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.     

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.     

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.     

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.     

Heat inactivation of enteric viruses in dewatered wastewater sludge.

The effect of moisture content on the rates of heat inactivation of enteric viruses in wastewater sludge was determined. The protective effect of raw sludge on poliovirus previously observed (R. L. Ward, C. S. Ashley, and R. H. Moseley, Appl. Environ. Microbiol. 32:339--346, 1976) was found to be greatly enhanced in sludge dewatered by evaporation. Other enteroviruses responded in a similar fashion. This effect did not appear to be due merely to the state of dryness of the sludge samples because in humus-deficient soil, a relatively inert material, the rate of poliovirus inactivation by heat was not significantly altered through dewatering. Instead, this effect appeared to have been caused by protective substances in the sludge, such as detergents, which are concentrated through dewatering. As reported previously (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681-688, 1977; R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol 36:889--897, 1978) raw sludge is not protective of reovirus, but, instead, the ionic detergents in sludge cause the rate of heat inactivation of this virus to be accelerated. Dewatering of sludge, however, was found to partially reverse this virucidal effect. Evidence is presented indicating that this reversal is caused by an unidentified protective substance in sludge also concentrated through dewatering. Finally, it was shown that the effects of raw sludge on heat inactivation of poliovirus and reovirus are greatly reduced by composting, a result that correlated with the degradation of detergents.     

Heat inactivation of enteric viruses in dewatered wastewater sludge.

The effect of moisture content on the rates of heat inactivation of enteric viruses in wastewater sludge was determined. The protective effect of raw sludge on poliovirus previously observed (R. L. Ward, C. S. Ashley, and R. H. Moseley, Appl. Environ. Microbiol. 32:339--346, 1976) was found to be greatly enhanced in sludge dewatered by evaporation. Other enteroviruses responded in a similar fashion. This effect did not appear to be due merely to the state of dryness of the sludge samples because in humus-deficient soil, a relatively inert material, the rate of poliovirus inactivation by heat was not significantly altered through dewatering. Instead, this effect appeared to have been caused by protective substances in the sludge, such as detergents, which are concentrated through dewatering. As reported previously (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681-688, 1977; R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol 36:889--897, 1978) raw sludge is not protective of reovirus, but, instead, the ionic detergents in sludge cause the rate of heat inactivation of this virus to be accelerated. Dewatering of sludge, however, was found to partially reverse this virucidal effect. Evidence is presented indicating that this reversal is caused by an unidentified protective substance in sludge also concentrated through dewatering. Finally, it was shown that the effects of raw sludge on heat inactivation of poliovirus and reovirus are greatly reduced by composting, a result that correlated with the degradation of detergents.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

Role of lime treatment in the removal of bacteria, enteric viruses, and coliphages in a wastewater reclamation plant.

Lime flocculation/sedimentation in the first process unit of a 4,500-m3/day wastewater reclamation plant reduced numbers of microorganisms extensively when operated at pH 11.2. The efficiency was much less at lower pH values, and some bacteria even multiplied at pH 9.6. Data on reduction in the number of microorganisms in the lime treatment and subsequent units indicate that inactivation by hydroxide alkalinity plays an important role in the efficiency of lime treatment. Reductions in the numbers of enteric viruses were higher than those of coliphages, enterococci, and total plate and coliform bacteria, which indicate that lime treatment can be monitored by means of coliphage and conventional bacteriological tests. This paper illustrates the valuable role of high-pH lime treatment in reducing the load of pathogenic microorganisms on subsequent units, including ultimate disinfection processes, which is important in the multiple safety barrier concept of wastewater reclamation processes.     

Role of lime treatment in the removal of bacteria, enteric viruses, and coliphages in a wastewater reclamation plant.

Lime flocculation/sedimentation in the first process unit of a 4,500-m3/day wastewater reclamation plant reduced numbers of microorganisms extensively when operated at pH 11.2. The efficiency was much less at lower pH values, and some bacteria even multiplied at pH 9.6. Data on reduction in the number of microorganisms in the lime treatment and subsequent units indicate that inactivation by hydroxide alkalinity plays an important role in the efficiency of lime treatment. Reductions in the numbers of enteric viruses were higher than those of coliphages, enterococci, and total plate and coliform bacteria, which indicate that lime treatment can be monitored by means of coliphage and conventional bacteriological tests. This paper illustrates the valuable role of high-pH lime treatment in reducing the load of pathogenic microorganisms on subsequent units, including ultimate disinfection processes, which is important in the multiple safety barrier concept of wastewater reclamation processes.     

Reduction of interfering cytotoxicity associated with wastewater sludge concentrates assayed for indigenous enteric viruses.

Washing, freon extraction, and cationic polyelectrolyte precipitation were compared for their ability to reduce cytotoxicity associated with virus concentrates derived from beef extract eluates of wastewater sludges. Eluates concentrated by hydroextraction were usually much more toxic than those concentrated by organic flocculation. This difference may be due entirely to nondialyzable material naturally present in the beef extract which did not precipitate during flocculation at pH 3.5. Washing inoculated cell monolayers with saline containing calf serum before the addition of agar overlay media was most effective in reducing cytotoxicity, although it resulted in a greater virus loss, as compared with freon extraction and cationic polyelectrolyte precipitation.     

