Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.     

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.     

Virus transport and survival after land application of sewage sludge.

The survival and transport patterns of poliovirus 1 and echovirus 1 were studied in undisturbed soil cores which were treated with digested sludge and exposed to natural weather conditions prevailing in north central Florida. It was shown that, under those experimental conditions, enteroviruses are relatively rapidly inactivated in the soil. A more rapid virus decline was observed during the warm and dry fall season than during the warm and wet summer season. The monitoring of soil core leachates has shown that both viruses were effectively retained by the sludge-treated soil.     

A method to detect enteroviruses in sewage sludge-amended soil using the PCR.

PCR detection of seeded poliovirus type 1 in sludge-amended soil was made possible by utilizing Sephadex G-50 and Chelex-100 resins to remove compounds present in sludge-amended soil that may inhibit PCR. With this method, enteroviruses indigenous to an anerobically digested sludge were detected by PCR in 10 different soils amended with this sludge.     

Effect of sludge type on poliovirus association with and recovery from sludge solids

Sludge type was found to affect the degree of association between seeded poliovirus type 1 (LSc) and sludge solids. The mean percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested sludges was 57.2, 70.4, and 94.7, respectively. The degree of association between poliovirus and sludge solids was significantly greater for aerobically digested sludges than for the other two sludge types. Sludge solids associated viruses were eluted using 0.05 M glycine buffer, pH 10.5-11.0, and subsequently concentrated by organic flocculation. The effectiveness of the glycine method in the recovery of solids-associated viruses was also found to be affected by sludge type. Significantly lower mean poliovirus recovery was found for aerobically digested sludges (14.5%) than for mixed liquors or anaerobically digested sludges (72.3 and 60.2%, respectively). The eluent used in the method was not as effective in dissociating the virus from aerobic sludge solids as it was for the other two sludge types. All other virus adsorption-elution steps of the method (i.e., virus concentration steps) were equally effective in poliovirus recovery for all three sludge types. It is suggested that future methods developed for the recovery of viruses from sludges be evaluated for the various sludge types likely to be tested.     

Persistence of poliovirus 1 in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent.

Land disposal of sewage sludge and effluent is becoming a common practice in the United States. The fertilizer content and humus value of such wastes are useful for agricultural purposes, and the recycling of sewage onto the land eliminates many of our stream pollution problems. The potential exists for crops grown in such irrigated soil to be contaminated by viruses that may be present in the sewage. Studies were initiated to determine viral persistence in soil and on crops grown under natural conditions in field plots that had been flooded to a depth of 1 inch (2.54 cm) with poliovirus 1-inoculated sewage wastes. Lettuce and radishes were planted in sludge- or effluent-flooded soil. In one study, the vegetables were planted 1 day before flooding, and in another they were planted 3 days after the plots were flooded. Survival of poliovirus 1 in soil irrigated with inoculated sewage sludge and effluent was determined during two summer growing seasons and one winter period. The longest period of survival was during the winter, when virus was detected after 96 days. During the summer, the longest survival period was 11 days. Poliovirus 1 was recovered from the mature vegetables 23 days after flooding of the plots had ceased. Lettuce and radishes are usually harvested 3 to 4 weeks after planting.     

Persistence of poliovirus 1 in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent.

Land disposal of sewage sludge and effluent is becoming a common practice in the United States. The fertilizer content and humus value of such wastes are useful for agricultural purposes, and the recycling of sewage onto the land eliminates many of our stream pollution problems. The potential exists for crops grown in such irrigated soil to be contaminated by viruses that may be present in the sewage. Studies were initiated to determine viral persistence in soil and on crops grown under natural conditions in field plots that had been flooded to a depth of 1 inch (2.54 cm) with poliovirus 1-inoculated sewage wastes. Lettuce and radishes were planted in sludge- or effluent-flooded soil. In one study, the vegetables were planted 1 day before flooding, and in another they were planted 3 days after the plots were flooded. Survival of poliovirus 1 in soil irrigated with inoculated sewage sludge and effluent was determined during two summer growing seasons and one winter period. The longest period of survival was during the winter, when virus was detected after 96 days. During the summer, the longest survival period was 11 days. Poliovirus 1 was recovered from the mature vegetables 23 days after flooding of the plots had ceased. Lettuce and radishes are usually harvested 3 to 4 weeks after planting.     

Urea-lysine method for recovery of enteroviruses from sludge.

Enteroviruses added to sludge and indigenous viruses present in sludge were recovered by treating the sludge flocs with a 4 M urea solution buffered at pH 9 with 0.5 M lysine. Eluted viruses were absorbed to aluminum hydroxide flocs and collected by centrifugation. The flocs were solubilized with 0.1 M ethylenediaminetetraacetic acid-3% beef extract at pH 9. After dialysis to remove the ethylenediaminetraacetic acid, viruses were further concentrated by organic flocculation. Approximately 40% of poliovirus and coxsackievirus B-3 added to 500 to 1,000 ml of sludge could be recovered in final sample volumes of less than 10 ml. Polioviruses, echoviruses, and coxsackieviruses were recovered from different samples of wastewater sludge.     

Heat inactivation of poliovirus in wastewater sludge.

