of sand and water from 16 beaches of Sao Paulo State, Brazil, was undertaken during spring of 1997 and summer of 1998. Ninety six samples each of wet and dry sand and seawater were collected and analysed for fecal indicator bacteria. A parasitological examination and Candida albicans analysis were also performed in sand samples and F-specific bacteriophages were determined in seawater. Statistical analysis of the results demonstrated higher concentrations of fecal coliforms and fecal streptococci in dry sand during summer. Correlation analysis indicated a significant relationship between fecal indicator densities in wet sand and seawater. There was a significant correlation between the densities of fecal coliforms and fecal streptococci for both types of sand, and this correlation was higher in wet sand. Cysts and eggs of parasites were detected in 4.2% of the samples and Candida albicans was isolated in 18% of the samples. The high concentrations of fecal indicators detected in sand during summer demonstrate that there is a health risk to the users of these recreational areas and suggest the necessity of some criteria for microbiological control. Preventive measures, such as education campaings and some management actions are important precautionary measures.

Year-Round Screening of Noncommercial and Commercial Oysters for the Presence of Human Pathogenic Viruses

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In The Netherlands, oysters are cultured and imported both for consumption and export; therefore, the presence of noroviruses, rotaviruses, astroviruses, hepatitis A viruses, and enteroviruses was determined in 64 commercial and noncommercial oyster samples. Oysters were collected monthly for 13 months from four different harvesting areas in the Oosterschelde Delta. Oyster samples were classified by determining Escherichia coli levels according to the standards set by the Councils Directive (91/492/EEC). Two of 36 commercial and 2 of 28 noncommercial oyster samples were B-classified and therefore not ready for consumption. All other oyster samples were A-classified. For the detection of viral RNA, 150 mg of hepatopancreatic tissue was subjected to the Qiagen RNeasy Mini Kit, followed by reverse transcriptase (RT)–PCR and Southern blot hybridization. Enterovirus RNA was detected in 14 of 64 oyster samples, of which 4 were from noncommercial oyster harvesting areas and 10 were from commercial harvesting areas. None of the other human pathogenic viruses were detected. The levels of somatic coliphages and F-specific phages were also determined in all 64 oyster samples, with some samples containing high phage levels (>50 PFU/g of hepatopancreatic tissue), but with most samples containing low phage levels (<50 PFU/g of hepato-pancreatic tissue). However, independent of these high or low phage levels, enterovirus RNA could be detected. Thus, commercial oysters can be contaminated with pathogenic viruses, and monitoring only fecal indicators might not sufficiently protect human health.

Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% +/- 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.

Assessment of Sources and Diversity of Male-Specific Coliphages for Source Tracking

Watershed managers and utilities responsible for source water protection are looking for ways to minimize risk from both human and nonhuman pathogen reservoirs. The ability to accurately identify microbial input source(s) would allow water managers to develop and apply effective, cost-efficient corrective measures. In this study, the potential role of F-specific coliphages with a special focus on the F+DNA coliphages as delineators of sources of surface water microbial pollution was investigated. A variety of source samples from across the United States were analyzed including wastewater and septic system samples, animal slurry lagoon samples, and freshly voided animal fecal samples. Samples were analyzed for fecal coliforms, F-specific coliphages, and somatic coliphages. F-specific phages were isolated and typed to determine whether they were F+RNA or F+DNA coliphages, and were subsequently serotyped and genotyped. F-specific coliphages were detected more often in wastewater samples and slurry lagoon samples when compared to individual fecal samples, where they were rarely detected. F+DNA coliphages were detected in all wastewater samples, in two cow manure samples, and were absent from avian fecal samples. Serotyping and genotyping analysis of F+DNA coliphages did not exclusively associate one subtype with human or animal wastes. A significant proportion of F+DNA isolates demonstrated inconclusive serotyping results, although a majority of the solely M13 serotyped isolates were from domestic wastewater sources. In wastewater samples, 77% were F+DNA while 23% were F+RNA coliphages. Consistent with previous studies, F+RNA coliphage serotyping analysis demonstrated a predominance of group II and III F+RNA coliphages in the wastewater samples, whereas group I and group IV predominated in animal fecal samples and animal slurry lagoon samples. Overall, the results from this study suggests that the presence of F+DNA coliphages, and especially M13 type F+DNA coliphages, could be a potential indicator of human-related wastes.

Hygienization of municipal sludge in automatically operated chamber filter presses with thermal vacuum drying

This paper presents the state of the art of thermal vacuum drying in chamber filter presses for unattended automatic operation. The achieved results are exemplified by the treatment of the two stage digested combined primary, chemical and biological sludge created by the VEAS concept for nutrient removal from municipal wastewater at VEAS. The water removal rate in each stage of the drying process is described, with comments on the low energy needs. The advantages of one-sided heating, the capacity and the drying potential are discussed. The hygienization potential of the process is demonstrated by the effect on thermostable coliform bacteria, Escherichia coli, salmonella, the spores of sulfite reducing anaerobic bacteria, f-specific bacteriophages, the seeds of the weed, wild oat, Avena fatua, and the parasite eggs of the potato cyst nematode, Globodera rostochiensis. A more complete paper with the VEAS-concept is found on the VEAS homepage (www.veas.nu).

