Adsorbed to Sediments

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1984, p /84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 48, No. 2 Isolation of Enteroviruses from Water, Suspended Solids, and Sediments from Galveston Bay: Survival of Poliovirus and Rotavirus Adsorbed to Sediments V. CHALAPATI RAO, KARSTEN M. SEIDEL,t S. M. GOYAL,: THEODORE G. METCALF, AND JOSEPH L. MELNICK* Department of Virology and Epidemiology, Baylor College of Medicine, Hoiston, Texas Received 23 January 1984/Accepted 4 May 1984 The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water ph appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying ph adjustment and salt addition for processing. Over 8 x 109 gallons (3 x 1010 liters) of municipal sewage, of which only one-half receives secondary treatment, are discharged each day into the inshore waters of the United States. These effluents contain as many as 200 detectable infectious viruses per gallon (3.785 liters) (22, 26). Outbreaks of infectious hepatitis and nonbacterial gastroenteritis traced to the consumption of shellfish harvested from polluted coastal water emphasize the significance attached to the occurrence and persistence of human viruses in the marine environment. Affinity of viruses for mixed liquor-suspended solids during sewage treatment has been demonstrated (3, 17). The relative concentrations of viruses associated with solids in influents, effluents, and chlorinated effluents from two sewage plants have been determined (34). About 68% of viruses in raw sewage have been estimated to be associated with suspended solids (27). A high percentage of solids-associated viruses in sludge has also been demonstrated (16, 34). The percentage of total coliphage and animal virus numbers associated with solids in secondarily treated sewage discharges ranged from <1.0 to 26% and 3 to 100%, respectively (9). Association of bacteriophages and animal viruses with solids has a protective effect, resulting in resistance to inactivation by chlorine and an enhanced virus survival in natural waters (1, 2, 7, 30, 31, 33). A 10- to 10,000-foldgreater virus concentration in sediments than in overlay water was found in coastal Texas and Florida waters polluted by sewage discharges (8). This observation implies that sediments can not only act as reservoirs of human enteric viruses but also as a source from which virus can be released into a water column by storm action, dredging activities, etc. Recovery of high numbers of viruses in sediments in the absence of detectable virus in 400 liters of overlay water * Corresponding author. t Present address: Institute for Water, Soil and Air Hygiene, D1000, Berlin 33, Federal Republic of Germany. t Present address: Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, St. Paul, MN suggests that analysis of sediment may furnish more information about water quality than tests of overlay water (8). Previous studies with enteric bacteria showed that the greatest number occurred at or near the surface of sediments. Collection of fluffy sediments representing the uppermost layers of bottom sediment became possible after the development of a sediment-associated virus sampler (21). Development of this capability, coupled with established methods for the collection and separation of water, suspended solids, and compact sediments, made it feasible to determine the fate of viruses in a polluted estuary, which is the subject of this report. MATERIALS AND METHODS Sampling sites. Samples of sediment, fluffy sediment, suspended solids, and water were collected from two sites at Kemah and Seabrook in Galveston Bay (Fig. 1). The two areas receive secondarily treated, chlorinated sewage effluent (0.3 x 106 to 5 x 106 gallons [1.1 x 106 to 19 x 106 liters] per day). Water depth at these sites ranged from 1 to 2 m. Sampling at Kemah was conducted 30 meters offshore from a partially sunken barge, whereas samples at Seabrook were collected from the shore. During July 1981 through February 1982, a total of 103 samples were collected and processed. Water at the collection sites varied as follows: temperature, 14 to 32 C; ph, 7.6 to 8.3; turbidity, 10 to 115 nephelometric turbidity units; salinity, 