Survival of indicator organisms during enrichment on tetrachloroethene.

A laboratory study was performed as the basis for a full-scale bioaugmentation project at a site contaminated with chlorinated ethenes. The objectives of this study were to 1) develop a protocol to enrich for a tetrachloroethene (PCE)-dechlorinating culture from waste activated sludge and anaerobic digester biosolids and 2) monitor the survival of fecal coliform bacteria and bacteriophage, which model enteric viruses, during the enrichment process. A culture was enriched in 8 days with the ability to degrade 6-microM PCE to cis-dichloroethene. Using the enrichment protocol in two identical experiments, significant inactivation of fecal coliform bacteria (2 log) and somatic coliphage (0.33 log) was observed in one of the experiments; no inactivation occurred in the second experiment. The number of F-specific coliphage decreased in both experiments (0.87 and 1.26 log inactivation). Despite the decrease in some of the coliform and bacteriophage numbers, the quantity of organisms and phage particles present after enrichment was still high (approximately 7.5 x 10(5) most probable number/L, 6.9 x 10(6) plaque-forming units (PFU)/L, and 3.3 x 10(5) PFU/L, for fecal coliform bacteria, somatic coliphage, and F-specific coliphage, respectively). This may be cause for concern, depending on the current and future groundwater use at or near a site undergoing bioaugmentation with cultures derived from waste activated sludge and anaerobic digester biosolids.

Assessment of the viral removal capability of the International Space Station Water Recovery and Management system.

The development of the International Space Station (ISS) Water Reclamation and Management (WRM) system has been supported through integrated testing at NASA/Marshall Space Flight Center (MSFC). Assessment of the viral removal capabilities of the ISS water processor was performed as part of the hardware performance evaluation. For the Viral Challenge Test, a known concentration of viruses was mixed with human-generated wastewater and reclaimed using the ISS Water Processor. The composition and concentrations of the different wastewaters used for this test represented the combination of contaminants expected to be found in the wastewater that will be collected and recycled on board the ISS. The results from the Viral Challenge Test clearly showed that the ISS WRM has the capacity to reduce the concentration of viruses in the wastewater by 12 log10 units.

Differential Effect of Tetrazolium Dyes upon Bacteriophage Plaque Assay Titers.

This study examined whether the practice of incorporating either tetrazolium red or tetrazolium violet dye into plaque assay medium deleteriously influences plaque assay titers. Representative members of six different virus families were studied: Cystoviridae (varphi6), Leviviridae (MS2), Microviridae (varphiX174), Myoviridae (T2), Podoviridae (P22), and Siphoviridae (Denver, T1, and VD13). Each of the members of the Podoviridae and Siphoviridae families appeared to be suppressed by either one or both dyes at a 300-mug/ml concentration. The chosen representatives of the other bacteriophage families were not suppressed by either dye at a 300-mug/ml concentration. Subsequent trials revealed no suppression of Podoviridae or Siphoviridae plaque assay titers when members of these virus families were tested with the same two dyes at the lower concentrations of 150 and 50 mug/ml. Interestingly, the bacteriophage families whose members were affected by the dyes have additional commonality in that they are the two bacteriophage families whose members possess both double-stranded DNA genomes and noncontractile tails.

Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae.

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 degrees C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 degrees C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 degrees C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage.

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.

Presence of enteric viruses in freshwater and their removal by the conventional drinking water treatment process.

A review of results published in English or French between 1980 and 1990 was carried out to determine the levels of indigenous human enteric viruses in untreated surface and subsurface freshwaters, as well as in drinking water that had undergone the complete conventional treatment process. For this purpose, the conventional treatment process was defined as an operation that included coagulation followed by sedimentation, filtration, and disinfection. Also assessed was the stepwise efficiency of the conventional treatment process, as practised at full-scale facilities, for removing indigenous viruses from naturally occurring freshwaters. A list was compiled of statistical correlations relating to the occurrence of indigenous viruses in water.

Thermal and water source effects upon the stability of enteroviruses in surface freshwaters.

The long-term survival of three human enterovirus serotypes, Coxsackievirus B3, echovirus 7, and poliovirus 1 was examined in samples of surface freshwater collected from five sites of physically different character. These were an artificial lake created by damming a creek, a small groundwater outlet pond, both a large- and a medium-sized river, and a small suburban creek. Survival was studied at temperatures of -20, 1, and 22 degrees C. The average amount of viral inactivation was 6.5-7.0 log10 units over 8 weeks at 22 degrees C, 4-5 log10 units over 12 weeks at 1 degree C, and 0.4-0.8 log10 units over 12 weeks at -20 degrees C. The effect of incubation temperature upon viral inactivation rate was statistically significant (p less than 0.00001). As determined by pairing tests, survival was also significantly related to both viral serotype and water source at each of the three incubation temperatures (p less than or equal to 0.05). Efforts were made to determine whether the rate of viral inactivation observed at the different incubation temperatures was related to characteristics inherent to the water that was collected from the different locations. The characteristics examined included physical and chemical parameters, indigenous bacterial counts, and the amount of bacterial growth that the waters would support (measured as the maximum number of generations which seeded bacteria could undergo after being placed into either pasteurized or sterile-filtered water samples). Analysis of viral inactivation rate versus these characteristics revealed three apparent effectors of viral persistence.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of viral replication by guanidine: a comparison of human adenoviruses and enteroviruses.

