Elution of viruses from coastal sediments

Enteric viruses were eluted from estuarine sediments by using four organic mixtures; these solutions, with or without various supplements, were compared by determining their abilities to desorb virus from sediments taken from shellfish-harvesting sites. The least effective eluents consisted of glycine buffer, milk preparations, and beef extract paste. When virus type and sediment composition were taken into consideration, higher percentages of virus recovery were achieved with isoelectric casein, powdered beef extract, and nutrient broth mixtures. In addition to the type of eluent used, variations in virus recovery were due to the pH of the eluent, the composition of the sediment, and the type of virus being extracted. No clear distinction between the values of protein and inorganic ion supplements could be made.     

Elution of viruses from coastal sediments.

Enteric viruses were eluted from estuarine sediments by using four organic mixtures; these solutions, with or without various supplements, were compared by determining their abilities to desorb virus from sediments taken from shellfish-harvesting sites. The least effective eluents consisted of glycine buffer, milk preparations, and beef extract paste. When virus type and sediment composition were taken into consideration, higher percentages of virus recovery were achieved with isoelectric casein, powdered beef extract, and nutrient broth mixtures. In addition to the type of eluent used, variations in virus recovery were due to the pH of the eluent, the composition of the sediment, and the type of virus being extracted. No clear distinction between the values of protein and inorganic ion supplements could be made.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Low molecular weight substitutes for beef extract as eluents for poliovirus adsorbed to membrane filters.

Basic solutions of beef extract and casein were able to elute poliovirus adsorbed to four membrane filters with different chemical compositions. Hydrolyzed protein and individual amino acids were able to elute virus adsorbed to certain filters but were unable to elute virus adsorbed to other filters efficiently. A solution of 4 M urea buffered at pH 9 with 0.05 M lysine was able to elute greater than 60% of the virus adsorbed to each of the filters tested. Certain solutions of amino acids were capable of eluting virus adsorbed to one filter but permitted adsorption of virus to another filter with a different chemical composition. Acidic amino acids could interfere with elution of virus from membrane filters. Aromatic compounds with an amino group attached to the ring were good eluents for virus adsorbed to epoxy-fiberglass membrane filters. In contrast, aromatic compounds with other substituents were generally poor eluents.     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

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.     

Evaluation of methods for concentrating hepatitis A virus from drinking water.

By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture-adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission.     

Evaluation of methods for concentrating hepatitis A virus from drinking water

By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture-adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Macrobenthic–mud relations strengthen the foundation for benthic index development: A case study from shallow,temperate New Zealand estuaries

Globally the input of sediment to coastal systems, in particular to estuaries, is predicted to increase due to anthropogenic activities. Sediment mud content is a powerful driver of ecologically important, macrobenthic taxa in estuarine intertidal flats. Accordingly, forecasting species responses to increased sedimentation is fundamental for effective ecosystem management, particularly for productive, geologically young, and sand-dominated estuaries that characterise many countries, including New Zealand (NZ). Modelling studies have highlighted the non-linear, highly variable responses of taxa to mud concentration. However, existing taxon-specific models have not adequately accounted for the full mud gradient, the influence of potentially confounding variables (e.g. organic enrichment, heavy metal concentrations), or regional differences in species responses. Furthermore, such models are often based on qualitative expert consensus of the membership of taxa in ecological groups that characterise their sensitivity to mud content. In this study, data from 25 unmodified to highly disturbed, shallow NZ estuaries, were used to develop an ecologically relevant model to relate the responses of 39 taxa to sediment mud content for use in the intertidal flats of shallow, temperate estuaries. Preliminary analyses indicated that sediment mud content was the dominant driver of macroinvertebrate community composition among sites, total organic carbon was of secondary importance and heavy metals did not explain significant variation in composition. Regression analysis revealed a significant linear relationship between sediment mud and total organic carbon content, which permitted subsequent analyses to be based on mud content alone. Generalised additive models were used to develop taxon-specific models that, according to k-fold cross validation, accurately predicted both probability of presence (up to 79% deviance explained) and maximum density (up to 96% deviance explained) along the sediment mud gradient (0.1–92.3%). Estimates of “optimal mud range” and “distribution mud range” were quantitatively-derived for each taxon and used to categorise taxa into one of five ecological groups (identical to those used in existing biotic indices), based on their individual sensitivities to increasing mud content. By removing expert consensus from the grouping process, the classification methods established herein provide strong support for the use of quantitative indices for the assessment and management of estuarine condition in response to increasing sediment mud content. The findings indicate that NZ estuarine sediments (2–25% mud) support a more diverse and abundant macroinvertebrate assemblage and exhibit low organic enrichment (<1% total organic carbon) compared to estuaries with >25% mud content.     

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.     