2 to 20 parts per thousand. Virus concentration procedures. (i) Suspended solids. About 100 gallons (379 liters) of seawater at ambient ph (7.6 to 8.3) were filtered through a combination of 10-inch (25- cm), tubular, pleated Filterite filters of 3- and 0.45-[Lm porosity (Filterite Corp., Timonium, Md.) at a flow rate of 5 gallons (19 liters) per min with a 1.9-kW (2.5-horsepower) gasoline-engine water pump. The intake water hose was submerged 1 to 2 cm below the water surface to prevent agitation of sediments. Suspended solids trapped on the filters were backwashed with 1 liter of 3% beef extract prepared in 0.05 M glycine (ph 10.5). Eluates were neutral-

2 VOL. 48, 1984 ISOLATION OF ENTEROVIRUSES 405 IBED #4 GULF OF MEXICO FIG. 1. Sampling locations (Kemah and Seabrook) in Galveston Bay. ized to ph 7.0, carried to the laboratory on ice, stored in a cold room overnight, and the next day reconcentrated by the organic flocculation method (12). (ii) Fluffy sediment. Samples were collected on 3- and 0.45-,um Filterite filters by the sediment-associated virus sampler. The fluffy sediment, separated from collecting filters by backwash with sterile 0.01 M phosphate-buffered saline, was returned to the laboratory on ice and stored overnight in the cold room. The sample was centrifuged at 1,500 x g for 10 min. The deposit was shaken for 15 min with 3 volumes of 3% beef extract-glycine (ph 10.5) to extract the virus. The sample was centrifuged at 1,500 x g for 10 min. The sediment was discarded. The supernatant containing virus was reconcentrated by organic flocculation (12). (iii) Sediment. Samples of compact sediment were collected with an Ekman dredge from the same site and transferred into polyethylene bags. After overnight storage in the cold room, processing began on the following morning. A 300-g portion of the sample was shaken for 15 min with 3 volumes of beef extract-glycine (ph 10.5) and clarified by centrifugation at 1,500 x g for 10 min. The supernatant ph was adjusted to 3.5 by 1 N HCI, the sample was stirred for 15 min, and the formed floc was deposited by centrifugation at 2,100 x g for 15 min. The floc was resuspended in 10 ml of 0.05 glycine (ph 11), and after thorough mixing, the solution (having a ph of 9.5) was frozen for 20 min, thawed, and centrifuged at 2,100 x g for 30 min. The supernatant was collected and neutralized, and fetal calf serum was added to give a final concentration of 2%. (iv) Water. A 100-gallon (379-liter) water sample from which suspended solids had been removed by filtration through 3- and 0.45-,um cartridge filters was collected in a Nalgene tank. Viruses from this sample were concentrated by using a single 0.45-,um, pleated Filterite filter as described previously (6). Viruses in the primary eluate were reconcentrated by organic flocculation (12). Final eluate volumes of 10 ml each were obtained from water, suspended solids, and fluffy sediments, and 20-ml volumes were obtained from compact sediments. (v) Virus isolation and identification. Virus assays were performed by three methods. (i) Fifty percent of the sample concentrate (5 to 10 ml) was assayed by the plaque technique (20) on monolayer cultures of Buffalo green monkey (BGM) kidney cells propagated in 75-cm2 plastic flasks. Cells were grown on Eagle medium containing 10% fetal calf serum, 100 U of penicillin, and 100,g of streptomycin per ml and maintained on the same medium containing 2% fetal calf serum. Test inocula of 1.0 ml each were adsorbed for 1.5 h at 37 C, followed by the addition of overlay medium. (ii) A 1-ml portion of the sample was inoculated into 10 roller-tube cultures of Buffalo green monkey kidney cells (16 by 125 mm), at 0.1 ml per tube. (iii) Selected samples which were negative for virus in the above two methods were examined by the suspended cell-agar overlay technique (4). In this method, 0.5 ml of test inoculum was added to 1 ml of Buffalo green monkey kidney cell suspension containing 106 to 107 cells, followed by a 1-h incubation at 37 C, after which 2 to 3 ml of agar overlay medium was added, and the agar-cell suspension was plated out in 1-oz (30-ml) bottles. Agaroverlaid monolayers were examined for plaques for 14 to 21 