A comparison was made between the relative sensitivities of laboratory strain human adenoviruses and enteroviruses, and recently isolated human enteroviruses, to the presence of guanidine hydrochloride in cell culture media. The concentration of guanidine hydrochloride used was 100 micrograms per ml. Representatives of all six human Adenovirus subgenera were unaffected in their replication at this concentration of guanidine. The different human Enterovirus types examined varied in their sensitivity, with suppression ranging from less than 1 to 3 log10 units for laboratory strains, and from 2 to 7 log10 units for recently isolated viruses. The findings suggest a novel role for antiviral drugs; serving as an adjunct in facilitating selective isolation of specific virus groups which may be present as part of mixed viral populations.

Influence of aerobic microorganisms upon virus survival in soil.

Survival of human poliovirus type 1 in a sandy loam soil appeared to be deleteriously influenced by aerobic microorganisms. This effect was determined by comparing the survival of virus in soil under four different possible combinations of aerobic versus anaerobic (H2-CO2) atmosphere and sterile versus nonsterile condition. Storage of samples was done in humid chambers to prevent soil desiccation. The effect attributed to aerobic microorganisms was measurable and statistically significant at all three incubation temperatures used in the study (1, 23, and 37 degrees C), with the increase in inactivation rate attributable to aerobic microorganisms generally being two to threefold. No comparable effect was observed to occur for anaerobic microorganisms under the sets of conditions employed in the study.

Survival of indigenous enteric viruses during storage of waste water sludge samples.

The stability of indigenous enteric viruses in samples of settled primary and mixed-liquor activated sludges was studied at 2, 23, and -70 degrees C. Changes of virus titer which occurred in these samples were followed during an 84-day observation period, with rates of change then calculated by least-squares regression. Virus survival was found to be statistically dependent (p less than or equal to 0.05) upon storage temperature but not sludge solids content. Based upon the observed rates of inactivation, the average times which would be required for a 90% decrease in virus titer are 26 days at 23 degrees C, 180 days at 2 degrees C, and 163 days at -70 degrees C. As a group, the rates of virus inactivation observed at 2 degrees C were statistically different (p less than or equal to 0.05) from those observed at 23 degrees C, but not different from those observed at -70 degrees C. The three study temperatures were selected to approximate holding of samples in an air-conditioned room, on wet ice (H2O), and on dry ice (CO2).

Stability of viruses in waste water sludge eluates.

The survival of indigenous enteric viruses in samples of unconcentrated and concentrated waste water sludge eluates, which had been prepared using a combination beef extract elution - organic flocculation concentration procedure, was studied at 2, 23, and -70 degrees C. Changes of virus titer occurring in the samples were followed during an 84-day observation period, with rates of change then calculated by least-squares regression. Virus survival in both types of eluates was statistically dependent (p less than or equal to 0.05) upon storage temperature. Based upon the observed rates of inactivation the average times which would be required for a 90% decrease (one log10 unit) in virus titer for unconcentrated eluates are 27 days at 23 degrees C, 198 days at 2 degrees C, and 375 days at -70 degrees C. The calculated average times required for a 90% decrease in virus titer for concentrated eluates are 22 days at 23 degrees C, 132 days at 2 degrees C, and 246 days at -70 degrees C. In both types of eluates the rates of virus inactivation at 2 degrees C were statistically different from those observed at 23 degrees C, but not different from those observed at -70 degrees C. The three study temperatures were selected to approximate holding of samples in an air-conditioned room, fluid on wet ice (H2O), and frozen on dry ice (CO2).

Evaluation of mixed cell types and 5-iodo-2'-deoxyuridine treatment upon plaque assay titers of human enteric viruses.

