Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site

Aerosol samples collected at the Muskegon County Wastewater Management System Number 1 spray irrigation site in Michigan by using the Army prototype XM2 Biological Sampler/Collector were examined for the presence of animal viruses, coliphages, and bacteria. Air samples, collected in Earle lactalbumen hydrolysate, and wastewater samples were filtered through a 0.45- and 1.2-micron membrane filter sandwich, pretreated with 10% beef extract (pH 7.0), and assayed for animal viruses by the plaque method on Buffalo green monkey kidney cells. Untreated air and wastewater samples were assayed for coliphages by the soft agar overlay method with three Escherichia coli hosts (ATCC 13706, 15597, and 11303) and for bacteria by the heterotrophic plate count method. Filtered air samples were assayed for coliphages by the most-probable-number method with the same three hosts. Although no animal viruses were detected in the aerosol samples, coliphages and bacteria were recovered. E. coli ATCC 13706 coliphage were recovered more often and in greater numbers than either of the other two types of coliphages. Concentrations of animal viruses, coliphages, and bacteria detected in the raw influent decreased as the wastewater was aerated and stored in the lagoons. No animal viruses were detected in the wastewater at the pump station just before distribution to the spray irrigation rigs. The most-probable-number method was more sensitive and consistent than the overlay procedure in detecting low levels of coliphages in air samples.     

Practical direct plaque assay for coliphages in 100-ml samples of drinking water.

A practical single-agar-layer plaque assay for the direct detection of coliphages in 100-ml samples of water was designed and evaluated. With this assay a 100-ml sample of water, an agar medium containing divalent cations, and the host Escherichia coli C (ATCC 13706) were mixed in a single container, and the mixture was plated on 10 14-cm-diameter petri dishes. It was more sensitive, reliable, and accurate than various other methods and proved rapid, simple, and economic.     

Airborne enteric bacteria and viruses from spray irrigation with wastewater.

The relationship between bacterial concentrations in wastewater used for spray irrigation and in the air was examined. Aerosolized coliforms were detected when their concentration was 10(3)/ml or more in the wastewater. Relative humidity and solar irradiation appeared to affect viable bacteria in the air; a positive correlation was found between relative humidity and the number of aerosolized bacteria. The correlation between solar irradiation and bacterial level, on the other hand, was negative. During night irrigation, up to 10 times more aerosolized bacteria were detected than with day irrigation. Wind velocity did not play an important role in the survival of aerosolized bacteria. Echovirus 7 was isolated in 4 out of 12 air samples collected 40 m downwind from the sprinkler.     

Airborne enteric bacteria and viruses from spray irrigation with wastewater.

The relationship between bacterial concentrations in wastewater used for spray irrigation and in the air was examined. Aerosolized coliforms were detected when their concentration was 10(3)/ml or more in the wastewater. Relative humidity and solar irradiation appeared to affect viable bacteria in the air; a positive correlation was found between relative humidity and the number of aerosolized bacteria. The correlation between solar irradiation and bacterial level, on the other hand, was negative. During night irrigation, up to 10 times more aerosolized bacteria were detected than with day irrigation. Wind velocity did not play an important role in the survival of aerosolized bacteria. Echovirus 7 was isolated in 4 out of 12 air samples collected 40 m downwind from the sprinkler.     

Practical direct plaque assay for coliphages in 100-ml samples of drinking water

A practical single-agar-layer plaque assay for the direct detection of coliphages in 100-ml samples of water was designed and evaluated. With this assay a 100-ml sample of water, an agar medium containing divalent cations, and the host Escherichia coli C (ATCC 13706) were mixed in a single container, and the mixture was plated on 10 14-cm-diameter petri dishes. It was more sensitive, reliable, and accurate than various other methods and proved rapid, simple, and economic.     

Procedure for the recovery of airborne human enteric viruses during spray irrigation of treated wastewater.

Because of the relatively low number of indigenous enteric viruses recovered from secondary wastewater effluents, their presence in air (aerosols) as a result of wastewater spray irrigation requires extensive sampling. Methodology to allow the recovery of indigenous enteroviruses from aerosols generated at an operational wastewater irrigation site was tested under both laboratory and field conditions.     

Procedure for the recovery of airborne human enteric viruses during spray irrigation of treated wastewater.

Because of the relatively low number of indigenous enteric viruses recovered from secondary wastewater effluents, their presence in air (aerosols) as a result of wastewater spray irrigation requires extensive sampling. Methodology to allow the recovery of indigenous enteroviruses from aerosols generated at an operational wastewater irrigation site was tested under both laboratory and field conditions.     

Airborne coliphages from wastewater treatment facilities.