days. Roller-tube cultures were passed at least one time before attributing the cytopathic effect to a viral isolate. Viral isolates were subsequently identified by using LBM (Lim-Benyesh-Melnick) antiserum pools (19). (vi) Virus survival. The two viruses used in these experiments were poliovirus 1 (strain LSc) and simian rotavirus SAl1. The estuarine water, sediment, fluffy sediment, and suspended solids were collected at the Kemah sampling site. Poliovirus was assayed as above; rotavirus was assayed on MA104 fetal rhesus kidney cells in monolayer cultures. Experiments were initiated on the same day that the samples were collected. Four Nalgene bottles representing four systems were prepared. One bottle contained 100 ml of estuarine water minus suspended solids and the viral inoculum. The remaining three bottles contained 10-ml volumes of wet sediment, fluffy sediment, and suspended solids, respectively, to which known numbers of viruses were adsorbed in the laboratory, plus 90 ml of estuarine water. Initial virus numbers varied from 4.5 x 106 to 9.4 x 107 PFU/ml. Samples were held at room temperature (20 to 25 C) and examined for virus at 3-day intervals for 19 days. A bottle was shaken thoroughly before removal of a sample. A 1-ml TABLE 1. Recovery of poliovirus (6.9 x 106 PFU) adsorbed to suspended solids in estuarine water' Nominal Virus recovered % of suspended pore size of ilitae solids-associated % Recovery Filterite ith virus retained on of virusd filter (pum) (PFU) filtersc x 106 (78) x 105 (4.7) x i03 (0.1) x 104 (0.2) (0) x 104 (0.2) a Seawater solids collected by centrifugation were mixed with 50 ml of artificial seawater (Instant Ocean; ph, 7.4; salinity, 15%o) seeded with 7.8 x 106 PFU of virus. After a 30-min shaking at 250 cycles per min, 1 ml of the sample was removed and centrifuged at 2,200 x g for 10 min. The supernatant was assayed, and the quantity of virus adsorbed on the solids was determined. The remaining 49 ml of the sample was filtered through Filterite filters (diameter, 47 mm) of nominal porosities ranging from 8 to 0.25 p.m as shown in the table. Filtrates from each experiment were examined for virus. Each filter or filter combination was eluted with 10 ml of 3% beef extract-0.05 M glycine (ph 10.5), and eluates were assayed for virus. b Based on the quantity of virus (6.9 x 106) PFU adsorbed on suspended solids, the figures within parentheses are percentages. C Difference between virus adsorbed on suspended solids and percentage of virus detected in the filtrates. d Estimated from the quantity of suspended solids-associated virus retained on filters and virus recovered on elution.

3 406 RAO ET AL. APPL. ENVIRON. MICROBIOL. Date Sample no. TABLE 2. Virus types" recovered from Galveston Bay Virus types recovered from: Water Sediment Fluffy sediment Suspended solids 10/12/81 17 Coxsackievirus B4 10/19/81 18 Echovirus 7 Echovirus 7 19 Echovirus 29 Coxsackievirus B4 11/03/81 23 Coxsackievirus B4 23 Echovirus Coxsackievirus B3 Coxsackievirus B3 24 Poliovirus 2 11/10/81 25 Poliovirus 2 Poliovirus 2 26 Poliovirus 2 Poliovirus 2 11/18/81 27 Coxsackievirus B4 Poliovirus 1 Poliovirus 1 28 Poliovirus 1 Poliovirus 2 12/08/81 31 Poliovirus 2 Poliovirus 2 12/15/81 33 Poliovirus 2 Poliovirus 2 34 Poliovirus 1 1/06/82 35 Poliovirus 2 1/26/82 38 Poliovirus 3 2/17/82 39 Poliovirus 1 2/24/82 42 Coxsackievirus B3 " Number of occasions on which different virus types were recovered are as follows: poliovirus 2, 11; poliovirus 1, 5: coxsackievirus B4, 4; coxsackievirus B3, 3; echovirus 7, 2; echovirus 29, 2: poliovirus sample was removed, diluted in Tris saline, and frozen at -20 C until all samples could be assayed simultaneously on the same lot of cell cultures. RESULTS Quantitation of solids-associated virus. Results in Table 1 indicate that poliovirus adsorbed on naturally occurring suspended solids was retained on Filterite filters of 3.0- to 0.22-,um porosity (individually and in combination) with an efficiency of 95 to 100%. Recovery of virus from these filters ranged from 13 to 28%. A combination of 3- and 0.45-,um filters was chosen for the present investigation to facilitate rapid filtration of 100 gallons (379 liters) of water in 