Anaerobic methanethiol degradation in upflow anaerobic sludge bed reactors at high salinity (> or =0.5 M Na(+))

The feasibility of anaerobic methanethiol (MT) degradation at elevated sodium concentrations was investigated in a mesophilic (30 degrees C) lab-scale upflow anaerobic sludge bed (UASB) reactor, inoculated with estuarine sediment originating from the Wadden Sea (The Netherlands). MT was almost completely degraded (>95%) to sulfide, methane and carbon dioxide at volumetric loading rates up to 37 mmol MT x L(-1) x day(-1), 0.5 M sodium (NaCl or NaHCO(3)) and between pH 7.3 and 8.4. Batch experiments revealed that inhibition of MT degradation started at sodium (both NaCl and NaHCO(3)) concentrations exceeding 0.8 M. Sulfide inhibited MT degradation already around 3 mM (pH 8.3).     

Ghost cells as sorption matrix for virus concentration from water

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Ghost cells as sorption matrix for virus concentration from water.

Pig erythrocyte membranes were used as adsorbent material for the concentration of bacteriophage phi x-174, MS-2, and f2 from 5 ml of saline solution. The adsorption was carried out at pH 3.5, and the elution was carried out at pH 7.0. Compared with adsorption on 3% beef extract, bacteriophage adsorption on erythrocyte ghost cells yielded 93 to 100%, and elution was 92 to 100%, whereas beef extract organic flocculation yielded adsorption of 0 to 98.8% and elution of 61 to 86.6%. The same procedure but with poliovirus LSC-1 gave 100% adsorption and 91 to 129% elution.     

Comparison of four eluents in the recovery of indigenous viruses from raw sludge.

The efficiency of 3% casein hydrolysate (CH), 3% lactalbumin hydrolysate (LH), 3% beef extract (BE), and 10% fetal calf serum (FCS) was compared for the recovery of viruses from raw sludge. CH and LH proved to be inefficient and were eliminated from the study after initial testing. In tests with 20 different samples of raw sludge, beef extract eluted virus in 15 (75%) and FCS revealed virus in 19 (95%) of the samples using BS-C-1 cells. That different eluents were not eluting different viruses from the same sample was shown by the serologic and electron-microscopic examination of 43% (18/42) of the isolates. The identified viruses included members of the entero- (coxsackie B, and polio) and reo-virus groups.     

TRACE METAL CYCLING IN THE U.S. COASTAL ZONE: A SYNTHESIS

This synthesis of trace element research in estuarine communities of the U.S. coastline and the Caribbean provides a summary applicable to the shoreline of the tropical and temperate regions of the world which have mangroves, kelp beds, riverine marshes and seagrass communities. An inventory of sediments and leaf tissues shows Mn and Fe to be the most highly concentrated elements with Hg and Cd present in lowest concentrations. Generally, trace element concentrations in roots are much higher than in leaves and other tissues above the sediment. Tissue to sediment concentration ratios show that Cd is most likely to be bioamplified and that Cu, Hg, Sr and Zn may have relatively high concentration ratios which can exceed unity. A conceptual model was constructed to integrate the forcing functions, compartmental couplings, and dynamics common to these estuarine systems. Seasonality is important for changes in some trace element concentrations in plants and litter. Trace element additions to water or sediment increased certain trace element concentrations in plants and dead organic matter. It is clear that estuarine plant communities serve as living filters of estuarine trace elements. However, increased knowledge of trace element cycling in estuarine systems and relationships between trace element concentrations in plants and the estuarine food chain is needed, particularly food chains to man. There is a need for structured long-term estuarine research to allow direct comparison of results among estuarine study sites, to identify the similarity of population and system processes among estuaries and to define the geographical scale over which estuarine research results may be generalized.     

Some remarks on the presence of organic phosphates in sediments

This article describes a new method developed to assess the size and nature of the organic phosphate pool. Using sediment suspensions from the Rhone, Garonne and Po rivers, inorganic P compounds, Fe(OOH) and CaCO₃ were removed using mild extractants at sediment pH. The residual phosphate was then fractionated into an acid soluble organic phosphate pool and a residual organic phosphate pool by acid hydrolysis (0.5 M H⁺). Both pools were quantitatively important, accounting for between 16 and 54% and 16 and 51% of total phosphate respectively. Acid hydrolysis was chosen since it yielded a distinct plateau, with high reproducibility, within 30 minutes.This fractionation permits a further study of dynamics and bioavailability of sediment org-P, without interference of Fe(OOH) and CaCO₃.In many studies in which changes in the organic pool were examined after extraction of inorganic phosphate, 0.5 M HCl was used to extract apatite bound phosphate. The results presented here show that this is likely to result in a considerable underestimation of the organic phosphate pool.