The emission (from wastewater treatment plants) of airborne coliphages that form plaques on two strains of Escherichia coli was investigated. Two activated-sludge and two trickling-filter plants were studied. Field sampling procedures used large-volume air samplers with recirculation devices. Coliphages were enumerated by a most-probable-number (MPN) procedure. Temperature, relative humidity, windspeed, and presence of sunlight were monitored. Concurrent samples of sewage were taken during each air-sampling run. Average coliphage levels in the airborne emissions of trickling-filter beds and activated-sludge units were 2.84 X 10(-1) and 3.02 X 10(-1) MPN/m3, respectively, for all positive observations, and sewage liquor concentrations from the sources were 4.48 X 10(5) and 2.94 X 10(6) plaque-forming units/liter, respectively, depending upon the E. coli host used for assay. This work establishes minimal airborne-coliphage concentrations from the plants studied. The procedures employed will be useful in evaluating the animal virus levels in these emissions.     

Airborne coliphages from wastewater treatment facilities.

The emission (from wastewater treatment plants) of airborne coliphages that form plaques on two strains of Escherichia coli was investigated. Two activated-sludge and two trickling-filter plants were studied. Field sampling procedures used large-volume air samplers with recirculation devices. Coliphages were enumerated by a most-probable-number (MPN) procedure. Temperature, relative humidity, windspeed, and presence of sunlight were monitored. Concurrent samples of sewage were taken during each air-sampling run. Average coliphage levels in the airborne emissions of trickling-filter beds and activated-sludge units were 2.84 X 10(-1) and 3.02 X 10(-1) MPN/m3, respectively, for all positive observations, and sewage liquor concentrations from the sources were 4.48 X 10(5) and 2.94 X 10(6) plaque-forming units/liter, respectively, depending upon the E. coli host used for assay. This work establishes minimal airborne-coliphage concentrations from the plants studied. The procedures employed will be useful in evaluating the animal virus levels in these emissions.     

Concentration of coliphages from large volumes of water and wastewater.

Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.     

F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

Enhancement of enterovirus infectivity in vitro by pretreating host cell monolayers with the cationic polymer polyethyleneimine.

Laboratory strains of enteroviruses, as well as viruses isolated from raw wastewater, were found to exhibit enhanced infectivity in vitro when BGM cell monolayers were pretreated with the cationic polymer polyethyleneimine (PEI). Viruses were assayed by the cytopathic effect technique and as PFU under methylcellulose and agar overlays with monolayers treated with 0 to 5.0 x 10(-3)% (wt/vol) PEI in phosphate-buffered saline supplemented with 2% fetal bovine serum. Poliovirus type 1 cytopathic effect occurred at an enhanced rate in cells treated with 5.0 x 10(-3)% PEI compared with untreated cells. PEI-treated cells were found to adsorb viruses much more effectively than untreated cells did. When the methylcellulose overlay procedure was used, rates of infectivity were enhanced as follows: poliovirus type 1, 5.5-fold; echovirus type 1, 1.2-fold; echovirus type 5, 5.2-fold; and coxsackievirus type B5, 4.9-fold. Viruses concentrated from raw wastewater showed a 3.8-fold increase in titer when quantitated by the most-probable-number method and a 3.3-fold increase when quantitated as PFU under an agar overlay.     

Enhancement of enterovirus infectivity in vitro by pretreating host cell monolayers with the cationic polymer polyethyleneimine

Laboratory strains of enteroviruses, as well as viruses isolated from raw wastewater, were found to exhibit enhanced infectivity in vitro when BGM cell monolayers were pretreated with the cationic polymer polyethyleneimine (PEI). Viruses were assayed by the cytopathic effect technique and as PFU under methylcellulose and agar overlays with monolayers treated with 0 to 5.0 x 10(-3)% (wt/vol) PEI in phosphate-buffered saline supplemented with 2% fetal bovine serum. Poliovirus type 1 cytopathic effect occurred at an enhanced rate in cells treated with 5.0 x 10(-3)% PEI compared with untreated cells. PEI-treated cells were found to adsorb viruses much more effectively than untreated cells did. When the methylcellulose overlay procedure was used, rates of infectivity were enhanced as follows: poliovirus type 1, 5.5-fold; echovirus type 1, 1.2-fold; echovirus type 5, 5.2-fold; and coxsackievirus type B5, 4.9-fold. Viruses concentrated from raw wastewater showed a 3.8-fold increase in titer when quantitated by the most-probable-number method and a 3.3-fold increase when quantitated as PFU under an agar overlay.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

Fate of human enteric viruses, coliphages, and Clostridium perfringens during drinking-water treatment.