20 to 30 min. Virus types. A qualitative distribution of virus types isolated from the various sample types (Table 2) indicates that poliovirus 2 was recovered most often (39% of isolates), followed by poliovirus 1 (18% of isolates). Other virus types recovered were coxsackievirus B4 (14%), coxsackievirus B3 (10%), echovirus 7 (7%), echovirus 29 (7%), and poliovirus 3 (3%). It is of interest to point out that in four of six times when virus was recovered from two different samples collected on the same day, the same virus type was found. For example, on 19 October 1981 (sample 18), echovirus 7 was TABLE 3. Location of virus in the water column in a polluted estuary No. of times virus was detected from Sample types Total no. of all samples examined on the same day Water, suspended solids, fluffy sediment, and sediment Water and sediment.35 0 Water and suspended solids.18 4 Water and fluffy sediment.15 1 Sediment, fluffy sediment, and suspended solids.15 1 Fluffy sediment and suspended solids 15 3 Suspended solids only Water alone.35 5 isolated from water and suspended solids and, on 10 November 1981 (sample 26), poliovirus 2 was recovered from fluffy sediment and suspended solids. Virus location in the water column. Of 103 samples tested during 29 sampling trips, all four types of samples examined were never found positive for virus on any one day (Table 3). Virus was recovered from water and suspended solids in 4 of 18 samples and from suspended solids and fluffy sediment in 3 of 15 samples. Quantity of virus. The virus concentration per 100 gallons (379 liters) of water varied from 3 to 12 PFU, whereas the greatest number of viruses, 39 to 398 PFU/1,000 g, were recovered from fluffy sediments. Suspended solids obtained from 100 gallons (379 liters) of water yielded 4 to 40 PFU, whereas 7 to 10 PFU/1,000 g were recovered from compact bottom sediments (Table 4). The distribution of enteroviruses at the two combined sampling locations in Galveston Bay is summarized in Table 5. The results indicate that the highest number of viruspositive samples (72%) were obtained from suspended solids. Almost one-half of fluffy sediment samples (47%) contained virus. Water and compact bottom sediments indicated the presence of virus in 14 and 5% of samples, respectively. Improved virus recovery by suspended cell-agar overlay. The usefulness of the suspended cell-agar overlay method for examining low levels of viruses from environmental samples is indicated from the results in Table 6. Of nine water samples, three yielded virus (33%), and of five samples each of suspended solids, fluffy sediment, and sediment, one TABLE 4. Distribution of virus in the water column Sample type Vol or wt recovered Water 100 gallons ( liters) Suspended solids (obtained 100 gallons ( from water) liters) of water Fluffy sediment (obtained 100 gallons ( from water) liters) of water Sediment 1,000 g 7-10

4 VOL. 48, 1984 ISOLATION OF ENTEROVIRUSES 407 TABLE 5. Recovery of enteroviruses at Kemah and Seabrook Sample No. 5 type N. No... examined positive positive Water Sediment Fluffy sediment Suspended solids sample each (20%) turned out to be positive. All of these samples had been selected because they had been negative for virus when tested by the plaque method and roller-tube culture method. Virus survival. Survival of poliovirus and rotavirus SAil, when associated with different fractions of the water column, is summarized in Table 7. Results indicate that both viruses survived longer when associated with sedimentary material. The numbers of virus detected on suspended solids on day 15 were almost equal to the numbers recovered from the sediment. Both virus types could be detected in the sediment, fluffy sediment, and suspended solids even on day 19, whereas the same viruses could not be detected beyond day 9 when suspended in seawater. DISCUSSION Previous studies on viruses in Galveston Bay waters polluted by domestic sewage were related to their occurrence and persistence in water, sediment, and shellfish (10, 13, 14, 22, 28). The present report on the distribution and quantification of naturally occurring enteroviruses among water, suspended solids, fluffy sediments, and compact sediment