The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. In low concentrations and at relatively low inactivation temperatures, digested sludge is nearly as protective of poliovirus as raw sludge. However, at higher tempeatures and concentrations, digested sludge caused a significant acceleration of poliovirus inactivation. The difference between the protective capability of raw and digested sludge is not due to loss of protective material, because this component is present in the solids of digested sludge as well as in those of raw sludge. Instead, the difference is due to a virucidal agent acquired during digestion. Addition of this agent to the solids of either raw or digested sludge reverses the protective potential of these solids during heat treatment of poliovirus.     

Heat inactivation of poliovirus in wastewater sludge.

The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. In low concentrations and at relatively low inactivation temperatures, digested sludge is nearly as protective of poliovirus as raw sludge. However, at higher tempeatures and concentrations, digested sludge caused a significant acceleration of poliovirus inactivation. The difference between the protective capability of raw and digested sludge is not due to loss of protective material, because this component is present in the solids of digested sludge as well as in those of raw sludge. Instead, the difference is due to a virucidal agent acquired during digestion. Addition of this agent to the solids of either raw or digested sludge reverses the protective potential of these solids during heat treatment of poliovirus.     

Prevalence of Nonpolio Enteroviruses in the Sewage of Guangzhou City,China, from 2009 to 2012

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.     

High Incidence of Mammalian Orthoreovirus Identified by Environmental Surveillance in Taiwan

Wild poliovirus (WPV) persists in diverse locales worldwide, spreading outward from endemic areas. In response to the international threat of WPV transmission and changes in the national vaccination policy, we established an environmental surveillance system to monitor the circulation of wild and vaccine-related poliovirus in Taiwan. From July 2012 to December 2013, we collected sewage specimens every month from 10 sewage treatment plants located throughout Taiwan. The specimens were concentrated by the two-phase separation method and then inoculated into L20B, RD, and A549 cells for virus isolation. Viral isolates were identified and serotyped by immunofluorescence assay or molecular analysis. A total of 300 sewage samples were collected, and the results showed 163 samples (54.3%) were positive for virus, and 268 isolates were identified. Among these, 75 samples (25%) were positive for enterovirus (EV), but no poliovirus was found. In addition, 92 isolates were identified as enteroviruses and the most common serotypes were coxsackievirus B4, coxsackievirus B3, and coxsackievirus B2. Interestingly, 102 (34%) and 82 (27.3%) specimens were positive for mammalian orthoreovirus (MRV) and adenovirus, respectively. This study confirmed that sewage surveillance can be a useful additional modality for monitoring the possible presence of wild-type or vaccine-derived poliovirus in wastewater, and can indicate the current types of viruses circulating in the population. Furthermore, since MRV was found in children with acute necrotizing encephalopathy and meningitis, the high incidence of MRV detected by environmental surveillance warrants further investigation.     

One-year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated-sludge purification plant.

Samples of raw sewage, primary effluent, and secondary effluent from a large activated-sludge purification plant near Melbourne (Victoria, Australia) were collected every second week for 1 year. Viruses were detected in all secondary effluent samples and in six of seven samples obtained after final chlorination. Adenoviruses (85% reduction) and reoviruses (28% reduction) were removed less efficiently by this treatment process than were enteroviruses (93% reduction). In addition, 57 of 171 samples of effluent tested were positive for either adenoviruses or reoviruses, or both, when enteroviruses were not isolated. This clearly shows that the use of enteroviruses as sole indicators of viruses in water may miss up to one-third of instances of viral contamination. Enteroviruses and adenoviruses were isolated most frequently in HeLa-R cell cultures, whereas reoviruses were most often isolated in primary monkey kidney cells.     

Occurrence of enteroviruses in marine sediment along the coast of Barcelona, Spain

A survey of the occurrence of enteroviruses in marine sediment was undertaken in an area receiving polluted effluents. Enteroviruses were detected in 21 out of 38 samples (55%). Viruses were found as far as 5 km from the shoreline and at a depth of 82 m. Multiple correlations between enteroviruses and bacteria, detected in the same samples, were computed. No correlation could be demonstrated between virus numbers and any other parameter in sediment samples collected south of Barcelona. This lack of correlation is probably due to the different decay rates shown by bacteria and viruses. In contrast, the cases where pollution resulted from a more recent deposition, as in sediment samples collected north of Barcelona, enterovirus levels were correlated with fecal streptococci levels.     

Isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater

Samples of air and corresponding wastewater samples were taken at wastewater spray-irrigated fields. The concentrations of salmonellae and enteroviruses present in these samples were determined and compared with those of coliforms, and the ratios between them were calculated. The most common Salmonella serotype in the air was Salmonella ohio, whereas in the wastewater, Salmonella anatum was the most common. Enteroviruses isolated and identified were poliovirus, echovirus, and coxsackievirus type B. From the ratios of salmonellas to coliforms and enteroviruses to coliforms in the air, as compared to these ratios in the wastewater, it was concluded that the suitability of coliforms as an indication of airborne contamination caused by spray irrigation is questionable.     

Isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater.

Samples of air and corresponding wastewater samples were taken at wastewater spray-irrigated fields. The concentrations of salmonellae and enteroviruses present in these samples were determined and compared with those of coliforms, and the ratios between them were calculated. The most common Salmonella serotype in the air was Salmonella ohio, whereas in the wastewater, Salmonella anatum was the most common. Enteroviruses isolated and identified were poliovirus, echovirus, and coxsackievirus type B. From the ratios of salmonellas to coliforms and enteroviruses to coliforms in the air, as compared to these ratios in the wastewater, it was concluded that the suitability of coliforms as an indication of airborne contamination caused by spray irrigation is questionable.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland.

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.