Comparison of coliphage and bacterial aerosols at a wastewater spray irrigation site.

Microbiological aerosols were measured on a spray irrigation site at Fort Huachuca, Ariz. Indigenous bacteria and tracer bacteriophage were sampled from sprays of chlorinated and unchlorinated secondary-treatment wastewaters during day and night periods. Aerosol dispersal and downwind migration were determined. Bacterial and coliphage f2 aerosols were sampled by using Andersen viable type stacked-sieve and high-volume electrostatic precipitator samplers. Bacterial standard plate counts averaged 2.4 x 10(5) colony-forming units per ml in unchlorinated effluents. Bacterial aerosols reached 500 bacteria per m3 at 152 m downwind and 10,500 bacteria per m3 at 46m. Seeded coliphage f2 averaged 4.0 x 10(5) plaque-forming units per ml in the effluent and were detected 563 m downwind. Downwind microbial aerosol levels were somewhat enhanced by nighttime conditions. The median aerodynamic particle size of the microbial aerosols was approximately 5.0 micrometer. Chlorination reduced wastewater bacterial levels 99.97% and reduced aerosol concentrations to near background levels; coliphage f2 was reduced only 95.4% in the chlorinated effluent and was readily measured 137 m downwind. Microbiological source strength an meteorological data were used in conjunction with a dispersion model to generate mathematical predictions of aerosol strength at various sampler locations. The mean calculated survival of aerosolized bacteria (standard plate count) in the range 46 to 76 m downwind was 5.2%, and that of coliphage f2 was 4.3 %.     

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.     

Elution of enteric viruses from Mississippi estuarine sediments with lecithin-supplemented eluents.

Isoelectric casein supplemented with lecithin was tested for its ability to recover enteric viruses from estuarine sediments of varied sand, silt, and clay composition. Recoveries were higher when lecithin was incorporated into an eluent as compared with trials with only the casein solution. Semipurified soybean lecithin (3%) allowed the highest overall recovery of virus from all sediments tested; crude soybean lecithin produced the lowest recovery. A difference in the percentage of virus able to be recovered from a sediment was related to the percentage of clay in the sample. Correlational statistics indicated a trend toward lower virus recovery as the clay composition of a sediment increased. Virus adsorption to the four sediments tested revealed differences between poliovirus, coxsackievirus, and echovirus adsorption that could not be explained on the basis of the clay content of a sediment.     

Reduction of interfering cytotoxicity associated with wastewater sludge concentrates assayed for indigenous enteric viruses

Washing, freon extraction, and cationic polyelectrolyte precipitation were compared for their ability to reduce cytotoxicity associated with virus concentrates derived from beef extract eluates of wastewater sludges. Eluates concentrated by hydroextraction were usually much more toxic than those concentrated by organic flocculation. This difference may be due entirely to nondialyzable material naturally present in the beef extract which did not precipitate during flocculation at pH 3.5. Washing inoculated cell monolayers with saline containing calf serum before the addition of agar overlay media was most effective in reducing cytotoxicity, although it resulted in a greater virus loss, as compared with freon extraction and cationic polyelectrolyte precipitation.     

Copper-resistant enteric bacteria from United Kingdom and Australian piggeries.

Thirty-three enteric isolates from Australian (Escherichia coli only) and United Kingdom (U.K.) (Salmonella sp., Citrobacter spp., and E. coli) piggeries were characterized with respect to their copper resistance. The copper resistance phenotypes of four new Australian E. coli isolates were comparable with that of the previously studied E. coli K-12 strain ED8739(pRJ1004), in that the resistance level in rich media was high (up to 18 mM CuSO4) and resistance was inducible. Copper resistance was transferable by conjugation from the new Australian isolates to E. coli K-12 recipients. DNA similarity between the new Australian isolates and the pco copper resistance determinant located on plasmid pRJ1004 was strong as measured by DNA-DNA hybridization; however, the copper resistance plasmids were nonidentical as indicated by the presence of restriction fragment length polymorphisms between the plasmids. DNA-DNA hybridization and polymerase chain reaction analysis demonstrated DNA homology between the pco determinant and DNA from the U.K.E. coli, Salmonella sp., and Citrobacter freundii isolates. However, the copper resistance level and inducibility were variable among the U.K. strains. Of the U.K. E. coli isolates, 1 demonstrated a high level of copper resistance, 4 exhibited intermediate resistance, and 16 showed a low level of copper resistance; all of these resistances were expressed constitutively. A single U.K. C. freundii isolate, had a high level of copper resistance, inducible by subtoxic levels of copper. Transconjugants from one E. coli and one C. freundii donor, with E. coli K-12 strain UB1637 as a recipient, showed copper resistance levels and inducibility of resistance which differed from that expressed from plasmid pRJ1004.(ABSTRACT TRUNCATED AT 250 WORDS)