supports the view that solids-associated virus must be an important consideration in monitoring estuarine waters. Earlier studies (5, 8) recognized sediments as potential reservoirs of enteric viruses which can be released into the water column as a result of agitation of the sediment by storms, dredging, boating, etc. Results obtained in the present investigation indicate that a large fraction of virus associated with suspended solids constantly floating in the water represents a newly recognized source of viral hazard in coastal waters. We recognize that fluffy sediments contain suspended solids-associated virus that recently settled out of the water column, and resuspension of this portion of virus can be caused by mild turbulence or water movements. Attention has been called to solids-associated virus in recreational and shellfish-producing waters (8, 13, 18). Our present findings offer a new perspective on the amount of virus that might be transported and permit an estimate of TABLE 6. Comparative sensitivity of the suspended cell-agar overlay method for virus isolation from field samples found negative by the standard plaque method and by roller-tube assay Plaque method No. Suspended Sample type tested (standard by roller- cell-agar agar tube overlay overlay) asy Water Suspended solids Fluffy sediment Sediment how far viruses may be transported in an infectious state. Quantitative estimates reveal that fluffy sediment- and suspended solids-associated virus constitute approximately three-fourths of enteroviruses recovered (Table 5) and that poliovirus and rotavirus SAil survived for 19 days when adsorbed on these solids (Table 7). These findings attest to the possible transportation of considerable numbers of viruses to nonpolluted areas of Galveston Bay, depending on the water circulation pattern and prevailing winds. Of immediate importance is the location of shellfish beds not far from our sampling area (Fig. 1). Shellfish have been shown to accumulate more virus when virus is present as a crude suspension rather than as purified virus, indicating that virus associated with particulate matter is taken up more efficiently by shellfish (11). A recent study (15) on the accumulation of sediment-associated viruses in shellfish indicated that the incidence of virus uptake was higher when sediments were resuspended in the water column. The resuspended virus remained solids associated, indicating a firm attachment to the particles and confirming an earlier report (13) that viruses may remain adsorbed to marine sediments over a wide range of temperature, ph, and salinity conditions. It follows, therefore, that the viruses accumulated by shellfish are, for the most part, in the particle-associated phase. The public health safety of shellfish and of water in which shellfish are harvested is presently judged by bacteriological standards, but evidence for shellfish-mediated transmission of human virus diseases such as hepatitis (25) and Norwalk virus-induced gastroenteritis (24) emphasizes the need to recognize the potential hazard of solids-associated viruses in the waters overlying shellfish beds. Results from various field studies seeking to determine the virus types isolated from the coastal waters of Texas indicate that the predominant virus types vary over time. In the present investigation, poliovirus 2 was isolated on 11 occasions, followed by poliovirus 1 (five times), coxsackie B TABLE 7. Survival of poliovirus and rotavirus in the water column and different samples of sediment" Virus type Fraction Survival of virus6 at day: ' Poliovirus Water 4.5 x x x l Sediment 5.9 x x x 10i 2.5 x X x x 101 Suspended solids 3.3 X X X X X 10' 2.4 X X 101 Fluffy sediment 4.0 x x x x x x x 101 SA1l Water 7.7 x x x x Sediment 5.6 x x x x x x X 103 Suspended solids 9.4 x X X X X X X 104 Fluffy sediment 4.0 x x X x x X X 102 All experiments were performed at room temperature (ca. 25 C). b PFU of virus in 100 ml of sample. ' For poliovirus, numbers in this column represent PFU in the concentrated sample. A 94-ml amount of each sample was concentrated to 2 ml, and the total volume was assayed. For rotavirus, numbers represent PFU detected in unconcentrated samples.