Four continuous cell lines, BGM, L-132, HEL-299, and RD, were compared both when cultured separately and as mixtures for use in plaque assay titrations of human adenovirus 1 and six human enterovirus serotypes. The effect of incubating these cell cultures in media containing 5-iodo-2'deoxyuridine (IDU) prior to inoculation with virus was also studied. The use of mixed-cell cultures revealed cell line-dependent synergistic effects as well as inhibitory effects. These effects were strongly virus dependent. In particular, enterovirus 69 did not form plaques on any of the four cell lines when cultured independently. However, it did form plaques on nearly all of the cell lines when cultured as mixtures. Contrary to this effect, when BGM cells were used in combination with the other cell lines, plaque counts for adenovirus 1 were greatly reduced. The effect of IDU pretreatment was also virus and cell line specific and enabled some viruses to form plaques on cell lines when they otherwise would not. Overall, IDU pretreatment resulted in an approximate twofold increase in plaque titers over those obtained without treatment.

Comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples.

Microbiological- and food-grade beef extracts, protein hydrolytic, enzymatic and autolytic digestion products, and whole protein materials were examined for their potential effectiveness for eluting adsorbed enteroviruses from membrane filters with observed efficiencies ranging from less than 1 to 69%. Concentration of enteroviruses from solutions of these protein and protein-derived products by organic flocculation ranged in efficiency from 2 to 125%. Both elution and concentration were dependent upon virus type, as well as nature, source, and production lot of the material being tested. Determining the efficiency of virus concentration was complicated by virus aggregation and apparent virus inactivation by low pH. Effectiveness of concentrating viruses by organic flocculation from solutions prepared with the various test materials seemed independent of the amount of precipitate produced during the flocculation procedure. Quality assurance tests were proposed by which solutions prepared from beef extracts, whole protein, and protein-derived materials could be evaluated for use in eluting adsorbed viruses from membrane filters and for concentrating viruses by organic flocculation. Food-grade beef extract seemed equal to microbiological-grade beef extract in terms of both virus elution and concentration. Several of the nonbeef extract materials evaluated were as effective as beef extract for virus concentration, but were less effective for virus elution.

Round robin investigation of methods for recovering human enteric viruses from sludge.

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultrasensitive radioligand assay for IgG using the protein A on Staphylococcus aureus bacteria.

The purpose of this study was to develop a simple and sensitive assay to measure IgG. Human IgG was radiolabelled with 125Iodine and 7.5 ng was incubated with heat-killed Staphylococcus aureus bacteria (Cowan 1 strain). To replicate sets of tubes, increasing amounts of a standard IgG preparation were added. The samples were incubated at room temperature for two hours and separated by centrifugation. Using this assay it was found that the IgG concentration could readily be determined in one nanoliter or less of human serum. There was no significant cross-reactivity with IgA, IgE and IgM or the F(ab')2 fragment of IgG. Serial dilutions of normal human or SLE sera, rabbit or guinea pig sera, the Fc fragment of human IgG and a mouse monoclonal anti-human DNA antibody parallelled the dose response curve obtained with standard human IgG. The method correlated well (r=0.89) with a routinely used nephelometric method. The mean (+/- SD) IgG concentration in 20 normal subjects measured by this assay was 10 +/- 3.6 g/L.

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.

Effects of environmental variables and soil characteristics on virus survival in soil.

Because of the increasing emphasis placed upon land application as a means of wastewater disposal, it is important to evaluate the influences of different factors upon virus survival in soil. The objective of this study was to measure the effects of various environmental variables on virus persistence. Test samples of soil were placed in vials, and the soil was wetted with suspensions of virus in either distilled water, unchlorinated secondary sewage effluent, or mixtures of effluent and water. The viruses used were coxsackieviruses A9 and B3, echovirus 1, poliovirus 2, rotavirus SA11, and bacteriophages T2 and MS2. The rate of virus inactivation was evaluated statistically with regard to conditions under which the vials were incubated and to the soil characteristics. The factors that were found to influence virus survival were temperature, soil moisture content, presence of aerobic microorganisms, degree of virus adsorption to the soil, soil levels of resin-extractable phosphorus, exchangeable aluminium, and soil pH. Overall, temperature and virus adsorption to soil appeared to be the most important factors affecting virus survival.

Relation of cord blood thyroxine and thyrotropin levels to gestational age and birth weight.

A program of screening cord blood for evidence of primary neonatal hypothyroidism was implemented in a general hospital. In 13 months 3456 newborns were screened: the thyroxine (T4) and triiodothyronine (T3) concentrations were measured in cord blood samples, and when the T4 level was below 8.0 micrograms/dl thyrotropin was also assayed in the sample. The two-tier program was effective. One hypothyroid newborn was detected and treated. More boys than girls had T4 levels below 8.0 micrograms/dl (9.7% v. 4.7%). The T4 level correlated with birth weight slightly better in the boys (r = 0.28 v. 0.21), and in the boys this correlation was stronger when the birth weight was lower. Regression analysis of the data for 54 sets of twins indicated that the T4 level was more strongly related to gestational age than to birth weight.