The elimination of human enteric viruses, coliphages, and Clostridium perfringens was studied during a conventional complete drinking-water treatment process. The respective concentrations (geometric mean) of these microorganisms in 100-L samples of river water were, respectively, as follows: viruses, 79 mpniu (most probable number of infectious units) per 100 L, coliphages, 6565 pfu (plaque-forming units) per 100 L. and clostridia, 11,349 cfu (colony-forming units) per 100 L. After predisinfection, flocculation with alum, and settling, human enteric viruses were not detected in any of the 100-L samples (less than 4 mpniu/100 L), but coliphages were detected in 7 of 14 samples and clostridia in 15 of 16 samples. In filtered water samples, human enteric viruses were detected in 2 of 31 samples, coliphages in 10 of 33, and clostridia in 17 of 33. Finished water was free of human enteric viruses (0/162 samples), but coliphages were detected in one sample (1.5 pfu/100 L) and clostridia in three, at 1.0, 4.1, and 7.0 cfu/100 L. It thus appears that coliphages and clostridia, which are present in larger numbers than viruses in river water and which may have similar resistance to drinking-water treatments, may be useful for estimating the level of treatment attained when large volumes of water (1000 L or greater) are sampled.     

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan

Aims: To determine the genogroup distribution of F‐specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup‐specific real‐time PCR. Methods and Results: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F‐specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F‐specific RNA (F‐RNA) coliphage genogroups and 24 (11%) plaques were F‐specific DNA coliphages. Human genogroups II and III F‐RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary‐treated sewage samples were highly contaminated with genogroup I F‐RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. Conclusions: A wide range of F‐specific coliphage genogroups were successfully identified in wastewater and river water samples. Significance and Impact of the Study: Our results clearly show the usefulness of the genogroup‐specific real‐time PCR for determining the genogroups of F‐specific coliphages present in aquatic environments.     

Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses

Bacteria- and virus-containing aerosols were studied during the late summer and fall seasons in a midwestern suburb of the United States before and during the start-up and operation of an unenclosed activated sludge wastewater treatment plant. The study showed that the air in this suburban area contained low-level densities of indicator microorganisms. After the plant began operating, the densities of total aerobic bacteria-containing particles, standard plate count bacteria, total coliforms, fecal coliforms, fecal streptococci, and coliphages increased significantly in the air within the perimeter of the plant. Before plant operations, bacteria were detected from five genera, Klebsiella, Enterobacter, Serratia, Salmonella, and Aeromonas. During plant operations, the number of genera identified increased to 11. In addition to those genera found before plant operations, Escherichia, Providencia, Citrobacter, Acinetobacter, Pasteurella, and Proteus, were also identified. Enteric viruses were detected in low densities from the air emissions of this plant. Only standard plate count bacteria remained at significantly higher than base-line densities beyond 250 m downwind from the center of the aeration tanks. Fecal streptococci and coliphages appeared to be more stable in aerosols than the other indicator microorganisms studied. In general, the densities of microorganism-containing aerosols were higher at night than during the day. The techniques used in this study may be employed to establish microorganism-containing aerosol exposure during epidemiological investigations.     

Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses.

Bacteria- and virus-containing aerosols were studied during the late summer and fall seasons in a midwestern suburb of the United States before and during the start-up and operation of an unenclosed activated sludge wastewater treatment plant. The study showed that the air in this suburban area contained low-level densities of indicator microorganisms. After the plant began operating, the densities of total aerobic bacteria-containing particles, standard plate count bacteria, total coliforms, fecal coliforms, fecal streptococci, and coliphages increased significantly in the air within the perimeter of the plant. Before plant operations, bacteria were detected from five genera, Klebsiella, Enterobacter, Serratia, Salmonella, and Aeromonas. During plant operations, the number of genera identified increased to 11. In addition to those genera found before plant operations, Escherichia, Providencia, Citrobacter, Acinetobacter, Pasteurella, and Proteus, were also identified. Enteric viruses were detected in low densities from the air emissions of this plant. Only standard plate count bacteria remained at significantly higher than base-line densities beyond 250 m downwind from the center of the aeration tanks. Fecal streptococci and coliphages appeared to be more stable in aerosols than the other indicator microorganisms studied. In general, the densities of microorganism-containing aerosols were higher at night than during the day. The techniques used in this study may be employed to establish microorganism-containing aerosol exposure during epidemiological investigations.     

Replication of coliphage Qβ as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Qβ did not replicate when host cells were fewer than 10⁴ cfu ml⁻¹. Replication selected for insusceptible cells when Qβ was incubated with its E. coli host. Loss of Qβ, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Qβ replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.