5 408 RAO ET AL. viruses (seven times), and echoviruses 7 and 29 (two times each), whereas earlier studies reported in 1978 (10) yielded poliovirus 1 six times more often than all other virus types combined, and in 1974 (22) indicated echovirus 7 as the predominant virus. Prevalence of virus in wastewater reflects the viral infections within the community, whether they are caused by wild viruses (coxsackievirus, echovirus) or by vaccine viruses (poliovirus). Recovery of virus by the suspended cell-agar plaque assay in samples considered negative for virus by the conventional plaque and roller tube culture assays (Table 6) emphasizes the merit of this assay for environmental samples containing low levels of virus. Our findings confirm those of Morris and Waite (23) who found a 10- to 100-fold-greater number of viral isolates from river water samples with this assay as compared with the standard plaque technique. Further studies are indicated to compare the distribution of viruses in the water column, with emphasis on the solid components. Methods used at present require the adjustment of the water ph to 3.5 and the addition of AIC13 to a final molarity of for processing water samples through negatively charged filters (32), and this involves considerable time and expense. Although positively charged filters were credited with efficient virus recoveries at ambient ph levels (7.0 to 7.5) from tap water (29), estuarine waters commonly having ph levels between 7.6 and 8.3 need ph adjustment to near neutrality. In contrast, collection of solids at an ambient ph of estuarine water is not only easy and less expensive to carry out, but it should result in the recovery of greater numbers of viruses. Viruses recovered from estuarine waters in the present study may have limited significance from a public health point of view, but they also serve as indicators for the occurrence of other human viruses like hepatitis A virus (enterovirus 72) and gastroenteritis viruses. The latter agents are well recognized as potential health hazards to recreational water users and shellfish consumers. ACKNOWLEDGMENTS This study was supported by the Texas A&M University Sea Grant College program, by the National Oceanic and Atmospheric Administration Office of Sea Grant, Department of Commerce, and by a grant from the National Oceanic and Atmospheric Administration, Office of Marine Pollution Assessment. K.M.S. was aided by a Berlin Airlift Memorial Fellowship (Federal Republic of Germany) and by the Mutual Educational Exchange (Fulbright) Scholarship Program (United States). The technical assistance of Sandy Secor and Edwin Schorr is gratefully acknowledged. Appreciation is expressed to Neil Travis, Director of Shellfish Sanitation Control, Texas Health Department, for furnishing maps of shellfish harvest areas in the Galveston Bay. LITERATURE CITED 1. Bitton, G., and R. Mitchell Effects of colloids on the survival of bacteriophages in seawater. Water Res. 8: Carlson, G. F., Jr., F. E. Woodward, D. F. Wentworth, and 0. J. Sproul Virus inactivation on clay particles in natural waters. J. Water Pollut. Control Fed. 40:R80-R Clarke, N. A., R. E. Stevenson, S. L. Chang, and P. W. Kabler Removal of enteric viruses from sewage by activated sludge treatment. Am. J. Public Health 51: Cooper, P. D The plaque assay of animal viruses. Adv. Virus Res. 8: De Flora, S., G. P. De Renzi, and G. Badolati Detection of animal viruses in coastal seawater and sediments. Appl. Microbiol. 30: Farrah, S. R., S. M. Goyal, C. P. Gerba, C. Wallis, and J. L. Melnick Concentration of enteroviruses from estuarine APPL. ENVIRON. MICROBIOL. water. Appl. Environ. Microbiol. 33: Gerba, C. P., and G. E. Schaiberger Effect of particulates on virus survival in seawater. J. Water Pollut. Control Fed. 47: Gerba, C. P., E. M. Smith, G. E. Schaiberger, and T. D. Edmond Field evaluation of methods for the detection of enteric viruses in marine sediments, p In C. D. Litchfield and P. L. Seyfried (ed.), Methodology for biomass determinations and microbial activities in sediments. American Society for Testing and Materials, Philadelphia. 