Determination of naturally occurring noroviruses in coastal seawater by alkaline elution after acid rinse using negatively charged membrane

A new procedure for concentrating viruses from seawater using a negatively charged membrane eluting with alkaline solution (NaOH, pH 10.5) after acid rinse (H2SO4, pH 3.0) was applied to determine naturally occurring enteric viruses in seawater in Tokyo bay. The levels of total coliforms and fecal coliforms ranged from 40 to 68000 (cfu/100ml) and from 2 to 32000 (cfu/100ml), respectively. The F-specific phages were not detected from 5 ml of 53 samples out of 61 tested. The levels of indicator microbes were not found to be related to the tide in Tokyo bay. Enteroviruses were not detected by cell culture RTÐPCR, but detected by direct RTÐPCR from approximately 10% of the samples. Noroviruses were found positive from approx. 31% of the winter samples (n=29), whereas only 3% from the summer samples (n=32). These results of direct RTÐPCR were equivalent to determination of Norwalk viruses occuring in 50 ml of seawater. These results suggested that the levels of noroviruses in Tokyo bay were higher in winter than those of enteroviruses The virus concentration method used was proved to be useful for determination of naturally occurring viruses in seawater, especially when applied prior to PCR detection of nonculturable viruses.

Removal of fecal coliforms, somatic coliphages and F-specific bacteriophages in a stabilization pond and reservoir system in arid regions

The contents of FC, somatic coliphages and F-specific bacteriophages were measured in the stabilization pond and stabilization reservoir system of the City of Arad (Israel) in order to determine the efficiency of the treatment process regarding the microorganisms removal. Monitoring was conducted close to one year. Physical and chemical parameters (temperature, pH, BOD5, COD, SS) were also analyzed with the aim of finding factors that affect the microorganisms removal. The field results prove a very good performance of this treatment system. FC, somatic coliphages and F-specific bacteriophages were removed between 4.16 and 5.76 log units, during winter and in between 6.02 and 6.47 log units during summer. The microbial quality of the final effluent complies with the WHO guidelines for unrestricted irrigation. The results also indicate that retention time and temperature seem to be the most important factors for microorganisms removal. F-specific bacteriophages were removed at higher rates than FC and somatic coliphages. Consequently, it is suggested that F-specific bacteriophages might be less adequate viral indicators for this treatment system.

Investigation of potential surrogate organisms and public health risk in UV irradiated secondary effluent

Watercare's Mangere Wastewater Treatment Plant in Auckland, New Zealand treats sewage from a population equivalent of approximately 1,000,000. The treatment plant is currently undergoing a major upgrade, and as a part of this upgrade the largest UV disinfection plant in the world (at the time of award of the contract) is being constructed. Pilot scale investigations were undertaken at a purpose built facility. The pilot plant employed secondary treatment, and filtration, UV disinfection and a number of low pressure membrane systems. Investigations at the facility focussed on attempting to identify relationships between potential surrogate indicator organisms (including enterococci, faecal coliforms, Clostridium perfringens spores and F-specific bacteriophage) and pathogenic organisms (including culturable human enteric viruses, bacterial pathogens and parasites). The aim of the study was to identify a suitable indicator organism and an associated effluent concentration that would ensure that an acceptable level of public health risk was maintained in the environment. The results showed that no suitable surrogate indicator organism could be found. However the results did indicate that a two tiered operating strategy, based on the concentration of enteroviruses present in raw sewage and an appropriate UV dose, would ensure that an acceptable level of public health risk was maintained in the environment.

Removal of enteric viruses and selected microbial indicators by UV irradiation of secondary effluent

The Watercare Mangere Wastewater Treatment Facility, which treats wastewater from the greater Auckland New Zealand region, is undergoing a major expansion/upgrading to add advanced treatment and disinfection prior to discharge into a harbor. One important goal of this project is to protect the receiving water from microbial contamination. Since sufficient information on the fate of various microorganisms through wastewater treatment plants in New Zealand was not readily available, extensive pilot- and bench-scale studies were undertaken to develop specific design criteria for the treatment and disinfection systems. The specific objective of this study was to evaluate the removal and inactivation of enteric pathogens and other microbial indicators through treatment processes that employs UV irradiation as a final disinfection process. The removal of indicator organisms through secondary treatment was typically between 2.5-log (99.7% removal) and 2.8-log (99.8% removal) for fecal coliforms and enterococci, respectively. Indigenous F-specific bacteriophage exhibited a mean removal of 1.6-log (i.e. 97.7% removal) and Clostridium perfringens spores showed a mean removal of 1.3-log (i.e. 95% removal). The UV dose required to achieve a one log reduction in the concentration of indigenous F-specific bacteriophage was found to be approximately 20 mWs/cm2 per log removal. The concentration of enterovirus and adenovirus were consistently reduced to the limit of detection (1 TCID50/100L) at UV doses of 35 to 40 mWs/cm2 and 40 to 45 mWs/cm2, respectively. Clostridium perfringens spores were the most resistant indicator organisms, being reduced to less than 200 MPN/100 mL at a UV dose of 75 mWs/cm2.