Adsorption of viruses to soil: impact of anaerobic treatment.

The adsorption of viruses in untreated flushed dairy manure wastewater (FDMW), anaerobically digested flushed dairy manure wastewater (ADFDMW) and groundwater to sandy soil was investigated. Batch adsorption studies showed differential adsorption of viruses in groundwater to soil. Less than 75% of PRD1 and MS2 added to groundwater adsorbed after 1 h, but greater than 95% of phiX174 and poliovirus 1 adsorbed to the soil. Adsorption differences in groundwater were related to the isoelectric points of the viruses. Suspending phages in untreated and treated wastewater reduced adsorption compared with groundwater. For MS2, more phages were adsorbed using ADFDMW than with FDMW. Adsorption of poliovirus 1 was not affected by FDMW and ADFDMW. Small column studies (6 x 2.5 cm) produced a similar trend in that adsorption was observed with groundwater and both FDMW and ADFDMW reduced virus adsorption. Groundwater, FDMW or ADFDMW did not affect the adsorption of poliovirus 1 in column studies. The major difference between FDMW and ADFDMW was in mobilisation of adsorbed viruses. The application of FDMW to soil columns with adsorbed viruses caused significantly more viruses to be mobilised than did the application of rainwater or ADFDMW. These results showed that treating FDMW by anaerobic digestion increased the adsorption of viruses to soil and decreased detachment of adsorbed viruses. As the potential for new zoonotic pathogens becomes known, the treatment of animal wastes may become mandatory. The assessment and management of viruses in manure for addressing possible risk to animal and human health is of interest.

Selective growth of Staphylococcus aureus from flushed dairy manure wastewater using acriflavine-supplemented mannitol salt agar.

AIMS: To investigate the use of mannitol salt agar (MSA) supplemented with acriflavine for selective growth and quantification of Staphylococcus aureus from flushed dairy manure wastewater (FDMW). METHODS AND RESULTS: Minimal inhibitory concentrations of acriflavine in MSA were determined by comparing the growth of S. aureus subsp. aureus (ATCC 33591) and Staphylococcus epidermidis (ATCC 155) in pure culture. Acriflavine concentrations of 1.3, 1.4 and 1.5 mg l(-1) reduced CFU of S. epidermidis by 43%, 55% and 87%, respectively, while CFU of S. aureus subsp. aureus were only reduced by 15%, 20% and 26% at the respective concentrations of acriflavine. MSA supplemented with 1.5 mg l(-1) acriflavine was tested for selective growth of indigenous S. aureus from three grab samples of FDMW. Acriflavine concentrations of 1.5 mg l(-1) reduced background flora without significantly reducing (P < 0.05) indigenous S. aureus counts. CONCLUSIONS: Acriflavine-supplemented MSA provides an effective media for selective growth and quantification of indigenous S. aureus from FDMW in the presence of high levels of background microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: S. aureus is implicated for mastitis infections in dairy cows. Therefore, a reliable means for monitoring and detecting the organism in FDMW provides a tool for measuring the effectiveness of treatment for reducing S. aureus levels and implementing flushwater recycling without affecting herd health.

Geographical variation in antibiotic resistance profiles of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida.

AIMS: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. METHODS AND RESULTS: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. CONCLUSIONS: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.

Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes.

This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella Montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms.

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

Immunological methods for the study of Zoogloea strains in natural environments.

Since Zoogloea ramigera has been considered to be important in aerobic wastewater treatment, we have evaluated several methods for detecting and enumerating Z. ramigera in water and wastewater samples. Indirect immunoassay methods for the detection of Zoogloea strains were developed using polyclonal antibodies against the cells or the isolated exocellular polymer (EP) of the neotype Zoogloea ramigera strain 106 (ATCC 19544). The primary antibodies reacted with the cells and the exopolymer associated with finger-like zoogloeal projections, but not with other bacteria from natural samples. These antibodies allowed detection of Z. ramigera in environmental samples. Scanning electron microscopy (SEM) was used to show that the cells and the exocellular polymer of naturally occurring zoogloeal projections are antigenically and structurally related to those of Z. ramigera 106. Both immunological procedures and probes complementary to regions on the 16S rRNA could detect Z. ramigera in natural samples but the immunological procedures were easier to use. RT-PCR was also used to detect Z. ramigera in natural samples. These methods were also used to identify Z. ramigera in biofilms that developed over wastewater samples as part of an MPN procedure that was used to quantitate Z. ramigera at different stages of the wastewater treatment process and in different lakes. Z. ramigera could be found in all stages of wastewa ter treatment processes, from raw wastewater to chlorinated effluent, The highest concentration of Z. ramigera was found in the mixed liquor stage of the a wastewater treatment plant. Additionally, Z. ramigera was found in all eutrophic and mesotrophic lakes and in some oligotrophic lakes.

Reduction of enteric microorganisms at the Upper Occoquan Sewage Authority Water Reclamation Plant.

The Upper Occoquan Sewage Authority (UOSA) Water Reclamation Plant, Centreville, Virginia, is a state-of-the-art wastewater treatment plant that was created to treat area wastewater and provide protection for the Occoquan Reservoir. This study investigated UOSA's unit processes as barriers to pathogenic as well as altemative and traditional-indicator microorganisms. Samples were collected once a month for 1 year from eight sites within UOSA's advanced wastewater reclamation plant. The eight sites were monitored for indicator bacteria total and fecal coliforms, enterococci, Clostridium, coliphage (the virus that infects Escherichia coli), human enteroviruses, and enteric protozoa. Overall, the plant was able to achieve a 5- to 7-log10 reduction of bacteria, 5-log10 reduction of enteroviruses, 4-log10 reduction for Clostridium, and 4.6-log10 reduction of protozoa. Total coliforms, enterococci, Clostridium, coliphage, Cryptosporidium, and Giardia were all detected in four or fewer samples of the final effluent. No enteroviruses or fecal coliforms were detected in the final effluent. The microbiological quality of reclaimed water and the reservoir water were compared. In every case, the treated wastewater was of a better quality than the ambient water in the reservoir, thus indicating that the reclaimed water will not adversely affect the water quality for downstream users.

Influence of salts on virus adsorption to microporous filters.

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).

Viral tracer studies indicate contamination of marine waters by sewage disposal practices in key largo, Florida.

Domestic wastewater disposal practices in the Florida Keys are primarily limited to on-site disposal systems such as septic tanks, injection wells, and illegal cesspits. Poorly treated sewage is thus released into the highly porous subsurface Key Largo limestone matrix. To investigate the fate and transport of sewage in the subsurface environment and the potential for contamination of marine surface waters, we employed bacteriophages as tracers in a domestic septic system and a simulated injection well in Key Largo, Florida. Transport of bacteriophage (Phi)HSIC-1 from the septic tank to adjacent surface canal waters and outstanding marine waters occurred in as little as 11 and 23 h, respectively. Transport of the Salmonella phage PRD1 from the simulated injection well to a canal adjacent to the injection site occurred in 11.2 h. Estimated rates of migration of viral tracers ranged from 0.57 to 24.2 m/h, over 500-fold greater than flow rates measured previously by subsurface flow meters in similar environments. These results suggest that current on-site disposal practices can lead to contamination of the subsurface and surface marine waters in the Keys.

Presence of human immunodeficiency virus nucleic acids in wastewater and their detection by polymerase chain reaction.

The human immunodeficiency virus type 1 (HIV-1) released by infected individuals or present in human and hospital wastes can potentially cause contamination problems. The presence of HIV-1 was investigated in 16 environmental samples, including raw wastewater, sludge, final effluent, soil, and pond water, collected from different locations. A method was developed to extract total nucleic acids in intact form directly from the raw samples or from the viral concentrates of the raw samples. The isolated nucleic acids were analyzed for the presence of HIV-1 by using in vitro amplification of the target sequences by the polymerase chain reaction (PCR) method. HIV-1-specific proviral DNA and viral RNA were detected in the extracted nucleic acids obtained from three wastewater samples by this method. The specificity of the PCR-amplified products was determined by Southern blot hybridization with an HIV-1-specific oligonucleotide probe, SK19. The isolated nucleic acids from wastewater samples were also screened for the presence of poliovirus type 1, representing a commonly found enteric virus, and simian immunodeficiency virus, representing, presumably, rare viruses. While poliovirus type 1 viral RNA was found in all of the wastewater samples, none of the samples yielded a simian immunodeficiency virus-specific product. No PCR-amplified product was yielded when wastewater samples were directly used for the detection of HIV-1 and poliovirus type 1. The wastewater constituents appeared to be inhibitory to the enzymes reverse transcriptase and DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Salmonella enteritidis in environmental samples by monoclonal antibody-based ELISA.

We have developed a enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ASCII) for the detection of Salmonella enteritidis in environmental samples. ELISA was used to test for sensitivity and specificity of ASCII. 38 other species of bacteria, including 31 Salmonella species were included in cross-reactivity testing with ELISA. ASCII showed no reactivity with any other species tested. ASCII was found to be an IgG1 specific for S. enteritidis lipopolysaccharide (LPS). The lower limits for S. enteritidis detection was 10(5) cells/ml for pure cultures and in 10% sludge (w/v). Environmental samples (raw wastewater, wastewater effluents, mixed liquor and aerobically digested sludge) were obtained twice from five sites and ELISA tested for the presence of S. enteritidis. ELISA results compared to the American Public Health Association (APHA) method of Salmonella detection were not significantly different (P greater than 0.05). The ELISA took 24 h for completion compared to 96-120 h for the APHA procedure. Results demonstrate the reliability of the ELISA and, more importantly, provides a rapid means of detection of S. enteritidis in environmental samples.

Use of hydrogen peroxide treatment and crystal violet agar plates for selective recovery of bacteriophages from natural environments.

Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of Escherichia coli C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).

Detection of nucleic acids homologous to human immunodeficiency virus in wastewater.

Raw wastewaters were obtained from the cities of Belle Glade, Ocala and Gainesville in the state of Florida and were concentrated using several established methods for the recovery of human enteroviruses. The nucleic acids were then extracted from the wastewater concentrates, suspended in 2 x SSC with and without 2 N NaOH (for the detection of DNA and both DNA and RNA, respectively), and dot blotted onto hybridization membranes. These membranes were then hybridized with three 32P-end-labeled 18-mer oligonucleotides directed against the LTR, gag, and env regions of the human immunodeficiency virus type 1 (HIV-1). Autoradiographic analyses of these blots indicate that sequences homologous to HIV-1 genomic RNA and proviral DNA were found in Belle Glade wastewater but not in wastewater from Ocala and Gainesville. These findings may have implications in the wastewater treatment system as well as for detection of HIV-1 in clinical samples.

The production of antibacterial tubing, sutures, and bandages by in situ precipitation of metallic salts.

Two procedures were used to modify gauze bandages, polyester sutures, silicone tubing, and polyvinyl chloride tubing. In one procedure, the materials were first modified by in situ precipitation of metallic hydroxides and then used to adsorb silver ions. In the second procedure, the materials were soaked in sodium pyrophosphate or sodium chloride, dried, and then soaked in silver nitrate. These procedures produced materials with silver deposited on the surface of the tubing and sutures and both on the surface and within the gauze fibers. The modified materials inhibited the growth of Pseudomonas aeruginosa. Escherichia coli, and Staphylococcus aureus in vitro.

Development and application of new positively charged filters for recovery of bacteriophages from water.

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.

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.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization.

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage phi X174. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples.

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.

Activation thermodynamics of virus adsorption to solids.

The kinetics of bacteriophage MS2, T2, and f2 adsorption to powdered nitrocellulose and disrupted Seitz S1 filters at pH 7 were determined as a function of temperature. Data from these studies were combined with data produced in a previous study on MS2 adsorption to clay by Stagg et al. (Appl. Environ. Microbiol. 33:385-391, 1977). These workers studied the adsorption of MS2 to bentonite clay as a function of temperature. Data from both this previous study and the current one were used to calculate the thermodynamic parameters of virus adsorption. The results show that adsorption of bacteriophages to the solids tested is a physical process (energy of activation, less than 40 kcal [168 J]/mol) rather than a chemical process (energy of activation, greater than 40 kcal/mol). The free energy of activation showed a high negative correlation (r = -0.904, r2 = 0.817) with the percentage of virus adsorption to the solids tested. The energy of activation was highly negatively correlated with the percentage of virus adsorption to nitrocellulose and clay (r = -0.913, r2 = 0.834) but poorly correlated with the percentage of virus adsorption to disrupted Seitz S1 filters (r = -0.348, r2 = 0.121). In general, under conditions in which the percentage of virus adsorption was low, the energy of activation, the free energy of activation, and the entropy of activation were high. Increasing the percentage of virus adsorbed by changing the adsorbing conditions or changing the adsorbing solid decreased the energy of activation, the free energy of activation, and the entropy of activation.