9. Gerba, C. P., C. H. Stagg, and M. C. Abadh Characterization of sewage solid-associated viruses and behaviour in natural waters. Water Res. 12: Goyal, S. M., C. P. Gerba, and J. L. Melnick Prevalence of human enteric viruses in coastal canal communities. J. Water Pollut. Control Fed. 50: Hoff, J. C., and R. C. Becker The accumulation and elimination of crude and clarified poliovirus suspension by shellfish. Am. J. Epidemiol. 90: Katzenelson, E., B. Fattal, and T. Hostovesky Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. Environ. Microbiol. 32: LaBelle, R. L., and C. P. Gerba Influence of ph, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment. Appl. Environ. Microbiol. 38: LaBelle, R. L., and C. P. Gerba Influence of estuarine sediment on virus survival under field conditions. Appl. Environ. Microbiol. 39: Landry, E. F., J. M. Vaughn, T. J. Vicale, and R. Mann Accumulation of sediment-associated viruses in shellfish. AppI. Environ. Microbiol. 45: Lund, E., and V. Ronne On the isolation of virus from sewage treatment plant sludges. Water Res. 7: Malina, J. F., Jr., K. R. Ranganadhan, B. P. Sagik, and B. E. Moore Poliovirus inactivation by activated sludge. J. Water Pollut. Control Fed. 47: Melnick, J. L., and C. P. Gerba Viruses in water and soil. Public Health Rep. 9: Melnick, J. L., V. Rennick, B. Hampil, N. J. Schmidt, and H. H. Ho Lyophilized combination pools of enterovirus equine antisera: preparation and test procedures for the identification of field strains of 42 enteroviruses. Bull. W.H.O. 48: Melnick, J. L., H. A. Wenner, and C. A. Phillips Enteroviruses, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial and chlamydial infections, 5th ed. American Public Health Association, Washington, D.C. 21. Metcalf, T. G., and J. L. Melnick Sample apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus. Appl. Environ. Microbiol. 45: Metcalf, T. G., C. Wallis, and J. L. Melnick Virus enumeration and public health assessments in polluted surface water contributing to transmission of virus in nature, p In J. F. Malina and B. P. Sagik (ed.), Virus survival in water and waste water systems. Center for Research in Water Resources, The University of Texas, Austin. 23. Morris, R., and W. M. Waite Evaluation of procedures for recovery of viruses from water. ll. Detection systems. Water Res. 14: Murphy, A. M., G. S. Grahmann, P. T. Christopher, W. A. Lopez, G. R. Davey, and R. H. Millsom An Australia-wide outbreak of gastroenteritis from oysters caused by Norwalk virus. Med. J. Aust. 2: Portnoy, B. L., P. A. Machowiak, C. T. Caraway, J. A. Walker, T. W. McKinley, and C. A. Klein Oyster associated hepatitis: failure of shellfish certification programs to prevent outbreaks. J. Am. Med. Assoc. 233: Rao, V. C., S. B. Lakhe, S. V. Waghmare, and P. Dube Virus removal in activated sludge sewage treatment. Prog. Water Technol. 9: Rao, V. C., S. B. Lakhe, S. V. Waghmare, and V. Raman Virus removal in primary settling of raw sewage. J. Environ.

6 VOL ISOLATION OF ENTEROVIRUSES 409 Eng. Div. Am. Soc. Civ. Eng. 107: Smith, E. M., C. P. Gerba, and J. L. Melnick Role of sediment in the persistence of enteroviruses in the estuarine environment. Appl. Environ. Microbiol. 35: Sobsey, M. D., and B. L. Jones Concentration of poliovirus from tap water using positively charged microporous filters. Appl. Environ. Microbiol. 37: Stagg, C. H., C. Wallis, and C. H. Ward Inactivation of clay-associated bacteriophage MS-2 by chlorine. Appl. Environ. Microbiol. 33: Trask, J. D., J. L. Melnick, and H. A. Wenner Chlorination of human. monkey-adapted. and mouse strains of poliomyelitis virus. Am. J. Hyg. 41: Wallis, C., J. L. Melnick, and C. P. Gerba Concentration of viruses from water by membrane chromatography. Annu. Rev. Microbiol. 33: Ward, R. L., C. S. Ashley, and R. H. Moseley Heat inactivation of poliovirus in wastewater sludge. Appl. Environ. Microbiol. 32: Wellings, F. M., A. L. Lewis, and C. W. Mountain Demonstration of solids-associated virus in wastewater and sludge. Appl. Environ. Microbiol. 31: